Difference: CemHeliosInstructions (1 vs. 18)

Revision 1819 Jan 2015 - Main.BillRice

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Sample prep

  • Samples come in resin blocks

Trim block size

  • These generally need to be trimmed to ~1cm height so they will fit in SEM
  • Put in vise, hold with tweezers and use diamond blade to cut the top off of the sample
  • when done, clean the dust with Kimwipe soaked in ethanol/isopropanol
  • smaller blocks fit easier in microscope, and there is less plastic to charge up

Attach to stub

  • Put a small piece of double sided carbon sticky tape on a stub
  • Attach block to tape
  • Paint sample with conductive silver paint
    • try to paint as much as possible, being careful not to paint top of sample
    • do this in fume hood
  • When done, place sample in 60 degree over for 1h or more to remove solvent

Metal coating samples

  • samples need to be pre-coated with metal prior to imaging, otherwise excessive charging prevents good imaging
  • Since spring 2013, we have been using a gold-palladium source, and doing this in an Ar environment
  • Using a pure platinum source seemed to cause a lot of trouble: lots of charging even with a thick coat
  • standard protocol (Jan 2014, in place since spring 2013):
    • Remove freshly baked sample from oven and put in coater
    • start mechanical pump
    • Open pump valve
    • Pump down to ~100 mTorr (<2 minutes)
    • turn on rotation so you don't forget
    • Open Argon gas tank valve then turn on gas in control panel
      • Vacuum will immediately degrade to ~800 mTorr
    • Wait for vacuum to recover to 50-60 mTorr
      • Wait a few minutes before adjusting gas valve on coater. Do NOT adjust valves on Ar tank
    • When vacuum is ~50-60 mTorr, turn on sputter coater at 25% power
    • coat for 5 minutes, monitor vacuum and adjust gas valve if necessary
    • turn off sputtering
    • Stop rotation
    • Turn off gas valve
    • Close Ar tank
    • Close mechanical pump valve
    • Turn off mechanical pump and system will vent
  • Sample is now ready to go into microscope

  • This gives a thick conductive coat: approximately 600nm is deposited evenly under these conditions
  • You should be able to punch through this coat by increasing SEM voltage to ~15-25 keV.
  • may get a finer coat by doing ~5 min at 10% (?)

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • Standard account: supervisor
  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Useful information

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

E-beam 0.78 pA 1.6 pA 3.1 pA 6.3 pA 13 pA 25 pA 50 pA 100 pA 0.2 nA 0.4 nA 0.8 nA 1.6 nA 3.2 nA 6.4 nA 13 nA 26 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms

Starting a project

Prep

  • Vent
  • Insert sample
  • Pump down
  • I generally wait until vacuum is in 10^-6 mbar range before opening beam valves
  • Jan 2014: ultimate vacuum generally 1.8*10^-6 mbar, without cold trap
    • with cold trap: 1.4*10^-6 mbar
    • with cryo stage and anticontaminator cold: ~8*10^-7 mbar

Check IGP4

  • Mouse over vacuum cartoon of machine and check all pumps running.
  • If IGP4 does not have a value:
    1. Stop UI
    2. Log off windows
    3. Log on to support
    4. Start FEI system Control
    5. Right click bar and choose tstMdlVacuum
    6. Select manual mode
    7. Click IGP4 to start
    8. If not:
      • ClVi?
      • PlVi?
      • IGP4
      • Close ClVi? / PlVi?
    9. Manual mode
    10. Close
    11. Log off windows and log on to Supervisor
    12. FEI system Control > Start UI
    13. Right click > tiny view

Find an area

Goal: Find an area using the E-beam

Notes:

  • "Wake up" will start up both beams
  • "Sleep" will shut down both beams. Note: Ion beam start up will be equivalent to 2 days of useage.
  • "Beam on" is similar to a gun valve.

  • Upper left panel E-beam > Beam on - F6 to live view and F9 for Brightness/Contrast
  • View block - 2kV, 100pA. Detector - ETD > Secondary electrons
      • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • Reduced area to help focus
  • Link sample to working distance -> 4mm
    • Link 2nd time with reduced area (2nd red halo)
  • Find Eucentric height
    • Double click on feature to move stage
    • Navigation > Tilt > (user specified angle to tilt sample)
    • Change Z height
      • Lower right panel > Middle click and drag up

Slice and View Prep (after finding area)

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.2mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV (will likely need to move to refind area of interest)
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum deposition

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
    • Area x 2-6pA/um2 is a rule of thumb, but time also a consideration
    • Use ruler in full view to help gauge dimensions
  • View area - Change view area 512x442, 1us - defocus to blurry *optional
  • Use reduced area below deposition site (where trench milling will occur) to focus and fix stigmators
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench *optional

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV 1us dwell time
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Patterning > Regular cross section > Si
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift and is focused
    • Reposition box as necessary
  • Charge reducing beam is very helpful here to prevent drift and minimize redeposition
Charge reducing beam
  • turn on charge reducing beam (in detector panel)
  • beam will start off blanked
  • cannot view with any detector other than ICE detector when beam is unblanked. Attempting to do so will blank beam
  • change i-beam detector to ICE, leave as secondary ions
  • increase current to desired level
  • unblank beam
  • take a quick image
  • turn selected area on and place in part of sample to be dug
  • live view selected area
  • adjust beam parameters (focus, stig) and get good contrast
  • when charge reduction beam settings are right, there should be no drift even during long views
  • image also tends to have a herringbone pattern and look bad when settings are right
  • If you need to adjust, change voltage first. Be sure to read the manual and talk to an expert before changing anything!
  • when happy, start patterning
    • remember to not use i-spy or any e-beam imaging, or beam will blank
  • when done, blank the beam. Leave on until you are sure you are done, as sometimes it cannot be turned on again after turning off
  • filament takes several minutes to cool after being turned off

  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4-5um deep
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min
  • Charge reducing beam will let you clean a large area in a short time with a high current

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 1 um deep and 750 nm wide
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Use of charge reducing beam will minimize redeposition and wall formation!
  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material
  • Save position as I-beam milling view

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
    • Mirror - Reduced Area and adjust to brightest signal
    • 2kV, 2000-400nA
  • Direct Adjustments to beam
    • Direct Adjustments > Beam - make sure image will not move when focusing
      • Crossover > center brighter/best part of the beam
      • Lens modulator > up /down to minimize movement - for 2-3kV and above use lens modulator for 1-2 kV and below use HV modulator
    • Correct focus / astigmatism
      • Stigmator centering > X + Y, may manually wobbe stigmator for fine adjustment
  • Find settings that gives rise to best E-beam image
    • To help with view - Stage > Tilet correction > Dynamic Focus > Automatic (cross section)
  • Save position as E-beam face view

Add final fiducial marker and setting up positions

  • E-beam view > find area - 2048x1768, 3us, 2kV 0.20pA 12,000x * 300 ns
  • Go to mill position, mode 1 Fieldfree
  • Tilt 0 degrees
  • Coordinate > relative > deltaR 180 - Make sure XCompucentric Rotation
  • Tilt -9 degrees to draw O
  • I beam -> ETD Patterning > Circle, Si 4um outer diameter 2 um inner diameter z 0.5 um 30kV, 80pA, 2500x ~6min
    • Mill position low end - 80pA 0.23 nA 0.43nA 0.79na 2.5 nA - high end - select currect to give 0.5-2min/slice
    • Make sure position higher than desired, block drifts up during setup

Start slice and view program

  • New project
  • 210 : ION beam alignment
    • Start + Next
    • Stig, Stig Sin/Cos, Quad - minimize settings
    • End with Sig to focus+stigmators -> will save these settings for slice and view
    • Finish
  • Ibeam - Yellow box
    • 2048x1768 1-3us F6+F9 in reduced area slice thickness 20nm depth 4um (will mill ~8x deeper depending on resin)
    • Make sure mill width is larger than view and not milling O fiducial >20um
  • Ibeam - Red box
    • Drift correction > Use pre-existing -> Red box > Find
  • Sample prep > uncheck all - Do it yourself outside program
  • E-beam setup
    • E-beam view 3us 2048x1768, stage Processing > LUT > Inverse Integrate 4 frames F3 + brightness/contrast
    • Image 1 2048x1768, 3us, immersion -> tile if want larger area
    • Enable Alignment
      • Refresh + ... close
    • Enable Autofocus + ...
      • Autofocus > Course scan settings - reduced are 75% HFW 1024x884 3us 6-10 steps 18-30um - target 3um
      • Fine scan settings 75% HFW 2048x1768 4-6 steps 4 um - target 1 um
    • No Enable Active no Enable Y shift correction
  • Next > est time and Run
  • CNTL + F5 will show Bif view of live window
      • settings: E-beam 2kV TLD 200 pA 21.31 um 2048x1768 3us dwell time 4.03mm working distance
Added:
>
>

FIB Milling of cryo samples

Materials needed

  • 230 PSI nitrogen tank
  • clean lN2
  • frozen grids
  • autogrids
  • tools for cryo: tweezers
  • grid shuttle (holds 2 grids)
  • good lighting

Set up for cryo

Attach cryo stage

  • Put cryo stage in LOAD position (x=0; y=0; z=0; R=137.5 deg)
  • open door
  • remove RT stage and set aside on top of microscope
  • Attach lower part of cryo stage (stored in back of microscope) to platform using 2.5 mm Hex screw
    • three ink marks should be facing you directly
  • Attach upper part of cryo-stage to lower part using 2 mm Hec screws (orang hex screwdrivers)
    • three ink marks should line up
  • Put cryo dewar into microscope if it is not already there
  • pump microscope down
  • Move stage back to LOAD position since things probably moved when attaching cryo stage

Set up Quorum

  • Turn on Quorum computer
  • start Arctic control application
  • Fill Quorum dewar to maximum to ensure system will stay cold a long time
  • Choose to start system using auto settings
  • Follow instructions it gives you to start up
    • For best Vacuum, wait until chamber is in E-5 range before cooling
    • Cool down microscope dewar once it is 3E-6 range or lower to ensure best ultimate vacuum
 
  • Set ALLOWTOPICVIEW =

-- BillRice - 29 Aug 2011

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 1707 Feb 2014 - Main.BillRice

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Sample prep

  • Samples come in resin blocks

Trim block size

  • These generally need to be trimmed to ~1cm height so they will fit in SEM
  • Put in vise, hold with tweezers and use diamond blade to cut the top off of the sample
  • when done, clean the dust with Kimwipe soaked in ethanol/isopropanol
  • smaller blocks fit easier in microscope, and there is less plastic to charge up

Attach to stub

  • Put a small piece of double sided carbon sticky tape on a stub
  • Attach block to tape
  • Paint sample with conductive silver paint
    • try to paint as much as possible, being careful not to paint top of sample
    • do this in fume hood
  • When done, place sample in 60 degree over for 1h or more to remove solvent

Metal coating samples

  • samples need to be pre-coated with metal prior to imaging, otherwise excessive charging prevents good imaging
  • Since spring 2013, we have been using a gold-palladium source, and doing this in an Ar environment
  • Using a pure platinum source seemed to cause a lot of trouble: lots of charging even with a thick coat
  • standard protocol (Jan 2014, in place since spring 2013):
    • Remove freshly baked sample from oven and put in coater
    • start mechanical pump
    • Open pump valve
    • Pump down to ~100 mTorr (<2 minutes)
    • turn on rotation so you don't forget
    • Open Argon gas tank valve then turn on gas in control panel
      • Vacuum will immediately degrade to ~800 mTorr
    • Wait for vacuum to recover to 50-60 mTorr
      • Wait a few minutes before adjusting gas valve on coater. Do NOT adjust valves on Ar tank
    • When vacuum is ~50-60 mTorr, turn on sputter coater at 25% power
    • coat for 5 minutes, monitor vacuum and adjust gas valve if necessary
    • turn off sputtering
    • Stop rotation
    • Turn off gas valve
    • Close Ar tank
    • Close mechanical pump valve
    • Turn off mechanical pump and system will vent
  • Sample is now ready to go into microscope

  • This gives a thick conductive coat: approximately 600nm is deposited evenly under these conditions
  • You should be able to punch through this coat by increasing SEM voltage to ~15-25 keV.
  • may get a finer coat by doing ~5 min at 10% (?)

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

Added:
>
>
  • Standard account: supervisor
 
  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Useful information

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

E-beam 0.78 pA 1.6 pA 3.1 pA 6.3 pA 13 pA 25 pA 50 pA 100 pA 0.2 nA 0.4 nA 0.8 nA 1.6 nA 3.2 nA 6.4 nA 13 nA 26 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms

Starting a project

Prep

  • Vent
  • Insert sample
  • Pump down
  • I generally wait until vacuum is in 10^-6 mbar range before opening beam valves
  • Jan 2014: ultimate vacuum generally 1.8*10^-6 mbar, without cold trap
    • with cold trap: 1.4*10^-6 mbar
    • with cryo stage and anticontaminator cold: ~8*10^-7 mbar

Check IGP4

  • Mouse over vacuum cartoon of machine and check all pumps running.
  • If IGP4 does not have a value:
    1. Stop UI
    2. Log off windows
    3. Log on to support
    4. Start FEI system Control
    5. Right click bar and choose tstMdlVacuum
    6. Select manual mode
    7. Click IGP4 to start
    8. If not:
      • ClVi?
      • PlVi?
      • IGP4
      • Close ClVi? / PlVi?
    9. Manual mode
    10. Close
    11. Log off windows and log on to Supervisor
    12. FEI system Control > Start UI
    13. Right click > tiny view

Find an area

Goal: Find an area using the E-beam

Notes:

  • "Wake up" will start up both beams
  • "Sleep" will shut down both beams. Note: Ion beam start up will be equivalent to 2 days of useage.
  • "Beam on" is similar to a gun valve.

  • Upper left panel E-beam > Beam on - F6 to live view and F9 for Brightness/Contrast
  • View block - 2kV, 100pA. Detector - ETD > Secondary electrons
      • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • Reduced area to help focus
  • Link sample to working distance -> 4mm
    • Link 2nd time with reduced area (2nd red halo)
  • Find Eucentric height
    • Double click on feature to move stage
    • Navigation > Tilt > (user specified angle to tilt sample)
    • Change Z height
      • Lower right panel > Middle click and drag up

Slice and View Prep (after finding area)

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.2mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV (will likely need to move to refind area of interest)
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum deposition

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
    • Area x 2-6pA/um2 is a rule of thumb, but time also a consideration
    • Use ruler in full view to help gauge dimensions
  • View area - Change view area 512x442, 1us - defocus to blurry *optional
  • Use reduced area below deposition site (where trench milling will occur) to focus and fix stigmators
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench *optional

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV 1us dwell time
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Patterning > Regular cross section > Si
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift and is focused
    • Reposition box as necessary
  • Charge reducing beam is very helpful here to prevent drift and minimize redeposition
Charge reducing beam
  • turn on charge reducing beam (in detector panel)
  • beam will start off blanked
  • cannot view with any detector other than ICE detector when beam is unblanked. Attempting to do so will blank beam
  • change i-beam detector to ICE, leave as secondary ions
  • increase current to desired level
  • unblank beam
  • take a quick image
  • turn selected area on and place in part of sample to be dug
  • live view selected area
  • adjust beam parameters (focus, stig) and get good contrast
  • when charge reduction beam settings are right, there should be no drift even during long views
  • image also tends to have a herringbone pattern and look bad when settings are right
  • If you need to adjust, change voltage first. Be sure to read the manual and talk to an expert before changing anything!
  • when happy, start patterning
    • remember to not use i-spy or any e-beam imaging, or beam will blank
  • when done, blank the beam. Leave on until you are sure you are done, as sometimes it cannot be turned on again after turning off
  • filament takes several minutes to cool after being turned off

  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4-5um deep
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min
  • Charge reducing beam will let you clean a large area in a short time with a high current

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 1 um deep and 750 nm wide
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Use of charge reducing beam will minimize redeposition and wall formation!
  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material
  • Save position as I-beam milling view

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
    • Mirror - Reduced Area and adjust to brightest signal
    • 2kV, 2000-400nA
  • Direct Adjustments to beam
    • Direct Adjustments > Beam - make sure image will not move when focusing
      • Crossover > center brighter/best part of the beam
      • Lens modulator > up /down to minimize movement - for 2-3kV and above use lens modulator for 1-2 kV and below use HV modulator
    • Correct focus / astigmatism
      • Stigmator centering > X + Y, may manually wobbe stigmator for fine adjustment
  • Find settings that gives rise to best E-beam image
    • To help with view - Stage > Tilet correction > Dynamic Focus > Automatic (cross section)
  • Save position as E-beam face view

Add final fiducial marker and setting up positions

  • E-beam view > find area - 2048x1768, 3us, 2kV 0.20pA 12,000x * 300 ns
  • Go to mill position, mode 1 Fieldfree
  • Tilt 0 degrees
  • Coordinate > relative > deltaR 180 - Make sure XCompucentric Rotation
  • Tilt -9 degrees to draw O
  • I beam -> ETD Patterning > Circle, Si 4um outer diameter 2 um inner diameter z 0.5 um 30kV, 80pA, 2500x ~6min
    • Mill position low end - 80pA 0.23 nA 0.43nA 0.79na 2.5 nA - high end - select currect to give 0.5-2min/slice
    • Make sure position higher than desired, block drifts up during setup

Start slice and view program

  • New project
  • 210 : ION beam alignment
    • Start + Next
    • Stig, Stig Sin/Cos, Quad - minimize settings
    • End with Sig to focus+stigmators -> will save these settings for slice and view
    • Finish
  • Ibeam - Yellow box
    • 2048x1768 1-3us F6+F9 in reduced area slice thickness 20nm depth 4um (will mill ~8x deeper depending on resin)
    • Make sure mill width is larger than view and not milling O fiducial >20um
  • Ibeam - Red box
    • Drift correction > Use pre-existing -> Red box > Find
  • Sample prep > uncheck all - Do it yourself outside program
  • E-beam setup
    • E-beam view 3us 2048x1768, stage Processing > LUT > Inverse Integrate 4 frames F3 + brightness/contrast
    • Image 1 2048x1768, 3us, immersion -> tile if want larger area
    • Enable Alignment
      • Refresh + ... close
    • Enable Autofocus + ...
      • Autofocus > Course scan settings - reduced are 75% HFW 1024x884 3us 6-10 steps 18-30um - target 3um
      • Fine scan settings 75% HFW 2048x1768 4-6 steps 4 um - target 1 um
    • No Enable Active no Enable Y shift correction
  • Next > est time and Run
  • CNTL + F5 will show Bif view of live window
      • settings: E-beam 2kV TLD 200 pA 21.31 um 2048x1768 3us dwell time 4.03mm working distance

  • Set ALLOWTOPICVIEW =

-- BillRice - 29 Aug 2011

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 1615 Jan 2014 - Main.BillRice

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Changed:
<
<

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%
>
>

Sample prep

  • Samples come in resin blocks

Trim block size

  • These generally need to be trimmed to ~1cm height so they will fit in SEM
Added:
>
>
  • Put in vise, hold with tweezers and use diamond blade to cut the top off of the sample
  • when done, clean the dust with Kimwipe soaked in ethanol/isopropanol
  • smaller blocks fit easier in microscope, and there is less plastic to charge up

Attach to stub

  • Put a small piece of double sided carbon sticky tape on a stub
  • Attach block to tape
  • Paint sample with conductive silver paint
    • try to paint as much as possible, being careful not to paint top of sample
    • do this in fume hood
  • When done, place sample in 60 degree over for 1h or more to remove solvent

Metal coating samples

  • samples need to be pre-coated with metal prior to imaging, otherwise excessive charging prevents good imaging
  • Since spring 2013, we have been using a gold-palladium source, and doing this in an Ar environment
  • Using a pure platinum source seemed to cause a lot of trouble: lots of charging even with a thick coat
  • standard protocol (Jan 2014, in place since spring 2013):
    • Remove freshly baked sample from oven and put in coater
    • start mechanical pump
    • Open pump valve
    • Pump down to ~100 mTorr (<2 minutes)
    • turn on rotation so you don't forget
    • Open Argon gas tank valve then turn on gas in control panel
      • Vacuum will immediately degrade to ~800 mTorr
    • Wait for vacuum to recover to 50-60 mTorr
      • Wait a few minutes before adjusting gas valve on coater. Do NOT adjust valves on Ar tank
    • When vacuum is ~50-60 mTorr, turn on sputter coater at 25% power
    • coat for 5 minutes, monitor vacuum and adjust gas valve if necessary
    • turn off sputtering
    • Stop rotation
    • Turn off gas valve
    • Close Ar tank
    • Close mechanical pump valve
    • Turn off mechanical pump and system will vent
  • Sample is now ready to go into microscope

  • This gives a thick conductive coat: approximately 600nm is deposited evenly under these conditions
  • You should be able to punch through this coat by increasing SEM voltage to ~15-25 keV.
  • may get a finer coat by doing ~5 min at 10% (?)
 

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Useful information

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

E-beam 0.78 pA 1.6 pA 3.1 pA 6.3 pA 13 pA 25 pA 50 pA 100 pA 0.2 nA 0.4 nA 0.8 nA 1.6 nA 3.2 nA 6.4 nA 13 nA 26 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms

Starting a project

Prep

Deleted:
<
<
  • Plasma clean chamber
 
  • Vent
  • Insert sample
Changed:
<
<
  • Pump down 2x10^-6 mbar or less
>
>
  • Pump down
Added:
>
>
  • I generally wait until vacuum is in 10^-6 mbar range before opening beam valves
  • Jan 2014: ultimate vacuum generally 1.8*10^-6 mbar, without cold trap
    • with cold trap: 1.4*10^-6 mbar
    • with cryo stage and anticontaminator cold: ~8*10^-7 mbar
 

Check IGP4

  • Mouse over vacuum cartoon of machine and check all pumps running.
  • If IGP4 does not have a value:
    1. Stop UI
    2. Log off windows
    3. Log on to support
    4. Start FEI system Control
    5. Right click bar and choose tstMdlVacuum
    6. Select manual mode
    7. Click IGP4 to start
    8. If not:
      • ClVi?
      • PlVi?
      • IGP4
      • Close ClVi? / PlVi?
    9. Manual mode
    10. Close
    11. Log off windows and log on to Supervisor
    12. FEI system Control > Start UI
    13. Right click > tiny view

Find an area

Goal: Find an area using the E-beam

Notes:

  • "Wake up" will start up both beams
  • "Sleep" will shut down both beams. Note: Ion beam start up will be equivalent to 2 days of useage.
  • "Beam on" is similar to a gun valve.

  • Upper left panel E-beam > Beam on - F6 to live view and F9 for Brightness/Contrast
  • View block - 2kV, 100pA. Detector - ETD > Secondary electrons
      • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • Reduced area to help focus
  • Link sample to working distance -> 4mm
    • Link 2nd time with reduced area (2nd red halo)
  • Find Eucentric height
    • Double click on feature to move stage
    • Navigation > Tilt > (user specified angle to tilt sample)
    • Change Z height
      • Lower right panel > Middle click and drag up

Slice and View Prep (after finding area)

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
Changed:
<
<
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
>
>
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.2mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV (will likely need to move to refind area of interest)
 
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA
Changed:
<
<

I-beam platinum depostion

>
>

I-beam platinum deposition

 
  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
    • Area x 2-6pA/um2 is a rule of thumb, but time also a consideration
    • Use ruler in full view to help gauge dimensions
  • View area - Change view area 512x442, 1us - defocus to blurry *optional
  • Use reduced area below deposition site (where trench milling will occur) to focus and fix stigmators
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench *optional

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV 1us dwell time
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Patterning > Regular cross section > Si
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift and is focused
    • Reposition box as necessary
Changed:
<
<
    • May use ICE detector for charge reducing.
>
>
  • Charge reducing beam is very helpful here to prevent drift and minimize redeposition
Added:
>
>
Charge reducing beam
  • turn on charge reducing beam (in detector panel)
  • beam will start off blanked
  • cannot view with any detector other than ICE detector when beam is unblanked. Attempting to do so will blank beam
  • change i-beam detector to ICE, leave as secondary ions
  • increase current to desired level
  • unblank beam
  • take a quick image
  • turn selected area on and place in part of sample to be dug
  • live view selected area
  • adjust beam parameters (focus, stig) and get good contrast
  • when charge reduction beam settings are right, there should be no drift even during long views
  • image also tends to have a herringbone pattern and look bad when settings are right
  • If you need to adjust, change voltage first. Be sure to read the manual and talk to an expert before changing anything!
  • when happy, start patterning
    • remember to not use i-spy or any e-beam imaging, or beam will blank
  • when done, blank the beam. Leave on until you are sure you are done, as sometimes it cannot be turned on again after turning off
  • filament takes several minutes to cool after being turned off

 
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
Deleted:
<
<
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle
 

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4-5um deep
Changed:
<
<
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min
>
>
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min
Added:
>
>
  • Charge reducing beam will let you clean a large area in a short time with a high current
 

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 1 um deep and 750 nm wide
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

Added:
>
>
  • Use of charge reducing beam will minimize redeposition and wall formation!
 
  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material
  • Save position as I-beam milling view

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
    • Mirror - Reduced Area and adjust to brightest signal
    • 2kV, 2000-400nA
  • Direct Adjustments to beam
    • Direct Adjustments > Beam - make sure image will not move when focusing
      • Crossover > center brighter/best part of the beam
      • Lens modulator > up /down to minimize movement - for 2-3kV and above use lens modulator for 1-2 kV and below use HV modulator
    • Correct focus / astigmatism
      • Stigmator centering > X + Y, may manually wobbe stigmator for fine adjustment
  • Find settings that gives rise to best E-beam image
    • To help with view - Stage > Tilet correction > Dynamic Focus > Automatic (cross section)
  • Save position as E-beam face view

Add final fiducial marker and setting up positions

  • E-beam view > find area - 2048x1768, 3us, 2kV 0.20pA 12,000x * 300 ns
  • Go to mill position, mode 1 Fieldfree
  • Tilt 0 degrees
  • Coordinate > relative > deltaR 180 - Make sure XCompucentric Rotation
  • Tilt -9 degrees to draw O
  • I beam -> ETD Patterning > Circle, Si 4um outer diameter 2 um inner diameter z 0.5 um 30kV, 80pA, 2500x ~6min
    • Mill position low end - 80pA 0.23 nA 0.43nA 0.79na 2.5 nA - high end - select currect to give 0.5-2min/slice
    • Make sure position higher than desired, block drifts up during setup

Start slice and view program

  • New project
  • 210 : ION beam alignment
    • Start + Next
    • Stig, Stig Sin/Cos, Quad - minimize settings
    • End with Sig to focus+stigmators -> will save these settings for slice and view
    • Finish
  • Ibeam - Yellow box
    • 2048x1768 1-3us F6+F9 in reduced area slice thickness 20nm depth 4um (will mill ~8x deeper depending on resin)
    • Make sure mill width is larger than view and not milling O fiducial >20um
  • Ibeam - Red box
    • Drift correction > Use pre-existing -> Red box > Find
  • Sample prep > uncheck all - Do it yourself outside program
  • E-beam setup
    • E-beam view 3us 2048x1768, stage Processing > LUT > Inverse Integrate 4 frames F3 + brightness/contrast
    • Image 1 2048x1768, 3us, immersion -> tile if want larger area
    • Enable Alignment
      • Refresh + ... close
    • Enable Autofocus + ...
      • Autofocus > Course scan settings - reduced are 75% HFW 1024x884 3us 6-10 steps 18-30um - target 3um
      • Fine scan settings 75% HFW 2048x1768 4-6 steps 4 um - target 1 um
    • No Enable Active no Enable Y shift correction
  • Next > est time and Run
  • CNTL + F5 will show Bif view of live window
      • settings: E-beam 2kV TLD 200 pA 21.31 um 2048x1768 3us dwell time 4.03mm working distance

  • Set ALLOWTOPICVIEW =

-- BillRice - 29 Aug 2011

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 1519 Dec 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Useful information

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA
Changed:
<
<
Custom Strip 1.6 pA 3.1 pA 6.3 pA 13 pA 25 pA 50 pA 100 pA 0.2 nA 0.4 nA 0.8 nA 1.6 nA 3.2 nA 6.4 nA 13 nA 26 nA
>
>
E-beam 0.78 pA 1.6 pA 3.1 pA 6.3 pA 13 pA 25 pA 50 pA 100 pA 0.2 nA 0.4 nA 0.8 nA 1.6 nA 3.2 nA 6.4 nA 13 nA 26 nA
  110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms

Starting a project

Prep

  • Plasma clean chamber
  • Vent
  • Insert sample
  • Pump down 2x10^-6 mbar or less

Check IGP4

  • Mouse over vacuum cartoon of machine and check all pumps running.
  • If IGP4 does not have a value:
    1. Stop UI
    2. Log off windows
    3. Log on to support
    4. Start FEI system Control
    5. Right click bar and choose tstMdlVacuum
    6. Select manual mode
    7. Click IGP4 to start
    8. If not:
      • ClVi?
      • PlVi?
      • IGP4
      • Close ClVi? / PlVi?
    9. Manual mode
    10. Close
    11. Log off windows and log on to Supervisor
    12. FEI system Control > Start UI
    13. Right click > tiny view

Find an area

Goal: Find an area using the E-beam

Notes:

  • "Wake up" will start up both beams
  • "Sleep" will shut down both beams. Note: Ion beam start up will be equivalent to 2 days of useage.
  • "Beam on" is similar to a gun valve.

  • Upper left panel E-beam > Beam on - F6 to live view and F9 for Brightness/Contrast
  • View block - 2kV, 100pA. Detector - ETD > Secondary electrons
      • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • Reduced area to help focus
  • Link sample to working distance -> 4mm
    • Link 2nd time with reduced area (2nd red halo)
  • Find Eucentric height
    • Double click on feature to move stage
    • Navigation > Tilt > (user specified angle to tilt sample)
    • Change Z height
      • Lower right panel > Middle click and drag up

Slice and View Prep (after finding area)

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
    • Area x 2-6pA/um2 is a rule of thumb, but time also a consideration
    • Use ruler in full view to help gauge dimensions
  • View area - Change view area 512x442, 1us - defocus to blurry *optional
  • Use reduced area below deposition site (where trench milling will occur) to focus and fix stigmators
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench *optional

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV 1us dwell time
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Patterning > Regular cross section > Si
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift and is focused
    • Reposition box as necessary
    • May use ICE detector for charge reducing.
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4-5um deep
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 1 um deep and 750 nm wide
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material
  • Save position as I-beam milling view

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
    • Mirror - Reduced Area and adjust to brightest signal
    • 2kV, 2000-400nA
  • Direct Adjustments to beam
    • Direct Adjustments > Beam - make sure image will not move when focusing
      • Crossover > center brighter/best part of the beam
      • Lens modulator > up /down to minimize movement - for 2-3kV and above use lens modulator for 1-2 kV and below use HV modulator
    • Correct focus / astigmatism
      • Stigmator centering > X + Y, may manually wobbe stigmator for fine adjustment
  • Find settings that gives rise to best E-beam image
    • To help with view - Stage > Tilet correction > Dynamic Focus > Automatic (cross section)
  • Save position as E-beam face view

Add final fiducial marker and setting up positions

  • E-beam view > find area - 2048x1768, 3us, 2kV 0.20pA 12,000x * 300 ns
  • Go to mill position, mode 1 Fieldfree
  • Tilt 0 degrees
  • Coordinate > relative > deltaR 180 - Make sure XCompucentric Rotation
  • Tilt -9 degrees to draw O
  • I beam -> ETD Patterning > Circle, Si 4um outer diameter 2 um inner diameter z 0.5 um 30kV, 80pA, 2500x ~6min
    • Mill position low end - 80pA 0.23 nA 0.43nA 0.79na 2.5 nA - high end - select currect to give 0.5-2min/slice
    • Make sure position higher than desired, block drifts up during setup

Start slice and view program

  • New project
  • 210 : ION beam alignment
    • Start + Next
    • Stig, Stig Sin/Cos, Quad - minimize settings
    • End with Sig to focus+stigmators -> will save these settings for slice and view
    • Finish
  • Ibeam - Yellow box
    • 2048x1768 1-3us F6+F9 in reduced area slice thickness 20nm depth 4um (will mill ~8x deeper depending on resin)
    • Make sure mill width is larger than view and not milling O fiducial >20um
  • Ibeam - Red box
    • Drift correction > Use pre-existing -> Red box > Find
  • Sample prep > uncheck all - Do it yourself outside program
  • E-beam setup
    • E-beam view 3us 2048x1768, stage Processing > LUT > Inverse Integrate 4 frames F3 + brightness/contrast
    • Image 1 2048x1768, 3us, immersion -> tile if want larger area
    • Enable Alignment
      • Refresh + ... close
    • Enable Autofocus + ...
      • Autofocus > Course scan settings - reduced are 75% HFW 1024x884 3us 6-10 steps 18-30um - target 3um
      • Fine scan settings 75% HFW 2048x1768 4-6 steps 4 um - target 1 um
    • No Enable Active no Enable Y shift correction
  • Next > est time and Run
  • CNTL + F5 will show Bif view of live window
      • settings: E-beam 2kV TLD 200 pA 21.31 um 2048x1768 3us dwell time 4.03mm working distance

  • Set ALLOWTOPICVIEW =

-- BillRice - 29 Aug 2011

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 1419 Dec 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Useful information

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA
Added:
>
>
Custom Strip 1.6 pA 3.1 pA 6.3 pA 13 pA 25 pA 50 pA 100 pA 0.2 nA 0.4 nA 0.8 nA 1.6 nA 3.2 nA 6.4 nA 13 nA 26 nA

 110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms

Starting a project

Prep

  • Plasma clean chamber
  • Vent
  • Insert sample
  • Pump down 2x10^-6 mbar or less

Check IGP4

  • Mouse over vacuum cartoon of machine and check all pumps running.
  • If IGP4 does not have a value:
    1. Stop UI
    2. Log off windows
    3. Log on to support
    4. Start FEI system Control
    5. Right click bar and choose tstMdlVacuum
    6. Select manual mode
    7. Click IGP4 to start
    8. If not:
      • ClVi?
      • PlVi?
      • IGP4
      • Close ClVi? / PlVi?
    9. Manual mode
    10. Close
    11. Log off windows and log on to Supervisor
    12. FEI system Control > Start UI
    13. Right click > tiny view

Find an area

Goal: Find an area using the E-beam

Notes:

  • "Wake up" will start up both beams
  • "Sleep" will shut down both beams. Note: Ion beam start up will be equivalent to 2 days of useage.
  • "Beam on" is similar to a gun valve.

  • Upper left panel E-beam > Beam on - F6 to live view and F9 for Brightness/Contrast
  • View block - 2kV, 100pA. Detector - ETD > Secondary electrons
      • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • Reduced area to help focus
  • Link sample to working distance -> 4mm
    • Link 2nd time with reduced area (2nd red halo)
  • Find Eucentric height
    • Double click on feature to move stage
    • Navigation > Tilt > (user specified angle to tilt sample)
    • Change Z height
      • Lower right panel > Middle click and drag up

Slice and View Prep (after finding area)

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
    • Area x 2-6pA/um2 is a rule of thumb, but time also a consideration
    • Use ruler in full view to help gauge dimensions
  • View area - Change view area 512x442, 1us - defocus to blurry *optional
  • Use reduced area below deposition site (where trench milling will occur) to focus and fix stigmators
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench *optional

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV 1us dwell time
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Patterning > Regular cross section > Si
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift and is focused
    • Reposition box as necessary
    • May use ICE detector for charge reducing.
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4-5um deep
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 1 um deep and 750 nm wide
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material
  • Save position as I-beam milling view

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
    • Mirror - Reduced Area and adjust to brightest signal
    • 2kV, 2000-400nA
  • Direct Adjustments to beam
    • Direct Adjustments > Beam - make sure image will not move when focusing
      • Crossover > center brighter/best part of the beam
      • Lens modulator > up /down to minimize movement - for 2-3kV and above use lens modulator for 1-2 kV and below use HV modulator
    • Correct focus / astigmatism
      • Stigmator centering > X + Y, may manually wobbe stigmator for fine adjustment
  • Find settings that gives rise to best E-beam image
    • To help with view - Stage > Tilet correction > Dynamic Focus > Automatic (cross section)
  • Save position as E-beam face view

Add final fiducial marker and setting up positions

  • E-beam view > find area - 2048x1768, 3us, 2kV 0.20pA 12,000x * 300 ns
  • Go to mill position, mode 1 Fieldfree
  • Tilt 0 degrees
  • Coordinate > relative > deltaR 180 - Make sure XCompucentric Rotation
  • Tilt -9 degrees to draw O
  • I beam -> ETD Patterning > Circle, Si 4um outer diameter 2 um inner diameter z 0.5 um 30kV, 80pA, 2500x ~6min
    • Mill position low end - 80pA 0.23 nA 0.43nA 0.79na 2.5 nA - high end - select currect to give 0.5-2min/slice
    • Make sure position higher than desired, block drifts up during setup

Start slice and view program

  • New project
  • 210 : ION beam alignment
    • Start + Next
    • Stig, Stig Sin/Cos, Quad - minimize settings
    • End with Sig to focus+stigmators -> will save these settings for slice and view
    • Finish
  • Ibeam - Yellow box
    • 2048x1768 1-3us F6+F9 in reduced area slice thickness 20nm depth 4um (will mill ~8x deeper depending on resin)
    • Make sure mill width is larger than view and not milling O fiducial >20um
  • Ibeam - Red box
    • Drift correction > Use pre-existing -> Red box > Find
  • Sample prep > uncheck all - Do it yourself outside program
  • E-beam setup
    • E-beam view 3us 2048x1768, stage Processing > LUT > Inverse Integrate 4 frames F3 + brightness/contrast
    • Image 1 2048x1768, 3us, immersion -> tile if want larger area
    • Enable Alignment
      • Refresh + ... close
    • Enable Autofocus + ...
      • Autofocus > Course scan settings - reduced are 75% HFW 1024x884 3us 6-10 steps 18-30um - target 3um
      • Fine scan settings 75% HFW 2048x1768 4-6 steps 4 um - target 1 um
    • No Enable Active no Enable Y shift correction
  • Next > est time and Run
  • CNTL + F5 will show Bif view of live window
      • settings: E-beam 2kV TLD 200 pA 21.31 um 2048x1768 3us dwell time 4.03mm working distance

  • Set ALLOWTOPICVIEW =

-- BillRice - 29 Aug 2011

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 1316 Oct 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Useful information

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms

Starting a project

Prep

  • Plasma clean chamber
  • Vent
  • Insert sample
  • Pump down 2x10^-6 mbar or less

Check IGP4

  • Mouse over vacuum cartoon of machine and check all pumps running.
  • If IGP4 does not have a value:
    1. Stop UI
    2. Log off windows
    3. Log on to support
    4. Start FEI system Control
    5. Right click bar and choose tstMdlVacuum
    6. Select manual mode
    7. Click IGP4 to start
    8. If not:
      • ClVi?
      • PlVi?
      • IGP4
      • Close ClVi? / PlVi?
    9. Manual mode
    10. Close
    11. Log off windows and log on to Supervisor
    12. FEI system Control > Start UI
    13. Right click > tiny view

Find an area

Goal: Find an area using the E-beam

Notes:

  • "Wake up" will start up both beams
  • "Sleep" will shut down both beams. Note: Ion beam start up will be equivalent to 2 days of useage.
  • "Beam on" is similar to a gun valve.

  • Upper left panel E-beam > Beam on - F6 to live view and F9 for Brightness/Contrast
  • View block - 2kV, 100pA. Detector - ETD > Secondary electrons
      • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • Reduced area to help focus
  • Link sample to working distance -> 4mm
    • Link 2nd time with reduced area (2nd red halo)
  • Find Eucentric height
    • Double click on feature to move stage
    • Navigation > Tilt > (user specified angle to tilt sample)
    • Change Z height
      • Lower right panel > Middle click and drag up

Slice and View Prep (after finding area)

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
    • Area x 2-6pA/um2 is a rule of thumb, but time also a consideration
    • Use ruler in full view to help gauge dimensions
  • View area - Change view area 512x442, 1us - defocus to blurry *optional
  • Use reduced area below deposition site (where trench milling will occur) to focus and fix stigmators
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench *optional

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV 1us dwell time
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Patterning > Regular cross section > Si
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift and is focused
    • Reposition box as necessary
    • May use ICE detector for charge reducing.
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4-5um deep
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 1 um deep and 750 nm wide
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material
  • Save position as I-beam milling view

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
    • Mirror - Reduced Area and adjust to brightest signal
    • 2kV, 2000-400nA
  • Direct Adjustments to beam
    • Direct Adjustments > Beam - make sure image will not move when focusing
      • Crossover > center brighter/best part of the beam
      • Lens modulator > up /down to minimize movement - for 2-3kV and above use lens modulator for 1-2 kV and below use HV modulator
    • Correct focus / astigmatism
      • Stigmator centering > X + Y, may manually wobbe stigmator for fine adjustment
  • Find settings that gives rise to best E-beam image
    • To help with view - Stage > Tilet correction > Dynamic Focus > Automatic (cross section)
  • Save position as E-beam face view

Add final fiducial marker and setting up positions

  • E-beam view > find area - 2048x1768, 3us, 2kV 0.20pA 12,000x * 300 ns
  • Go to mill position, mode 1 Fieldfree
  • Tilt 0 degrees
  • Coordinate > relative > deltaR 180 - Make sure XCompucentric Rotation
  • Tilt -9 degrees to draw O
  • I beam -> ETD Patterning > Circle, Si 4um outer diameter 2 um inner diameter z 0.5 um 30kV, 80pA, 2500x ~6min
    • Mill position low end - 80pA 0.23 nA 0.43nA 0.79na 2.5 nA - high end - select currect to give 0.5-2min/slice
    • Make sure position higher than desired, block drifts up during setup

Start slice and view program

  • New project
  • 210 : ION beam alignment
    • Start + Next
    • Stig, Stig Sin/Cos, Quad - minimize settings
    • End with Sig to focus+stigmators -> will save these settings for slice and view
    • Finish
  • Ibeam - Yellow box
    • 2048x1768 1-3us F6+F9 in reduced area slice thickness 20nm depth 4um (will mill ~8x deeper depending on resin)
    • Make sure mill width is larger than view and not milling O fiducial >20um
  • Ibeam - Red box
    • Drift correction > Use pre-existing -> Red box > Find
  • Sample prep > uncheck all - Do it yourself outside program
  • E-beam setup
    • E-beam view 3us 2048x1768, stage Processing > LUT > Inverse Integrate 4 frames F3 + brightness/contrast
Added:
>
>
    • Image 1 2048x1768, 3us, immersion -> tile if want larger area
 
    • Enable Alignment
Added:
>
>
      • Refresh + ... close
    • Enable Autofocus + ...
      • Autofocus > Course scan settings - reduced are 75% HFW 1024x884 3us 6-10 steps 18-30um - target 3um
      • Fine scan settings 75% HFW 2048x1768 4-6 steps 4 um - target 1 um
    • No Enable Active no Enable Y shift correction
  • Next > est time and Run
  • CNTL + F5 will show Bif view of live window
      • settings: E-beam 2kV TLD 200 pA 21.31 um 2048x1768 3us dwell time 4.03mm working distance
 
  • Set ALLOWTOPICVIEW =

-- BillRice - 29 Aug 2011

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 1216 Oct 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Added:
>
>

Useful information

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms
 

Starting a project

Prep

  • Plasma clean chamber
  • Vent
  • Insert sample
  • Pump down 2x10^-6 mbar or less
Added:
>
>
 

Check IGP4

  • Mouse over vacuum cartoon of machine and check all pumps running.
  • If IGP4 does not have a value:
    1. Stop UI
    2. Log off windows
    3. Log on to support
    4. Start FEI system Control
    5. Right click bar and choose tstMdlVacuum
    6. Select manual mode
    7. Click IGP4 to start
    8. If not:
      • ClVi?
      • PlVi?
      • IGP4
      • Close ClVi? / PlVi?
    9. Manual mode
    10. Close
    11. Log off windows and log on to Supervisor
    12. FEI system Control > Start UI
    13. Right click > tiny view

Find an area

Changed:
<
<
  • Find an area using the E-beam
>
>		Goal: Find an area using the E-beam
 
Added:
>
>		Notes:
 
  • "Wake up" will start up both beams
  • "Sleep" will shut down both beams. Note: Ion beam start up will be equivalent to 2 days of useage.
  • "Beam on" is similar to a gun valve.

  • Upper left panel E-beam > Beam on - F6 to live view and F9 for Brightness/Contrast
Added:
>
>
  • View block - 2kV, 100pA. Detector - ETD > Secondary electrons
      • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • Reduced area to help focus
  • Link sample to working distance -> 4mm
    • Link 2nd time with reduced area (2nd red halo)
  • Find Eucentric height
    • Double click on feature to move stage
    • Navigation > Tilt > (user specified angle to tilt sample)
    • Change Z height
      • Lower right panel > Middle click and drag up

 

Slice and View Prep (after finding area)

Deleted:
<
<		Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms
d117 1
 

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
Changed:
<
<
  • View area - Change view are 512x442, 1us - defocus to blurry
>
>
    • Area x 2-6pA/um2 is a rule of thumb, but time also a consideration
Added:
>
>
    • Use ruler in full view to help gauge dimensions
  • View area - Change view area 512x442, 1us - defocus to blurry *optional
  • Use reduced area below deposition site (where trench milling will occur) to focus and fix stigmators
 
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench
Changed:
<
<

Dig trench

>
>

Dig trench *optional

 Need to mill area to be able to view area - 2x long in y as you want to view down in z
Changed:
<
<
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV
>
>
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV 1us dwell time
 
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
Added:
>
>
    • Patterning > Regular cross section > Si
 
    • Make sure below area of Pt dep / area of interest, can mill excess later
Changed:
<
<
  • Reduced Area view to make sure no drift
>
>
  • Reduced Area view to make sure no drift and is focused
 
    • Reposition box as necessary
Added:
>
>
    • May use ICE detector for charge reducing.
 
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
Changed:
<
<
  • Patterning tab - Cleaning cross section, Application - Si, Z-4um
>
>
  • Patterning tab - Cleaning cross section, Application - Si, Z-4-5um deep
 
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
Added:
>
>
    • 1 um deep and 750 nm wide
 
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material
Added:
>
>
  • Save position as I-beam milling view
 
Deleted:
<
<

Add final fiducial marker

d170 4
 

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
Deleted:
<
<
  • Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V
 
    • Mirror - Reduced Area and adjust to brightest signal
Added:
>
>
    • 2kV, 2000-400nA
 
  • Direct Adjustments to beam
Changed:
<
<
  • Correct focus / astigmatism
>
>
    • Direct Adjustments > Beam - make sure image will not move when focusing
Added:
>
>
      • Crossover > center brighter/best part of the beam
      • Lens modulator > up /down to minimize movement - for 2-3kV and above use lens modulator for 1-2 kV and below use HV modulator
    • Correct focus / astigmatism
      • Stigmator centering > X + Y, may manually wobbe stigmator for fine adjustment
 
  • Find settings that gives rise to best E-beam image
Changed:
<
<
  • Set ALLOWTOPICVIEW =
>
>
    • To help with view - Stage > Tilet correction > Dynamic Focus > Automatic (cross section)
  • Save position as E-beam face view
Added:
>
>

Add final fiducial marker and setting up positions

  • E-beam view > find area - 2048x1768, 3us, 2kV 0.20pA 12,000x * 300 ns
  • Go to mill position, mode 1 Fieldfree
  • Tilt 0 degrees
  • Coordinate > relative > deltaR 180 - Make sure XCompucentric Rotation
  • Tilt -9 degrees to draw O
  • I beam -> ETD Patterning > Circle, Si 4um outer diameter 2 um inner diameter z 0.5 um 30kV, 80pA, 2500x ~6min
    • Mill position low end - 80pA 0.23 nA 0.43nA 0.79na 2.5 nA - high end - select currect to give 0.5-2min/slice
    • Make sure position higher than desired, block drifts up during setup

Start slice and view program

  • New project
  • 210 : ION beam alignment
    • Start + Next
    • Stig, Stig Sin/Cos, Quad - minimize settings
    • End with Sig to focus+stigmators -> will save these settings for slice and view
    • Finish
  • Ibeam - Yellow box
    • 2048x1768 1-3us F6+F9 in reduced area slice thickness 20nm depth 4um (will mill ~8x deeper depending on resin)
    • Make sure mill width is larger than view and not milling O fiducial >20um
  • Ibeam - Red box
    • Drift correction > Use pre-existing -> Red box > Find
  • Sample prep > uncheck all - Do it yourself outside program
  • E-beam setup
    • E-beam view 3us 2048x1768, stage Processing > LUT > Inverse Integrate 4 frames F3 + brightness/contrast
    • Enable Alignment

  • Set ALLOWTOPICVIEW =
 -- BillRice - 29 Aug 2011

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 1115 Oct 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Added:
>
>

Starting a project

 
Added:
>
>

Prep

  • Plasma clean chamber
  • Vent
  • Insert sample
  • Pump down 2x10^-6 mbar or less

Check IGP4

  • Mouse over vacuum cartoon of machine and check all pumps running.
  • If IGP4 does not have a value:
    1. Stop UI
    2. Log off windows
    3. Log on to support
    4. Start FEI system Control
    5. Right click bar and choose tstMdlVacuum
    6. Select manual mode
    7. Click IGP4 to start
    8. If not:
      • ClVi?
      • PlVi?
      • IGP4
      • Close ClVi? / PlVi?
    9. Manual mode
    10. Close
    11. Log off windows and log on to Supervisor
    12. FEI system Control > Start UI
    13. Right click > tiny view

Find an area

  • Find an area using the E-beam

  • "Wake up" will start up both beams
  • "Sleep" will shut down both beams. Note: Ion beam start up will be equivalent to 2 days of useage.
  • "Beam on" is similar to a gun valve.

  • Upper left panel E-beam > Beam on - F6 to live view and F9 for Brightness/Contrast
 

Slice and View Prep (after finding area)

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms
d117 1

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
  • View area - Change view are 512x442, 1us - defocus to blurry
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift
    • Reposition box as necessary
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4um
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material

Add final fiducial marker

d170 4

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
  • Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
  • Find settings that gives rise to best E-beam image

  • Set ALLOWTOPICVIEW =

-- BillRice - 29 Aug 2011

Added:
>
>
  * helkios vacuum page:
Deleted:
<
<
META FILEATTACHMENT attachment="vacuum_page.PNG" attr="" comment="helkios vacuum page" date="1379961492" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" stream="vacuum_page.PNG" user="Main.BillRice" version="1"
  vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 1023 Sep 2013 - Main.BillRice

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • support Feico95-001
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Slice and View Prep (after finding area)

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms
d117 1

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
  • View area - Change view are 512x442, 1us - defocus to blurry
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift
    • Reposition box as necessary
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4um
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material

Add final fiducial marker

d170 4

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
  • Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
  • Find settings that gives rise to best E-beam image

Added:
>
>
  • Set ALLOWTOPICVIEW =

-- BillRice - 29 Aug 2011

  * helkios vacuum page:
Added:
>
>
META FILEATTACHMENT attachment="vacuum_page.PNG" attr="" comment="helkios vacuum page" date="1379961492" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" stream="vacuum_page.PNG" user="Main.BillRice" version="1"
  vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 913 Aug 2013 - Main.BillRice

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
Added:
>
>
  • support Feico95-001
 
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Slice and View Prep (after finding area)

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms
d117 1

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
  • View area - Change view are 512x442, 1us - defocus to blurry
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift
    • Reposition box as necessary
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4um
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
    • 2 rectangles at and angle to form an "X"

Expose milling face and remove any walls that may have formed

  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material

Add final fiducial marker

d170 4

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
  • Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
  • Find settings that gives rise to best E-beam image

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 824 Jun 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Slice and View Prep (after finding area)

Useful shortcuts - F6 - view F9 - brightness and contrast

Aperature strip

Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms
d117 1

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
  • View area - Change view are 512x442, 1us - defocus to blurry
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift
    • Reposition box as necessary
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4um
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min
Added:
>
>

Finish fiducial with I-beam

  • Cleaning cross-section - x,y,z : 0.67, 5.74, 0.75 µm = ~4 min/rectangle ~8min
 
    • 2 rectangles at and angle to form an "X"
Added:
>
>

Expose milling face and remove any walls that may have formed

  • Z - 2µm or 4µm for wall, 0.79nA X,Y,Z:34.5,1.63,2 µm = 16 min
    • Can use 0.23 nA | 0.43 nA | 0.79 nA for fine milling
  • 0.20nA 4µm to expose dark sample, free of resin and redeposited material

Add final fiducial marker

d170 4

View face

  • Image mode 2nd
    • E-beam -> ETD mode 1 - fieldfree is ageneral mode
    • TLD backscatter mode2 - Immersion for imaging * Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V to reduce secondary electron signal, backscatter have higher energy
  • Detector Settings = Secondary Electrons - > Backscatter
    • Suction Tube Voltage -245V
 
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
  • Find settings that gives rise to best E-beam image

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 724 Jun 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Slice and View Prep (after finding area)

Changed:
<
<		Useful shortcuts - F6 - view F9 - brightness and contrast
>
>		Useful shortcuts - F6 - view F9 - brightness and contrast
Added:
>
>		Aperature strip
Standard Strip 1.1 pA 7.7 pA 24 pA 40 pA 80 pA 80 pA 0.23 nA 0.43 nA 0.79 nA 0.79 nA 2.5 nA 9.3 nA 21 nA 47 nA 65 nA
2013.06.24 1.0 pA 7.6 pA 25 pA 42 pA 87 pA 82 pA 0.23 nA 0.53 nA 1.03 nA 0.81 nA 2.7 nA 9.9 nA 22 nA 48 nA 75 nA

110 - Ion Beam: Aperture alignment at 30 000V Slice and View only sees first aperature of duplicates = 1st and 2nd 0.79nA and 80pA switched

Volatage - defaults

500 V 1.0 kV 2.0kV 5.0 kV 8.0 kV 16.0 kV 30.0 kV

Field of view

512x442 1024x884 2048x1768 4096x3536

Time

50 ns 100 ns 300ns 1µs 3µs 10µs 30µs 100µs 300µs 500µs 1ms
 d117 1

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
  • View area - Change view are 512x442, 1us - defocus to blurry
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift
    • Reposition box as necessary
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4um
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min
    • 2 rectangles at and angle to form an "X"
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
  • Find settings that gives rise to best E-beam image

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 621 Jun 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Slice and View Prep (after finding area)

Useful shortcuts - F6 - view F9 - brightness and contrast d117 1

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
  • View area - Change view are 512x442, 1us - defocus to blurry
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift
    • Reposition box as necessary
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
Added:
>
>
    • Click on E beam tab and hit F6
  • Lower current to 80pA and check area
  • Extend trench if needed
  • Pt dep on trench - draw rectangle over trench leaving gap on top
    • Z:0.2um, X:59um, Y:48um 2.5nA 30kV - 7.5 min
  • Retract Pt dep needle

Clean surface

  • 0.79nA view in I beam, reposition area and use Reduced area to focus
  • Patterning tab - Cleaning cross section, Application - Si, Z-4um
  • Set rectangle large enough to be half out/in current face e.g. 2.5 nA - 34x1x4um - 8.5min
 
    • 2 rectangles at and angle to form an "X"
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
  • Find settings that gives rise to best E-beam image

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 521 Jun 2013 - Main.EdEng

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

Helios general information

  • factory ID: Brno02-QE
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

Alignment notes - Brandon Van Leer's visit (Nov 2011)

Added:
>
>

Slice and View Prep (after finding area)

Useful shortcuts - F6 - view F9 - brightness and contrast d117 1

E-beam platinum deposition

Goal : soft deposition to protect surface
  • Cool cryo holder - Chamber Pressure: 2.19 e-6 mbar 6/13 (not necessary)
  • Viewing : 15-25kV (finding cells) Lower voltage for normal viewing : 1-200pA (E-beam Live view 1-10us)
  • After finding area of interest sample should be ~4mm working distance. Lower to 4.5mm - Navigation tab : Z height
  • Lower voltage to 2.0 kV
  • In Patterning tab : In platinum dep (Click on green box) - Make sure Heat is Warm, if cold warm up
  • Draw green box over area of deposition (Reduced Area tool in menu bar)
  • Flow open for 5 min. Increase to ~1.6 nA and check position has not moved. (Toggle between Reduced Area and full view)
  • Patterning: Gas Injection = Close flow, retract Pt dep, reduce current to ~200 pA

I-beam platinum depostion

  • Warm up I-beam if not on ~10min
  • Beam Control Tab : Tilt stage to 52 degress for I beam view (or CNTL+I) - (CNTL+E - zero)
  • 7.7-24 pA I beam viewing, Beam control Beam On button, Integrate (1frame mode)
  • Change Mag and recenter area
  • Patterning - Draw rectangle, Basic = Application - Pt dep - Z size 1.00 um
    • Set current. In Advanced tab 2 pA per um2 e.g. 561.4 um2 24 min at 0.79 nA (target 10 min, but try to keep below 2.5nA current)
  • View area - Change view are 512x442, 1us - defocus to blurry
  • Insert Pt dep needle and check area did not move
  • Start patterning (Arrow in menu bar).

Add fiducial Pt dep with I-beam

  • Patterning tab - Draw rectangle, select Pt dep - X,Y,Z - 5um,5um,0.5um e.g. 80pA 30.0kV - 5 min
  • Place Pt dep box for fiducial marker middle, above main area of interest where previously Pt dep
  • Milling of fiducial will be after trench

Dig trench

Need to mill area to be able to view area - 2x long in y as you want to view down in z
  • Reduced Area view, move some nonimportant area, refocus - 24 pA 30kV
  • Patterning : Regular cross section, draw rectangle - 5um in Z, X Y will be relative to volume desired (XYZ: 47,55,5 - 21nA - 42min)
    • Make sure below area of Pt dep / area of interest, can mill excess later
  • Reduced Area view to make sure no drift
    • Reposition box as necessary
  • To monitor turn on iSPI, Time Interval 30s to check on progress (optional)
 
    • 2 rectangles at and angle to form an "X"
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
  • Find settings that gives rise to best E-beam image

* helkios vacuum page:
vacuum_page.PNG

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 428 Feb 2012 - Main.MarienaSilvestry

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)
Changed:
<
<

helios general information

>
>

Helios general information

 
  • factory ID: Brno02-QE
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)
Added:
>
>

Alignment notes - Brandon Van Leer's visit (Nov 2011)

 
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
Added:
>
>
  • Find settings that gives rise to best E-beam image

* helkios vacuum page:
vacuum_page.PNG

 
META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 306 Sep 2011 - Main.BillRice

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Added:
>
>

Platinum coating samples

  • samples need to be pre-coated with platinum prior to imaging, otherwise excessive charging prevents good imaging
  • currently (Aug 2011) do 2 min at 25% using sputter coater in Helios room
  • may get a finer coat by doing ~5 min at 10%

 

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

helios general information

  • factory ID: Brno02-QE
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)

    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
Deleted:
<
<
 
META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 229 Aug 2011 - Main.BillRice

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)
Changed:
<
<

helios login information

  • Brno02-QE
>
>

helios general information

  • factory ID: Brno02-QE
Added:
>
>
  • working distance: 4mm
  • resolution: 0.8 nm (at 20KV)
  • 1 um Si milling ~ 15 um EPON (in one sample)
 
    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism
Added:
>
>
 
META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"

Revision 129 Aug 2011 - Main.BillRice

 
META TOPICPARENT name="CryoEM"
Contents

Fei Helios Instructions

Making a Fiducial Marker from a bitmap

  • Open image in Image J
  • Choose Colors --> split RGB
    • 3 windows will pop up, one for red, green, blue
  • Make red window all black
  • make green and blue windows white where pattern is
    • green is whether to mill (white=mill); blue is depth (white is 100% of set depth)

helios login information

  • Brno02-QE

    • Mirror - Reduced Area and adjust to brightest signal
  • Direct Adjustments to beam
  • Correct focus / astigmatism

META FILEATTACHMENT attr="" autoattached="1" comment="Omniprobe notes - Brandon's visit" date="1330459652" name="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" path="FIB_Alignment_notes_from_Brandon_Van_Leer_Omniprobe.doc" size="56832" user="Main.MarienaSilvestry" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="helkios vacuum page" date="1379961493" name="vacuum_page.PNG" path="vacuum_page.PNG" size="48098" user="Main.BillRice" version="1"
META FILEATTACHMENT attr="" autoattached="1" comment="Helios alignment notes - Brandon's visit" date="1330459864" name="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" path="FIB_Alignment_notes_from_Brandon_Van_Leer_day1.docx" size="17003" user="Main.MarienaSilvestry" version="1"
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