Difference: ZeissDensitometer (9 vs. 10)

Procedure with screen shots available on web site http://www.nysbc.org/facilities/CEM/Zeiss_PhotoScan_Usage.html

Download MSWord format with screen shots at end * Zeiss-Photoscan-Usage.doc:

Contents

General Guidelines

• Always use gloves
• Always have something between the glass plattens to prevent scratching
• Never use compressed air sources (which inevitably contain water or oil),
  • use brushes to remove dust or clean glass while wet with glass cleaner - experts only
• Transfer and Delete your images from the local disk immediately after scanning they won't be there when you return
• Turn off scanner when no foreseable use later that day (to preserve lamp hours)

Turning on Scanner

• Scanner should be left on, with the light bulb off
  • The on switch of the bulb is on the right side of the scanner, toward the back. Up/down = on/off
  • Turn the scanner off then on again using the main on/off switch on the left side of the scanner
  • Turn the bulb to the on position
  • Computer will beep a few times, then the scanner will do a self-test
  • Wait until self test has finished "Self Test OK"
  • If no beep and no self test initiated, please call for help
• Turn on Windows Computer (open black door underneath scanner and toggle power switch)
  • Note that "Carl Zeiss" SCSI device is identified during start up
• Log onto computer
• Open Hyperterminal window (Controller Hyperterminal icon on Desktop - should open automatically)
• Start the Zeiss Photoscan software
  • Should get a bunch of messages from the scanner in the Hyperterminal window

Loading the film

• Use gloves
• Open cover
• Raise upper glass platten, which can be held up by the cover
• Remove dust from film and glass with brush
  • Do not use glass cleaner without assistance from NYSBC staf)
• Place film on lower glass platten emulsion down
  • Cutout corner goes on the upper left
• Lower upper glass platten
  • Glass will break if cover is lowered first
• Lower cover

Starting program

• Open Photoscan program, if not already started
• Select File -> New Scan
• Scan Setup - select nysbc_def.par
• Save setup parameters in your own file by changing nysbc_def.par and hitting save

Preview film

• Select file name on "Scan Setup" tab (e.g., E:\stokes\test-mrcgrid.tif)
• Select course Resolution on "Scan Setup" tab (e.g., 28 um)
• Make sure "Overview" is set for "None"
• Should not need to change anything on "Camera Definition" tab
• Select wide range for OD on "Film/Output" tab (e.g., 2.0 to 0.2)
• Save setup parameters (program crashes periodically)
• Select Scan -> Scan to Disk from main menu (F7)

Check normalization data

• Upon initiation of first scan of the day, a normalization is automatically done.
• Look at Hyperterminal window specifically for the following data * Information appears on monitor on top of scanner. Watch closely as it will scroll off screen.

• Min values should be within 50-60 of max values
• Max values should be < 255
• Three channels should be within 20 counts of one another

• Max values should be < 10 with no zeros

• Number of values cut should all be zero
• Any anomalies may indicate time to change the light bulb

Select Scan Area

• Use "Define Scan Area" tool along top tool bar (or in "View" menu)
• Adjust size of box to that desired for final scan
• Select File -> Scan Properties
  • check Histogram tab and adjust OD range under Film/Output
  • adjust Resolution (e.g. 7 um)
  • adjust image size (select "pixels" and enter size)
• Save setup parameters
  • please use your own file
  • program will be crashing soon, but parameters will be saved

Final Scan

• Select Scan -> Scan to Disk (or hit F7)
• Select File -> Scan Properties (or hit alt-ENTER) and look at Histogram tab
• Adjust any parameters if you want to rescan the image
  • OD range usually adjusted so that the densities comfortably fit within the chosen OD window.
  • However, at 7 um scan, min OD >0.6 is likely to cause an error (scanning hangs at the end of the 1st swath). If so, wait 3-4 minutes for a SCSI timeout. Lower the min OD value to 0.5 and rescan. This does not appear to be a problem with 14 um scan.
• Save the setup parameter file (please use your own file)
• Close window with image and agree to save the corresponding file
  • (otherwise will be deleted)

Adjust levels

• After proper level adjustment, histogram should look like this:
  

• Above was a film at measured OD 0.965 scanned at 7 um
• min density=0.61; max density=1.15; film negative, image positive
• in this case, setting min density higher than 0.61 gave an error at 7 um scan interval
• therefore, films should have OD 0.9-1 at most for scanning at highest (7 um) resolution

Shutdown

• Exit Photoscan
• Remove film from scanner
• Replace rubberized insert between glass plates (brush off any dust first)
• Be mindful of upper glass platten before closing lid
• Shutdown computer
  • You can leave computer on if returning in foreseeable future.
  • However, SCSI connection between computer and scanner seems to get corrupted after significant periods of time.
• Turn off scanner light bulb (right side of scanner, towards back
  • Leave scanner itself on, as it sometimes has problems starting from a cold-state

Getting your scanned images

• in order of preference:

Transfer files to File Server

• Run the Winscp software (icon on Desktop)
• auto connections are available to zeus, orpheus, and minotaur

Burn CD

• Open door to lower computer (black door under scanner)
• Insert blank CD in the drive
• Open "Nero Express" on desktop
• Select Data -> Data Disk and select files to include on CD

Transfer files to USB stick

• Not recommended:
  1. USB Connection is at the back and hard to access
  2. Speed is slow (USB1)
  3. possibility of transferring virus to old unpatched Windows 2000 computer

Conversion for Processing

• Use tif2mrc (part of IMOD package)

8-bit images

• for 8-bit images, tif2mrc follows recent mrc conventions and saves numbers as signed bytes (-128 -to +127)
• this is not what you want (imod will internally still treat them as unsigned bytes; other software may not)
• after conversion, can convert to signed bytes using newstack with -byte 0 option:

   newstack -byte 0 myfile.mrc myfile_fix.mrc

• or, convert to integer (mode 1) files without changing range:

   newsatck -mode 1 -mult 1,0 myfile.mrc myfile_fix.mrc

• The program /cryoem/script/tif8tomrc16.py (attached) will convert all tif images in current directory to integer (mode 1) files

12-bit images

• If images were scanned at 12-bit, need -s option to save as signed 16-bit

   tif2mrc -s myfile.tif myfile.mrc

• To convert all tif files:

   foreach f (*.tif)
      tif2mrc -s $f $f:r.mrc
      end

Troubleshooting

• Call staff for any problems starting or completing scans
• If you cannot log in to a remote computer, just burn a CD
  • Contact staff if blank CD's are not available in scanner room
• Photoscan software crash:
  • In event of a crash, try restarting Photoscan program
  • If simple restart doesn't work, try logging out of Windows PC and logging back in
  • If logout/login doesn't work, try rebooting Windows PC
  • If you still cannot connect, turn off Windows PC, turn off scanner, and restart from scratch
• Zeiss scanner errors
  • Please contact staff if scans fail to complete or normalization fails

Testing Scanner

Visit Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

* 8bit scan 7um OD 0.965: