Difference: MicroscopyMicroanaylsis (1 vs. 2)

Revision 213 Sep 2006 - Main.KdDerr

 
META TOPICPARENT name="KdDerr"
Contents

Kent Notes

  • indicators of good freeze
    • smooth membranes
    • round organelles *indicators of bad freeze
    • Echlin Book has good section
  • Heart and Brain difficult to HPF
    • Biopsy is an especially difficult technique
  • Advocated for lots of good and bad images for training purposes

Andres/Alex Notes

  • replace exhaust aperture to bring down cooling time
  • Alex wants simple sample
  • Freeze fracture as a cantrol
  • Walther paper on FF
  • FF replicas Dual layer coat
    • Pt 2nm
    • C 5nm
    • Images with backscatter detector or TEM
  • I want pictures of ice crystal damage in FF samples
  • sublimate - raise temp to -110C or higher must be mindful of recrystalization
  • Use isopropanol for FF
  • 5-15kV for SEM imaging
  • extraction replicas
    • 70% sufuric acid clean for 20-30 min
    • water wash
    • 14% hypochloric acid (bleach(?))
    • wash 2 steps
    • not clean if it has dark spots
    • over cleaned if it falls apart
  • Burt Menko at Northwastern FF expert
  • acetone saturated after 8 hours - why shorter runs work
Added:
>
>
  • helpful to do biopsies into syrefoam
  • Capilary tubes
    • Broken tip of scalpel sanded works well to cut and seal tubes
    • Can glue pipette tip to tube
    • Use 200 micron deep wells for tubes
    • clip ends of tubes before filling
    • one open end on tube facilitates infiltration
    • Hoenburg paper describes making scalpel
  • Agar plate good for providing humid environment to work in
  • 5 part planchette uses:
    • 0.4mm spaces
    • saphire disk
    • 10 micron spacer
    • saphinre disk
    • 0.5mm spacer
  • 5 part planchette allows for quicker heat transfer
 
Deleted:
<
<
 

FS Users group

  • photoactivating osmium with green laser - Mike Reedy (really inovative ideas)

Possible control samples for HPF

  • oil emulsion
  • transvecton vessicles
Changed:
<
<

NYSBC electricity Notes (inregards to the RMC cryomicrotome)

>
>

NYSBC electricity Notes (in regards to the RMC cryomicrotome)

 
  • high frequency noise prevents voltage from ever reaching zero V
  • Microtome heater particularly sensitive due to amplifier that needs to turn on and off for the heating circuit

Marko Notes

  • indium - brittle/maliable at low temp
  • liver difficult sample - water content high
  • cryosectioning at Wadsworth
    • 60% of samples adequate for imaging
    • 10% of samples suitable for tomography
  • Use FIB to make pilar sample 360 reconstruction
  • Mulder 2003 frozen section in FIB
  • FIB stage has bevel gear for 360 rotation
  • beam must be parallel to sample to prevent preferential milling
  • claims crevasing is due to poor freezing
  • moly grids make smoother films due to expansion coefficient

Studer Notes

  • Pressure artifacts
    • lipid phase change
    • filaments not straight - could be due to thick sample interference
    • DNA no longer crystaline
  • Hoechli (1970(?)) pressure/survival of cell graph
  • Cannot vitrify native brain tissue
    • Cryoprotectants have dehydrating effects
    • really cannot win with brain

  • Set ALLOWTOPICVIEW =

-- KdDerr - 13 Sep 2006

Revision 113 Sep 2006 - Main.KdDerr

 
META TOPICPARENT name="KdDerr"
Contents

Kent Notes

  • indicators of good freeze
    • smooth membranes
    • round organelles *indicators of bad freeze
    • Echlin Book has good section
  • Heart and Brain difficult to HPF
    • Biopsy is an especially difficult technique
  • Advocated for lots of good and bad images for training purposes

Andres/Alex Notes

  • replace exhaust aperture to bring down cooling time
  • Alex wants simple sample
  • Freeze fracture as a cantrol
  • Walther paper on FF
  • FF replicas Dual layer coat
    • Pt 2nm
    • C 5nm
    • Images with backscatter detector or TEM
  • I want pictures of ice crystal damage in FF samples
  • sublimate - raise temp to -110C or higher must be mindful of recrystalization
  • Use isopropanol for FF
  • 5-15kV for SEM imaging
  • extraction replicas
    • 70% sufuric acid clean for 20-30 min
    • water wash
    • 14% hypochloric acid (bleach(?))
    • wash 2 steps
    • not clean if it has dark spots
    • over cleaned if it falls apart
  • Burt Menko at Northwastern FF expert
  • acetone saturated after 8 hours - why shorter runs work

FS Users group

  • photoactivating osmium with green laser - Mike Reedy (really inovative ideas)

Possible control samples for HPF

  • oil emulsion
  • transvecton vessicles

NYSBC electricity Notes (inregards to the RMC cryomicrotome)

  • high frequency noise prevents voltage from ever reaching zero V
  • Microtome heater particularly sensitive due to amplifier that needs to turn on and off for the heating circuit

Marko Notes

  • indium - brittle/maliable at low temp
  • liver difficult sample - water content high
  • cryosectioning at Wadsworth
    • 60% of samples adequate for imaging
    • 10% of samples suitable for tomography
  • Use FIB to make pilar sample 360 reconstruction
  • Mulder 2003 frozen section in FIB
  • FIB stage has bevel gear for 360 rotation
  • beam must be parallel to sample to prevent preferential milling
  • claims crevasing is due to poor freezing
  • moly grids make smoother films due to expansion coefficient

Studer Notes

  • Pressure artifacts
    • lipid phase change
    • filaments not straight - could be due to thick sample interference
    • DNA no longer crystaline
  • Hoechli (1970(?)) pressure/survival of cell graph
  • Cannot vitrify native brain tissue
    • Cryoprotectants have dehydrating effects
    • really cannot win with brain

  • Set ALLOWTOPICVIEW =

-- KdDerr - 13 Sep 2006

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