Cell Sciences Imaging Facility: Beckman Center

• Typically get 3 arrays of 40, 200nm sections per coverslip array.

• That is 120 sections, on 3 coverslips or approximately 24 um in 2.5 hrs.

• Standard processing of 1-4 samples
• Some samples may be higher due to special needs/processing

• Standard processing fee for each additional sample beyond the base set of 4 samples, processed at the same time
• Some samples may be higher due to special needs/processing

The Tousimis 815, Series A, Critical Point Dryer is a fully automated instrument with a chamber size of 1.25" (ID) and 1.25" (H) and Soter condensor unit to capture waste alcohol and eliminate purge exhaust noise. Samples need to be dehydrated into 100% ethanol before CPD drying with liquid CO2. Samples are mounted (using various custom-made accessories) in ethanol in the CPD chamber and after cooling the chamber, the process will automatically advance through steps of purging, heating till Tousimis equalibrium is reached, followed by gradual bleeding of CO2 gas and venting to ambient pressures.

Training on TEM or SEM takes 4 hrs, divided into 3hrs (assisted) and 1hr (independent) use

• Charged per hour
• Instrument time is separate cost

This is a flat-fee item that covers a training session by CSIF technician for three hours and the setup of a CSIF service account for one microscope instrument. This is a prerequisite for all independent usage of each microscope in the facility.

Please contact CSIF at 650-723-2449 or email microscopy@stanford.edu to discuss your imaging needs and our time availability.  After which, CSIF will book time under your name for the training on the specific instrument.  Thank you.  You can also fill out the email form here.

This charge is added whenever extended technician assistance is rendered.

Grid Staining: Uranyl acetate and lead citrate contrast staining ??- charge per each additional grid over first 8 grids

Contrast staining of sections on grids (up to 8) for best morphological analysis: 3.5% Uranyl Acetate in Acetone followed by 2% Lead Citrate.

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More than 8 grids will be at a charge per each additional grid.

Zeiss Schott glass high-performance cover glass #1.5 170+/- 5um (thickness) 18x18mm (LxW). Zeiss # 474030-9000-000.??Required for all super-resolution applications on OMX microscope.??Highly recommended for critical imaging applications, particularly deconvolution. 100/box = 1 each. $5/each for Internal Stanford users

The Leica EM PACT2 HPF can freeze samples 100 to 200nm thick by 1.5mm in diameter. It can also accomodate 1.2mm round sapphire coverslips which cells can be grown on. (Leica 100nm and 200nm specimen carriers as well as sapphire discs as well as other HPF supplies available at cost)

2 to 8 samples should be lightly fixed using 2 to 4% EM grade Formaldehyde along with Glutaraldehyde between 0.01 to 0.1% to improve the morphology in 0.1M Sodium Cacodylate or PO₄ buffer @ RT 1hr and cut into 1mm³ cubes then placed into 4^oC. Fixed cells should be scraped/quenched and washed with buffer then equilibrated with warm 10% gelatin/spun down and pelleted then chilled on ice before cutting into 1mm³ or smaller. They will be embedded equilibrated in 2.3M Sucrose and then frozen on pins for sectioning using the Cryo-ultramicrotome.??

??

Typical cost. Single antibody/grid on up to 8 grids, including controls. ??This includes contrast (UA and Pb) staining when appropriate. Additional antibodies, double labeling etc. will increase time and cost.??

NOTES:??Sections should be collected on nickel grids treated with sticky grid solution or Formvar/Carbon coated. Copper grids can be used but uncoated copper grids should not be used.

Sections are mounted on shiny side of grid, all steps below are done with shiny side up.

All incubations should be done in a humidity chamber.

Block, localization and wash are done in ceramic spot plate (Coors).

Reagents for IEM:

PBST: 140 mM NaCl, 3mM KCl, 8mM Na2HPO4, 1.5 mM KH2PO4, 0.05% Tween20, pH7.4

Standard block: PBST, 0.5% (w/v) ovalbumin (Sigma), 0.5 % (w/v) BSA (Sigma)

Sticky grid solution:

4ml 0.5% formvar (Ted Pella, Inc.) (final concentration will be 0.08%)

21ml of a 24:1 (v/v) dichloroethane : chloroform solution

2 to 8 samples should be lightly fixed using 2 to 4% EM grade Formaldehyde along with Glutaraldehyde between 0.01 to 0.1% to improve the morphology in 0.1M Sodium Cacodylate or PO₄ buffer @ RT 1hr and cut into 1mm³ cubes then placed into 4^oC. They will be embedded in acrylic resins such as LR White (EMS) or Lowicryl (EMS). These allow for easy storage/trimming/handling of samples at room temperature once embedded.

• up to 4 coverslips at the same time minimum
• separate charge will apply per additional coverslip at same time
• can do multiple antibodies if raised in different organisms (if no cross-reactivity).

• Fee for each additional coverslip, beyond the base session charge for first 4 coverslips, processed at the same time
• Separate minimum session charge will include first 4 coverslips
• can do multiple antibodies if raised in different organisms (if no cross-reactivity).

This is used to bill out cost recovery of variable consumable supplies such as specialty coverslips that may not be in existing add-on categories. Please comment and specify what this charge exactly covers per the order.

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NOTE: There is a minimum charge for Negative Staining that includes up to 5 grids.

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NOTE: A charge per grid will apply for each additional grid after first five.

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Items needed:

25uM is a good starting point for protein concentration

1% Uranyl Acetate in ddH₂O

Grids: Formvar and Carbon coated copper grids (300-400 mesh; Cat#F F300-Cu), glow discharged

DUMONT N4AC Dumoxel tweezers are anti-capillary (Cat# 72870-D www.emsdiasum.com)

Procedure for negative staining:

1. Place ~4ul of purified protein on glow discharged 300mesh carbon/formvar coated Cu grids.
2. Allow to settle 3 min.
3. Wash grids with 3 drops 1% Uranyl Acetate in ddH₂O then allow 3rd drop to sit on grid for 1 min.
4. Pull off most of drop with filter paper and allow to dry.
5. View samples at 120kV on the TEM.

SEM imaging is done by the staff electron microscopist, using the user/researcher's prepared samples. The user should preferably be present (at least partially) during the first imaging session to discuss and indicate the feautures of interest, and image magnifications required. Data can be downloaded from the SEM-PC on site, or remotely from CSIF servers, or sent to the user through Stanford Box.

Samples are aldehyde fixed and resin embedded for ultrathin sectioning similar to TEM analysis, but with additional en bloc staining techniques with heavt metals to improve conductivity of ultrahin sections on glass sides. Slides are carbon coated for improved conductivity, and scanned serially with FESEM to obtain a stack of images for 3D reconstruction.

PROCESS

Fix samples in 4%PFA/2%Glut in 0.1M NaCacodylate Buffer (4h+) Keep overnight or for extended periods of time at 4deg Celsius, fully submerged in fixatives and sealed with parafilm.

Wash in same buffer (2x5min)

OTO:

Post-fix in 1% OsO4 (2hrs) - highly toxic and volatile, keep in vacuum hood

Wash in H₂O (3x5min)

Post-fix in TCH: 30min

Wash 3-x5min

Post-fix OsO4: 1hr

Wash 3x5min

Post-fix 2% UranylAcetate: 2hrs (or 1% overnight @ 4C)

Wash 3x H₂O

Post-fix Pb-Citrate: 1-2min, as for section staining (closed container, pellet NaOH to absorb CO₂) not for 2^nd series of embedding

Wash H₂O

DEHYDRATE: 30-50-70-90-100-100% EtOH

Dehydrate in 100% Acetonitrile, replace with Acetone (2x) 2^nd embedding, prevent curling of membrane

Infiltrate Epon:Acetone 50%[1:1], 66% [2:1] (overnight)100% (4-6hrs) on rotator

Embed in fresh 100% resin, polymerize @ 60^oC 2days

Thiocarbohydrazide (1% (wt/vol) aqueous solution) Heat 25 ml of ddH₂O on a hot plate to 58^oC. Add 0.25 g of TCH to the heated water while stirring until the powder is dissolved, and then leave the solution for 2-3 h; the color will darken. Turn down the heat while stirring. Remove the solution from the hot plate and allow it to cool to room temperature (25^oC). Filter using a 0.45 um syringe filter. Store at room temperature to avoid precipitation for up to 1 week

Prepared specimens (CPD dried and/or cut to size) are mounted on 12 or 25mm aluminum pin stubs using colloidal graphite or Copper tape (or Carbon tabs for Hitachi 3400N VP-SEM only). Strictly NO carbon tabs are allowed in the Zeiss Sigma FESEM.

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For improved conductivity specimens are coated with gold/Palladium (Au/Pd) to a thickness of 50-100Angstroms, using the Denton Desk II sputter-coater. Non-conductive samples can be used uncoated with the Hitachi VP-SEM using low vacuum mode.

• typical time/costs??

Fix tissue (or cells on coverslips, or cells on hydrogels) 4 hrs to overnight in 2% Glutaraldehyde with 4% PFA (Paraformaldehyde) in 0.1M Na Cacodylate Buffer (pH 7.3). Samples should be either cut to appropriate size, grown on 12-22 mm coverslips, or centrifuged to concentrate small particles. Submerge samples in fixatives, close with parafilm, and keep cold (4^oC) for 1hr to weeks, before continuing processing. Never allow samples to dry out, or warm to room temperature in fixatives for longer than 1hr. Bacterial samples must be grown to late log pahse (OD~0.6) for sufficient numbers, or grow as biofilm on a suitable surface, or as cell culture on poly-L-lysine coated coverslip. Bacteria can also be fixed in eppendorf tubes, and 100ul aliquots pipetted onto PLL coated coverlips. Vacuum-filtration of bacteria through microporous filters (0.22-0.45um pore size) also allows for retention of small particels, cell clusters or univellular organisms on usable surfaces. Microporous capsules (70-200um pores) that are compatbile with CPD drying can also be used with great success to retain and easily handle small particles during processing.

Wash 5 min in same buffer

Post-fix 1 hr in 1% aqueous OsO₄ (Osmium Tetroxide) Highly toxic!! Use in hood!!.

Wash 2x 5 min in mQ H₂O

*Dehydrate in increasing ethanol series: 50-70-90-100-100% (10min each).

**CPD (Critical Point Dry) with liquid CO₂ with Tousimis Autosamdri 815.

Mount samples with double-sided carbon tape, or Colloidal Graphite, on Aluminum stubs.

Sputter-coat with Pd/Au - 100 Angstrom layer (3-5 min) with the Denton DeskII system in lab.

* Hydrogels and samples in VP-mode, using coolstage control: do not dehydrate, CPD or sputter-coat.

** CPD drying can be replaced by HMDS (Hexamethyldisalazane) drying in conditions where the size, orientation, or other physical parameters are not compatible with CPD conditions. Replace the final 100% ethanol with a 50% solution of HMDS in ethanol (15min), followed by 100% ethanol (2x15min). Remove all HMDS and allow sample to dry overnight in a desiccator.

Supplies needed

Suppliers:

Electron Microscopy Sciences http://www.emsdiasum.com/microscopy/default.aspx

Ted Pella http://www.tedpella.com/

Fixatives:

16% PFA (EMS Cat #15710)

8% Glut (EMS Cat #16020) with

0.2M NaCacodylate (EMS CAT #11652)

Mix equal volumes (1 vial each) of PFA and Glutaraldehyde with 20 ml Buffer for required fix conc

2% OsO4 (EMS Cat #19192) dilute with mQ water to 1% conc.

Colloidal Graphite: Ted Pella #16053

Specimen Mounts: Ted Pella #16111 (1/2 dm slotted head)

Carbon conductive Tape: Ted Pella #16073

Pelco Tabs 12mm: Ted Pella #16084-1

Pelco Tabs 25mm Ted Pella #16084-2

Carbon conductive sheet (x10): Ted Pella #16085-1

Microporous capsules: EMS # 70187 (30 and 78 micron)

The Denton Desk II sputtering unit is equipped for Gold/Palladium coating and includes a thickness monitor. The secimen chamber can accomodate ten 15mm (D) SEM stubs at a time. Ease of use and reliability are features valuable for use by students from all disciplines.

Epon Embedding of Cells on Coverslips (typically 4 to 8 samples)(TestJP 8-14-12)

Sample Fixation: (Preferably grown on 12mm round glass or 13mm round Thermanox coverslips)

1. Samples fixed in 2 % Glutaraldehyde(8% stock-EM grade) and 4% p-Formaldehyde in 0.1M NaCacodylate (or HEPES, Tris, PO₄) buffer pH~7.2 for 20 min @ RT then moved to 4^oC for 40 min to a couple of weeks.

Post-Fixation for Epon (Fume hood, Osmium is Toxic!!) All at 4^oC

1. Fix is then gently replaced with1% OsO4 in ddH2O, Rotated gently and left for 1 hr
2. Wash samples briefly 3X each in ddH2O
3. Change to 1% Uranyl acetate in ddH2O, stained for 2 hrs to Overnight.

III. Dehydration:

1. 50% EtOH, 5 min
2. 70% EtOH, 5 min
3. 95% EtOH, 10 min (allowed to warn to room temp)
4. 100% EtOH, 2X, 15 min each @ RT
5. Changed to Acetonitrile for 15 min

Epon* Infiltration: (Use gloves) *Epon = EMbed 812 using 20ml EMbed 812 and 16ml DDSA and 8ml NMA with 1.2ml BDMA

1. Change to 1:1 Epon/ Acetonitrile for 1hr
2. Change to 2:1 EPON/ Acetonitrile and leave 1 hr
3. Replace with 100% Epon 1 hr
4. Change to fresh Epon, place labels into beem capsules and fill with fresh resin, put on top of coverslip.
5. Polymerize in oven @ 65^oC O/N, I usually make about 2 labeled beem capsules/coverslip
6. Remove plastic from bottom of coverslip and dissolve glass w/HF (49%) for 20 min

I. Sample Fixation(2 to 8 samples):

1. Samples are usually fixed w/2% glutaraldehyde & 4% paraformaldehyde in 0.1M Sodium Cacodylate buffer, pH 7.4 for 1 hour @Room Temp and cut into blocks ~1mm³. Other buffers can be used: e.g. HEPES or PO_4. After fixation samples are then moved to 4^oC for immediate processing. Some people have left samples in fix for days to weeks with little change to the morphology.

To enrobe cells in gelatin spin cells down 5 min @ 4kX and wash in buffer 3X: re-suspend cells in warm gelatin, 10% in PBS, for 5 min/spin 5 min @ 4 kX, place on ice 5 minutes then cut into blocks. Cover with cold Osmium(step 2).

II. Post-Fixation @ 4^oC(Use Fume hood and gloves, Osmium is toxic)

2. Remove fixative and add 1% OsO₄ in ddH₂O Shake gently and leave for 1 hr @ 4^o
3. Wash samples 3X, 5 min each w/cold ddH₂O
4. Change to 1% Uranyl acetate in ddH₂O, stain for 2 hr to O/N

III. Dehydration:

5. 50% EtOH, 10 min
6. 70% EtOH, 10 min
7. 95% EtOH, 10 min (allow to warm to room temperature, rest performed at RT)
8. 100% EtOH, 2X, 10 min each @ RT
9. Acetonitrile 15 min

IV. Epon* Infiltration: (Use gloves):

10. Change to 1:1 Acetonitrile/Epon 1hr
11. Change to 1:2 Acetonitrile/Epon Overnight
12. Change to 100% Epon 2-3 hrs

Place in molds labeled and filled with 100% Epon and allow to settle for 4hr to Overnight (O/N)

13. Polymerize in 65^oC oven 24hrs

Tomographic reconstruction of 3-D data, based on images acquired as tilted projections, using intermediate voltage electrons interpreted by IMOD which is a set of image processing, modeling and display programs used for tomographic reconstruction and for 3D reconstruction of EM serial sections and optical sections. The package contains tools for assembling and aligning data within multiple types and sizes of image stacks, viewing 3-D data from any orientation, and modeling and display of the image files. IMOD was developed primarily by David Mastronarde, Rick Gaudette, Sue Held, Jim Kremer, and Quanren Xiong at the Boulder Laboratory for 3-D Electron Microscopy of Cells.

??

***Was originally name:??High Pressure Freezing/Freeze Substitution***

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Two to Eight samples are typically accepted as one run. The Leica EM PACT2 HPF can freeze samples 100 to 200nm thick by 1.5mm in diameter. It can also accomodate 1.2mm round sapphire coverslips which cells can be grown on. Samples are then freeze substituted/processed into an Eponate for morphological analysis or Acrylic resin for immuno-gold localization.

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• NOTE: more difficult samples may require additional time and cost.

• Note: more difficult samples may require additional time and cost.

Obtain Semi-thin (350 to 1000nm thick) and ultrathin (50 to 120nm thick) sections.

??

NOTE: More difficult blocks require additional time/cost.

??

• NOTE: More difficult blocks require additional time/cost.

The Denton Vacuum Bench Top Turbo (BTT) is a fast, clean, high vacuum bench top evaporator with simple automated operation for evporation or sputtering. It utilizes solid control electronics to control the pumpdown and venting sequences. The pumping package consists of a turbomolecular as well as a mechanical pump. It has a 1kV evaporation power supply for carbon coating, and 4000 volt AC glow, as well as a sputtering module for depositing noble metal. Also, a rotaing and tilting substrate table is included.

OPTIONS:

1. AC GLOW DISCHARGE: All materials exposed to our atmosphere tend to accumulate molecular lauers of oill and water on the surface. These few moleculaar latyers cause the surface to repel water. Carbon fioms so contaminated will cause aqueous solutions to bead rather than to spread over the surface. Contaminated griads will not pick up replicas readily. The AC glow discharge will clean the carbon support films and grids in vacuum of the molecular layers of oil and water.

2. CARBON EVAPORATION SOURCE: The carbon evaporation source is adjustable and is designed to provide carbon fims for support, replication or conduction. Rods 0.04mm (D) are evaporated for 30sec to 2 minutes.

Data processing computer installed with software for in image data quantification and 3D rendering.

• Typically get 3 arrays of 40, 200nm sections per coverslip array.

• That is 120 sections, on 3 coverslips or approximately 24 um in 2.5 hrs.

• Standard processing of 1-4 samples
• Some samples may be higher due to special needs/processing

• Standard processing fee for each additional sample beyond the base set of 4 samples, processed at the same time
• Some samples may be higher due to special needs/processing

• up to 4 coverslips at the same time minimum
• separate charge will apply per additional coverslip at same time
• can do multiple antibodies if raised in different organisms (if no cross-reactivity).

• Fee for each additional coverslip, beyond the base session charge for first 4 coverslips, processed at the same time
• Separate minimum session charge will include first 4 coverslips
• can do multiple antibodies if raised in different organisms (if no cross-reactivity).

The Tousimis 815, Series A, Critical Point Dryer is a fully automated instrument with a chamber size of 1.25" (ID) and 1.25" (H) and Soter condensor unit to capture waste alcohol and eliminate purge exhaust noise. Samples need to be dehydrated into 100% ethanol before CPD drying with liquid CO2. Samples are mounted (using various custom-made accessories) in ethanol in the CPD chamber and after cooling the chamber, the process will automatically advance through steps of purging, heating till Tousimis equalibrium is reached, followed by gradual bleeding of CO2 gas and venting to ambient pressures.

The Leica EM PACT2 HPF can freeze samples 100 to 200nm thick by 1.5mm in diameter. It can also accomodate 1.2mm round sapphire coverslips which cells can be grown on. (Leica 100nm and 200nm specimen carriers as well as sapphire discs as well as other HPF supplies available at cost)

The Denton Desk II sputtering unit is equipped for Gold/Palladium coating and includes a thickness monitor. The secimen chamber can accomodate ten 15mm (D) SEM stubs at a time. Ease of use and reliability are features valuable for use by students from all disciplines.

Obtain Semi-thin (350 to 1000nm thick) and ultrathin (50 to 120nm thick) sections.

The Denton Vacuum Bench Top Turbo (BTT) is a fast, clean, high vacuum bench top evaporator with simple automated operation for evporation or sputtering. It utilizes solid control electronics to control the pumpdown and venting sequences. The pumping package consists of a turbomolecular as well as a mechanical pump. It has a 1kV evaporation power supply for carbon coating, and 4000 volt AC glow, as well as a sputtering module for depositing noble metal. Also, a rotaing and tilting substrate table is included.

OPTIONS:

1. AC GLOW DISCHARGE: All materials exposed to our atmosphere tend to accumulate molecular lauers of oill and water on the surface. These few moleculaar latyers cause the surface to repel water. Carbon fioms so contaminated will cause aqueous solutions to bead rather than to spread over the surface. Contaminated griads will not pick up replicas readily. The AC glow discharge will clean the carbon support films and grids in vacuum of the molecular layers of oil and water.

2. CARBON EVAPORATION SOURCE: The carbon evaporation source is adjustable and is designed to provide carbon fims for support, replication or conduction. Rods 0.04mm (D) are evaporated for 30sec to 2 minutes.

• Charged per hour
• Instrument time is separate cost

Grid Staining: Uranyl acetate and lead citrate contrast staining ??- charge per each additional grid over first 8 grids

Contrast staining of sections on grids (up to 8) for best morphological analysis: 3.5% Uranyl Acetate in Acetone followed by 2% Lead Citrate.

??

More than 8 grids will be at a charge per each additional grid.

2 to 8 samples should be lightly fixed using 2 to 4% EM grade Formaldehyde along with Glutaraldehyde between 0.01 to 0.1% to improve the morphology in 0.1M Sodium Cacodylate or PO₄ buffer @ RT 1hr and cut into 1mm³ cubes then placed into 4^oC. Fixed cells should be scraped/quenched and washed with buffer then equilibrated with warm 10% gelatin/spun down and pelleted then chilled on ice before cutting into 1mm³ or smaller. They will be embedded equilibrated in 2.3M Sucrose and then frozen on pins for sectioning using the Cryo-ultramicrotome.??

??

Typical cost. Single antibody/grid on up to 8 grids, including controls. ??This includes contrast (UA and Pb) staining when appropriate. Additional antibodies, double labeling etc. will increase time and cost.??

NOTES:??Sections should be collected on nickel grids treated with sticky grid solution or Formvar/Carbon coated. Copper grids can be used but uncoated copper grids should not be used.

Sections are mounted on shiny side of grid, all steps below are done with shiny side up.

All incubations should be done in a humidity chamber.

Block, localization and wash are done in ceramic spot plate (Coors).

Reagents for IEM:

PBST: 140 mM NaCl, 3mM KCl, 8mM Na2HPO4, 1.5 mM KH2PO4, 0.05% Tween20, pH7.4

Standard block: PBST, 0.5% (w/v) ovalbumin (Sigma), 0.5 % (w/v) BSA (Sigma)

Sticky grid solution:

4ml 0.5% formvar (Ted Pella, Inc.) (final concentration will be 0.08%)

21ml of a 24:1 (v/v) dichloroethane : chloroform solution

2 to 8 samples should be lightly fixed using 2 to 4% EM grade Formaldehyde along with Glutaraldehyde between 0.01 to 0.1% to improve the morphology in 0.1M Sodium Cacodylate or PO₄ buffer @ RT 1hr and cut into 1mm³ cubes then placed into 4^oC. They will be embedded in acrylic resins such as LR White (EMS) or Lowicryl (EMS). These allow for easy storage/trimming/handling of samples at room temperature once embedded.

??

NOTE: There is a minimum charge for Negative Staining that includes up to 5 grids.

??

NOTE: A charge per grid will apply for each additional grid after first five.

??

Items needed:

25uM is a good starting point for protein concentration

1% Uranyl Acetate in ddH₂O

Grids: Formvar and Carbon coated copper grids (300-400 mesh; Cat#F F300-Cu), glow discharged

DUMONT N4AC Dumoxel tweezers are anti-capillary (Cat# 72870-D www.emsdiasum.com)

Procedure for negative staining:

1. Place ~4ul of purified protein on glow discharged 300mesh carbon/formvar coated Cu grids.
2. Allow to settle 3 min.
3. Wash grids with 3 drops 1% Uranyl Acetate in ddH₂O then allow 3rd drop to sit on grid for 1 min.
4. Pull off most of drop with filter paper and allow to dry.
5. View samples at 120kV on the TEM.

SEM imaging is done by the staff electron microscopist, using the user/researcher's prepared samples. The user should preferably be present (at least partially) during the first imaging session to discuss and indicate the feautures of interest, and image magnifications required. Data can be downloaded from the SEM-PC on site, or remotely from CSIF servers, or sent to the user through Stanford Box.

Samples are aldehyde fixed and resin embedded for ultrathin sectioning similar to TEM analysis, but with additional en bloc staining techniques with heavt metals to improve conductivity of ultrahin sections on glass sides. Slides are carbon coated for improved conductivity, and scanned serially with FESEM to obtain a stack of images for 3D reconstruction.

PROCESS

Fix samples in 4%PFA/2%Glut in 0.1M NaCacodylate Buffer (4h+) Keep overnight or for extended periods of time at 4deg Celsius, fully submerged in fixatives and sealed with parafilm.

Wash in same buffer (2x5min)

OTO:

Post-fix in 1% OsO4 (2hrs) - highly toxic and volatile, keep in vacuum hood

Wash in H₂O (3x5min)

Post-fix in TCH: 30min

Wash 3-x5min

Post-fix OsO4: 1hr

Wash 3x5min

Post-fix 2% UranylAcetate: 2hrs (or 1% overnight @ 4C)

Wash 3x H₂O

Post-fix Pb-Citrate: 1-2min, as for section staining (closed container, pellet NaOH to absorb CO₂) not for 2^nd series of embedding

Wash H₂O

DEHYDRATE: 30-50-70-90-100-100% EtOH

Dehydrate in 100% Acetonitrile, replace with Acetone (2x) 2^nd embedding, prevent curling of membrane

Infiltrate Epon:Acetone 50%[1:1], 66% [2:1] (overnight)100% (4-6hrs) on rotator

Embed in fresh 100% resin, polymerize @ 60^oC 2days

Thiocarbohydrazide (1% (wt/vol) aqueous solution) Heat 25 ml of ddH₂O on a hot plate to 58^oC. Add 0.25 g of TCH to the heated water while stirring until the powder is dissolved, and then leave the solution for 2-3 h; the color will darken. Turn down the heat while stirring. Remove the solution from the hot plate and allow it to cool to room temperature (25^oC). Filter using a 0.45 um syringe filter. Store at room temperature to avoid precipitation for up to 1 week

Prepared specimens (CPD dried and/or cut to size) are mounted on 12 or 25mm aluminum pin stubs using colloidal graphite or Copper tape (or Carbon tabs for Hitachi 3400N VP-SEM only). Strictly NO carbon tabs are allowed in the Zeiss Sigma FESEM.

??

For improved conductivity specimens are coated with gold/Palladium (Au/Pd) to a thickness of 50-100Angstroms, using the Denton Desk II sputter-coater. Non-conductive samples can be used uncoated with the Hitachi VP-SEM using low vacuum mode.

• typical time/costs??

Fix tissue (or cells on coverslips, or cells on hydrogels) 4 hrs to overnight in 2% Glutaraldehyde with 4% PFA (Paraformaldehyde) in 0.1M Na Cacodylate Buffer (pH 7.3). Samples should be either cut to appropriate size, grown on 12-22 mm coverslips, or centrifuged to concentrate small particles. Submerge samples in fixatives, close with parafilm, and keep cold (4^oC) for 1hr to weeks, before continuing processing. Never allow samples to dry out, or warm to room temperature in fixatives for longer than 1hr. Bacterial samples must be grown to late log pahse (OD~0.6) for sufficient numbers, or grow as biofilm on a suitable surface, or as cell culture on poly-L-lysine coated coverslip. Bacteria can also be fixed in eppendorf tubes, and 100ul aliquots pipetted onto PLL coated coverlips. Vacuum-filtration of bacteria through microporous filters (0.22-0.45um pore size) also allows for retention of small particels, cell clusters or univellular organisms on usable surfaces. Microporous capsules (70-200um pores) that are compatbile with CPD drying can also be used with great success to retain and easily handle small particles during processing.

Wash 5 min in same buffer

Post-fix 1 hr in 1% aqueous OsO₄ (Osmium Tetroxide) Highly toxic!! Use in hood!!.

Wash 2x 5 min in mQ H₂O

*Dehydrate in increasing ethanol series: 50-70-90-100-100% (10min each).

**CPD (Critical Point Dry) with liquid CO₂ with Tousimis Autosamdri 815.

Mount samples with double-sided carbon tape, or Colloidal Graphite, on Aluminum stubs.

Sputter-coat with Pd/Au - 100 Angstrom layer (3-5 min) with the Denton DeskII system in lab.

* Hydrogels and samples in VP-mode, using coolstage control: do not dehydrate, CPD or sputter-coat.

** CPD drying can be replaced by HMDS (Hexamethyldisalazane) drying in conditions where the size, orientation, or other physical parameters are not compatible with CPD conditions. Replace the final 100% ethanol with a 50% solution of HMDS in ethanol (15min), followed by 100% ethanol (2x15min). Remove all HMDS and allow sample to dry overnight in a desiccator.

Supplies needed

Suppliers:

Electron Microscopy Sciences http://www.emsdiasum.com/microscopy/default.aspx

Ted Pella http://www.tedpella.com/

Fixatives:

16% PFA (EMS Cat #15710)

8% Glut (EMS Cat #16020) with

0.2M NaCacodylate (EMS CAT #11652)

Mix equal volumes (1 vial each) of PFA and Glutaraldehyde with 20 ml Buffer for required fix conc

2% OsO4 (EMS Cat #19192) dilute with mQ water to 1% conc.

Colloidal Graphite: Ted Pella #16053

Specimen Mounts: Ted Pella #16111 (1/2 dm slotted head)

Carbon conductive Tape: Ted Pella #16073

Pelco Tabs 12mm: Ted Pella #16084-1

Pelco Tabs 25mm Ted Pella #16084-2

Carbon conductive sheet (x10): Ted Pella #16085-1

Microporous capsules: EMS # 70187 (30 and 78 micron)

Epon Embedding of Cells on Coverslips (typically 4 to 8 samples)(TestJP 8-14-12)

Sample Fixation: (Preferably grown on 12mm round glass or 13mm round Thermanox coverslips)

1. Samples fixed in 2 % Glutaraldehyde(8% stock-EM grade) and 4% p-Formaldehyde in 0.1M NaCacodylate (or HEPES, Tris, PO₄) buffer pH~7.2 for 20 min @ RT then moved to 4^oC for 40 min to a couple of weeks.

Post-Fixation for Epon (Fume hood, Osmium is Toxic!!) All at 4^oC

1. Fix is then gently replaced with1% OsO4 in ddH2O, Rotated gently and left for 1 hr
2. Wash samples briefly 3X each in ddH2O
3. Change to 1% Uranyl acetate in ddH2O, stained for 2 hrs to Overnight.

III. Dehydration:

1. 50% EtOH, 5 min
2. 70% EtOH, 5 min
3. 95% EtOH, 10 min (allowed to warn to room temp)
4. 100% EtOH, 2X, 15 min each @ RT
5. Changed to Acetonitrile for 15 min

Epon* Infiltration: (Use gloves) *Epon = EMbed 812 using 20ml EMbed 812 and 16ml DDSA and 8ml NMA with 1.2ml BDMA

1. Change to 1:1 Epon/ Acetonitrile for 1hr
2. Change to 2:1 EPON/ Acetonitrile and leave 1 hr
3. Replace with 100% Epon 1 hr
4. Change to fresh Epon, place labels into beem capsules and fill with fresh resin, put on top of coverslip.
5. Polymerize in oven @ 65^oC O/N, I usually make about 2 labeled beem capsules/coverslip
6. Remove plastic from bottom of coverslip and dissolve glass w/HF (49%) for 20 min

I. Sample Fixation(2 to 8 samples):

1. Samples are usually fixed w/2% glutaraldehyde & 4% paraformaldehyde in 0.1M Sodium Cacodylate buffer, pH 7.4 for 1 hour @Room Temp and cut into blocks ~1mm³. Other buffers can be used: e.g. HEPES or PO_4. After fixation samples are then moved to 4^oC for immediate processing. Some people have left samples in fix for days to weeks with little change to the morphology.

To enrobe cells in gelatin spin cells down 5 min @ 4kX and wash in buffer 3X: re-suspend cells in warm gelatin, 10% in PBS, for 5 min/spin 5 min @ 4 kX, place on ice 5 minutes then cut into blocks. Cover with cold Osmium(step 2).

II. Post-Fixation @ 4^oC(Use Fume hood and gloves, Osmium is toxic)

2. Remove fixative and add 1% OsO₄ in ddH₂O Shake gently and leave for 1 hr @ 4^o
3. Wash samples 3X, 5 min each w/cold ddH₂O
4. Change to 1% Uranyl acetate in ddH₂O, stain for 2 hr to O/N

III. Dehydration:

5. 50% EtOH, 10 min
6. 70% EtOH, 10 min
7. 95% EtOH, 10 min (allow to warm to room temperature, rest performed at RT)
8. 100% EtOH, 2X, 10 min each @ RT
9. Acetonitrile 15 min

IV. Epon* Infiltration: (Use gloves):

10. Change to 1:1 Acetonitrile/Epon 1hr
11. Change to 1:2 Acetonitrile/Epon Overnight
12. Change to 100% Epon 2-3 hrs

Place in molds labeled and filled with 100% Epon and allow to settle for 4hr to Overnight (O/N)

13. Polymerize in 65^oC oven 24hrs

Tomographic reconstruction of 3-D data, based on images acquired as tilted projections, using intermediate voltage electrons interpreted by IMOD which is a set of image processing, modeling and display programs used for tomographic reconstruction and for 3D reconstruction of EM serial sections and optical sections. The package contains tools for assembling and aligning data within multiple types and sizes of image stacks, viewing 3-D data from any orientation, and modeling and display of the image files. IMOD was developed primarily by David Mastronarde, Rick Gaudette, Sue Held, Jim Kremer, and Quanren Xiong at the Boulder Laboratory for 3-D Electron Microscopy of Cells.

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***Was originally name:??High Pressure Freezing/Freeze Substitution***

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Two to Eight samples are typically accepted as one run. The Leica EM PACT2 HPF can freeze samples 100 to 200nm thick by 1.5mm in diameter. It can also accomodate 1.2mm round sapphire coverslips which cells can be grown on. Samples are then freeze substituted/processed into an Eponate for morphological analysis or Acrylic resin for immuno-gold localization.

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• NOTE: more difficult samples may require additional time and cost.

• Note: more difficult samples may require additional time and cost.

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NOTE: More difficult blocks require additional time/cost.

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• NOTE: More difficult blocks require additional time/cost.

Training on TEM or SEM takes 4 hrs, divided into 3hrs (assisted) and 1hr (independent) use

This is a flat-fee item that covers a training session by CSIF technician for three hours and the setup of a CSIF service account for one microscope instrument. This is a prerequisite for all independent usage of each microscope in the facility.

Please contact CSIF at 650-723-2449 or email microscopy@stanford.edu to discuss your imaging needs and our time availability.  After which, CSIF will book time under your name for the training on the specific instrument.  Thank you.  You can also fill out the email form here.

This charge is added whenever extended technician assistance is rendered.

Data processing computer installed with software for in image data quantification and 3D rendering.

Zeiss Schott glass high-performance cover glass #1.5 170+/- 5um (thickness) 18x18mm (LxW). Zeiss # 474030-9000-000.??Required for all super-resolution applications on OMX microscope.??Highly recommended for critical imaging applications, particularly deconvolution. 100/box = 1 each. $5/each for Internal Stanford users

This is used to bill out cost recovery of variable consumable supplies such as specialty coverslips that may not be in existing add-on categories. Please comment and specify what this charge exactly covers per the order.