Equipment: http://cryoem.nysbc.org/equipment.html
Overview of EM techniques: http://cryoem.nysbc.org/overview.html
Considerations before beginning a project: http://cryoem.nysbc.org/considerations.html

2) Determine your eligibility to access NYSBC: http://cryoem.nysbc.org/about.html

Member institutions:

Albert Einstein College of Medicine of Yeshiva University
City University of New York
Columbia University
Memorial Sloan-Kettering Cancer Center
Mount Sinai School of Medicine
New York University
Rockefeller University
Wadsworth Center of the Department of Health
Joan and Sanford Weill Medical College of Cornell University

3) Create an account.

Create an account on the NYSBC Intranet for yourself. (http://www.nysbc.net/twiki/register.html)

Note: You must create an account from a member institution IP address. Once created you may then log in from anywhere.

4) Register your project.

Register your project by logging into the NYSBC Intranet (TWiki - http://www.nysbc.net/twiki/bin/view/Main/CemProposalMainEdit) and using the following intranet form: EM Proposal Submission

5) Review our information.

Read the resources and capabilities of the EM facility on the website.

6) Schedule a meeting.

Arrange a meeting with the EMG staff (email: emg@nysbc.org) and notify your institution's Operations Committee Member (see: http://cryoem.nysbc.org/about.html or http://www.nysbc.net/twiki/bin/view/Main/CemOpCom).

7) Meet with the EM Staff to discuss your project and schedule time on the equipment.

Read and sign the New User form, which includes Safety Procedures, Facility Use, User guidelines and responsibilities, and Publication/authorship agreement.

Back to the Table of Contents

New user workflow

The goal of the EM Facility is to train you to become an electron microscopist capable of preparing samples and collecting data on the microscopes. The EMG staff will be available to help train and advise you. The hope would be that you would become your laboratory’s EM ambassador — able to contribute EM expertise to your lab’s research program.

Macintosh HD:Users:ed:Desktop:EMG:k2warning:Slide3.jpg

Back to the Table of Contents

User Responsibilities

Back to the Table of Contents

Entering the facility

Hours of Operation

NYSBC hours of operation are normally 9:00 am - 5:00 pm, Monday-Friday, with the exception of Federal Holidays. NYSBC may reduce hours of operation because of weather or other emergencies, staff limitations, or other factors.

Users are not allowed in the facility without a staff member present.

Back to the Table of Contents

Sign In and Out

To enter you must announce your presence to the security guard and the guard will contact us to pick you up from the front entrance.

A daily sign in/sign out sheet is used to maintain an accurate record of people at NYSBC and to ensure everyone has evacuated the building in the event of an emergency. It is your responsibility to use these sheets whenever you enter or leave the building.

Back to the Table of Contents

Identification Badges

Users will be issued an identification badge upon orientation or after microscope training has completed. These badges will only open doors to areas necessary to access the microscopes. These cards will not allow you into the building outside normal hours of operation.

Back to the Table of Contents

Visitors

If you are expecting a visitor, please notify the EM staff. All visitors must first check in at the reception area. Visitors are not allowed in any area of the building without being accompanied by an authorized staff member. Under no circumstances will visitors be allowed in confidential, unauthorized or potentially hazardous areas.

Back to the Table of Contents

Personal Property

NYSBC is not responsible for loss or damage to personal property. Valuable personal items should not be left in areas where theft might occur.

Back to the Table of Contents

Liquid nitrogen dispensing

Please use caution when dispensing liquid nitrogen. We provide personal protective equipment for use including but not limited to: lab coats, cryo gloves and goggles/face shield.

Back to the Table of Contents

Care of equipment

You are expected to demonstrate proper care when using NYSBC instrumentation.

Back to the Table of Contents

EM toolbox/kit

If EM is a major part of your project or you become a regular EM user, you should have your own EM supplies and build an EM toolbox. You may be required to have your own EM toolkit to make full use of the EM facility.

One does not have to have separate toolboxes for negative stain and cryo work. As you become more familiar with EM you should customize and supplement this toolbox.

For your convenience the EM Facility offers toolkits, however you are not obligated to purchase our EMG User toolkit. Again, this is a basic toolkit and we encourage you to customize your toolkit with tools suited to your project. If you are interested in a toolkit please ask EMG staff for details.

Macintosh HD:Users:ed:Desktop:Screen Shot 2015-03-31 at 12.18.20 PM.png

Ed’s EM Toolkit

Back to the Table of Contents

EMG User Toolkit Item List:

Ted Pella Item #	EMS Item #	Name/Description	Qty	Price Estimate
13215	72940	4 ½” SCISSORS	1	$8.00ea
160	71150	GRID BOX	2	$5.00ea
510-5	0202-N5-PO	DUMONT ANTI-CAP BIO TWEEZER	1	$50.00ea
504	0209-4-PO	DUMONT STAINLESS STEEL TWEEZER	1	$40.00ea
160-40	71166-10	CRYO GRID BOX WITH LID	3	$10.00ea
5002-29	77937-26	DISSECTING FORCEPS	1	$20.00ea

Other items for cryo-EM (not included in standard toolkit):

CP3690 CP3 CRYOPLUNGE PLUNGING TWEEZER x1 $140.00ea

Q250-CR2 QUANTIFOIL, 200#, 2um, Copper x2 $300.00/50

CF222C-100 C-FLAT GRIDS, 2um/2um-2C, Copper x2 $230.00/25

Back to the Table of Contents

EM Toolkit Parts Links:

Forceps:

Empty grids:

Grids with carbon on them:

Grid boxes:

We typically use grids overlayed with carbon that contains regularly spaced holes, such as Quantifoil. Our most frequently used C-flats or Quantifoil grids are 300 mesh, R2/2: 2µm hole separated by 2µm. For higher resolution you may choose R1.2/1.3. You may purchase them from :

Dissecting forceps:

Cryo grid boxes:

Wiha hex screwdriver for NYSBC cryo grid boxes:

http://www.wihatools.com/200seri/264serie.htm

Back to the Table of Contents

Plunge freezer tweezers

The facility has common plunge freezer tweezers, which users may sign out if they are scheduled to use the plunge freezer. Users planning on doing Cryo EM should obtain their own pair of tweezers. We have three plunge freezers: a custom manual vitrobox, a GatanCp3 (http://www.gatan.com/products/specimen_prep/products/CryoPlunge3.php), and an FEI Vitrobot.

We may be able to custom adapt your DUMONT L5 tweezers (http://www.dumonttweezers.com/Tweezer/TweezerType/2) to fit our Cp3 plunge freezer (see below).

Cp3 plunge freezer tweezers

Back to the Table of Contents

Examples of EM toolkits

Example1: Standard components of an EM toolkit.

Example2: What’s in Ed's EM toolbox (Negative-stain and Cryo).

Back to the Table of Contents

EM Vendors

There are several vendors where you may obtain the necessary equipment. We do not endorse a particular vendor and are free to purchase your tools from vendors of your choosing.

A few suppliers that we use often are:

Electron Microscopy Sciences - http://www.emsdiasum.com/microscopy/
TedPella - http://www.tedpella.com/
SPI - http://www.microscopy.cc
Protochips - http://www.protochips.com

Back to the Table of Contents

Examples of EM Grids

Macintosh HD:Users:ed:Desktop:Screen Shot 2015-03-31 at 2.51.26 PM.png

EM grid: 3mm diameter metal mesh

Macintosh HD:Users:ed:Desktop:C-Flat_Large.jpg

C-flat grids: Reference - www.protochips.com

Back to the Table of Contents

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

A sample has to be prepared and optimized for EM imaging.
Several EM images or micrographs of your sample are collected.
The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
The images are further analyzed and combined to create a reconstruction of your sample.

Back to the Table of Contents

How to prepare samples?

Back to the Table of Contents

General considerations

In order to visualize a sample in a transmission electron microscope the sample must (a) be thin enough such that a beam of electrons can penetrate it (<250nm, ideally <100nm), (b) be deposited onto an EM grid, which is a thin circular copper grid that is 3mm in diameter, and (c) withstand high vacuum and electron radiation within the microscope column. For tissue, samples are prepared by cutting thin sections (Sectioning). For aqueous suspensions of macromolecules, including 2D crystals and ordered helical arrays, 1-5 microliters of the solution is pipetted onto the EM grid, which is then subjected to either negative staining , plunge freezing , or a combination of these sample preservation techniques, cryo-negative staining.

Back to the Table of Contents

Negative Stain or Frozen-hydrated?

As informative as stained samples might be, it is desirable eventually to collect data from specimens frozen in their native buffers. To this end, once the ideal conditions for negative staining have been determined, we can freeze solutions of sample for imaging. These samples are visualized at low temperatures, to preserve the samples in a vitrified state, and to provide them some protection from the noxious effects of the electron bombardment.

Back to the Table of Contents

Negative staining

Negative staining involves the addition of a heavy metal salt solution that forms an electron-dense mould around individual macromolecular complexes. Normally, this mould is formed by simply air drying the EM grid. The resulting samples are easy to manipulate and can be stored for long periods. In the electron microscope, this mould produces a high contrast image and is resistant to radiation damage. Thus, negative staining is the preferred method for screening samples and can also be used for low-resolution structure determination of the molecular envelope.

Advantages

Disadvantages

high contrast

good signal-to-noise ratio of molecules <100 kD

simple to apply

resistant to radiation

Works well on heterogeneous preps

Can induce preferred orientation

3-D reconstruction is possible

prone to structural collapse

high background from surrounding stain

flattening/squashing artifacts

distortions due to ionic strength and pH

limited resolution (~25Å)

Back to the Table of Contents

Plunge freezing

Plunge freezing results in a sample that is preserved in a physiological buffer, thus preserving not only its native conformation but also high resolution structural information. The trade offs involve severe radiation sensitivity and substantially lower contrast of the native biological material as well as the technical demands of handling frozen samples and ensuring their mechanical stability while in the microscope. For this technique, samples are pipetted onto an EM grid and, after blotting away excess solution, the grid is then plunged into liquid ethane. This procedure vitrifies the aqueous solvent, thus preserving the hydrogen-bonding networks that normally surround a macromolecule in liquid water. However, freezing must be rapid enough to prevent ice crystal formation, which will displace these hydrogen bonds and produce severe physical damage as the ice crystals push on neighboring biological material.

Advantages

Disadvantages

No artifacts due to fixation, dehydration or staining

preservation of native conformation

Random orientation

Works well on homogeneous sample

Good contrast at high defocus

Higher resolution info than negative stain

low background

low contrast

low signal-to-noise-ratio

sensitive to radiation

technically challenging

Difficult to visualize smaller than 100 kD

Difficult to distinguish between different orientations vs. conformations

Freezing artifacts

Back to the Table of Contents

Negative staining vs. Plunge freezing

While preserving a sample in an unstained frozen-hydrated state is desirable, the use of negative staining is more practical at the beginning of new projects when dealing with small macromolecular complexes. Screening of samples prepared under different conditions is considerably faster by negative stain and the resulting high-contrast images are easier to evaluate.

Negative staining

Plunge freezing

quick and easy to learn

low tech microscope

resistant to radiation damage

high contrast and signal-to-noise ratio

samples as small as 100kDa

structural artifacts due to staining and dehydration

low resolution

surface contour only

sophisticated equipment required

involves learning curve

high-end microscope with cryo accessories

image dose limited by radiation damage

low signal-to-noise ratio

sample size >300kDa

preservation of native structure

preservation of high resolution

internal details revealed in reconstructions

Back to the Table of Contents

Sectioning

Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Back to the Table of Contents

Negative Staining

Negative staining is an easy, rapid, qualitative method for examining the structure of isolated organelles, individual macromolecules and viruses at the EM level. However, the method does not allow the high resolution examination of samples – for this more technically demanding methods, using rapid freezing and sample vitrification are required. Also, because negative staining involves deposition of heavy atom stains, structural artefacts such as flattening of spherical or cylindrical structures are common. Nevertheless, negative staining is a very useful technique because of its ease and rapidity, and also because it requires no specialized equipment other than that found in a regular EM laboratory.

(Reference: http://web.path.ox.ac.uk/~bioimaging/bitm/instructions_and_information/em/neg_stain.pdf)

Back to the Table of Contents

Principle of negative stains:

Ideally, the negative stain should not react with the specimen in a ‘positive staining’ manner (i.e. it should not bind to the specimen). However, uranyl ions will bind to proteins and sialic acid carboxyl groups and to lipid and nucleic acid phosphate groups. One effect of this is to induce aggregation of the material.

Samples should be suspended in a suitable buffer (e.g. 10 mM HEPES or PIPES), in 1% ammonium acetate, or in distilled water. It is best not to use phosphate buffer or PBS as they may contaminate the grid with salt residues that have to be washed off after staining resulting in a loss of contrast. Uranyl salts, in particular, react with phosphate ions to produce a fine crystalline precipitate that obscures the specimen. [The precipitation of uranyl ions by phosphate ions is also a potential problem when using uranyl acetate as a third fixative/en bloc stain during processing of specimens for TEM].

Fixed or unfixed samples may be used. With unfixed specimens there is the potential problem of changes occurring due to osmotic shock (or to changes in ionic composition) since most negative stains are made up in distilled water. Also there may be safety implications when examining unfixed bacterial or viral samples.

To fix samples spin them down, remove the supernatant and replace it with 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.0). Immediately re-suspend the sample in the fix and leave for a minimum of 1 h at 4 ̊C. (If samples are left in a pellet they will much harder to put back into suspension disperse after fixation). After fixation, the samples should be gently pelleted, washed and re-suspended in distilled water or a suitable buffer.

Back to the Table of Contents

Commonly used negative stains:

Stain	Normal pH range for use
Sodium (K) phosphotungstate (PTA)	5-8
Uranyl acetate	4.2 – 4.5
Uranyl formate	4.2 – 4.5
Ammonium molybdate	5–7

Sodium (Potassium) Phosphotungstate (Pta)
PTA is one of the most commonly used negative stains although it does have a significant disruptive effect on many membrane systems. PTA does not act as a fixative and can destroy some viruses. It is also known to interact with lipoproteins and cause the formation of ‘myelin figures’. However, it can be used at physiological pH, and is less likely to precipitate with salts and biological media.

Neutral Phosphotungstic Acid
A 1- 3% solution of neutral PTA (buffered to pH 7 using sodium hydroxide) is a useful stain for many samples but is especially good for viruses that dissociate at low pH. The stain produces less contrast than the uranyl acetate.

Uranyl Acetate
A 1% to 3% solution of uranyl acetate dissolved in distilled water (pH 4.2 to 4.5) can be used to negatively stain many types of samples. The stain should be filtered through a 0.22 μm filter that has been pre-rinsed with large volumes of double distilled water. The filtered stain should be stored in the dark at 4 ̊C and can be used for >1 year.

Uranyl acetate solutions also act as a fixative for viruses. The advantage of uranyl acetate and uranyl formate is that they produce the highest electron density and image contrast as well as imparting a fine grain to the image. The finer grained image produced is particularly useful for smaller particulate specimens.

Because stain has a low pH it is not recommended for use with specimens that are unstable in acid conditions. Also, the stain precipitates at physiological pH and in the presence of many salts and great care is need when using it.

Uranyl Formate

A .75% solution of uranyl formate dissolved in distilled water can be used to negatively stain many samples. The stain should be filtered through a 0.22 μm filter that has been pre-rinsed with large volumes of double distilled water. Uranyl formate stain must be kept in the dark and as it will precipitate out of solution over time must be used immediately.

Ammonium Molybdate
Used as a 1-2% solution in distilled water with the pH adjusted with ammonium or sodium hydroxide to pH 7.0. Do not exceed pH 7.0 as crystallization may occur during drying. A 2% solution of ammonium molybdate is particularly useful for staining osmotically sensitive organelles. While this negative stain seems to give the best results for many types of specimen, it does produce a lower electron density than other stains.
(This stain has also been used to negatively stain thawed, thin cryosections of fixed cells.)

Other Stains
Other less common stains include: gold thioglucose, lanthanum acetate, lithium tungstate, methylamine tungstate, sodium zirconium glycollate, sodium silicotungstate, tungstoborate, uranyl acetate, aluminium formate, uranyl formate, uranyl oxalate, and uranyl sulphate.

Back to the Table of Contents

Negative stain protocols

The following procedures are from Ohi, et al's excellent 2004 paper "Negative Staining and Image Classification – Powerful Tools in Modern Electron Microscopy" (Biological Procedures Online • Vol. 6 No. 1 • March 19, 2004 • www.biologicalprocedures.com) which is a nice introductory overview of some of the techniques and issues in negative stain.

Back to the Table of Contents

Preparation of a 0.75% uranyl formate solution

Attention: uranyl formate is radioactive, toxic and light-sensitive

Weigh out 37.5 mg of uranyl formate into a small beaker
Add 5 ml of boiling deionized water and stir for 5 min in the dark
Add drops of 5 M NaOH until the stain solution becomes slightly darker yellow (too much NaOH will precipitate the stain) and stir for another 5 min in the dark
Filter the solution with a 0.2 μm syringe filter into a Falcon tube wrapped with aluminum foil and add deionized water to a final volume of 5 ml

Back to the Table of Contents

Conventional negative staining protocol

Place two 50 μl drops of deionized water and two 50 μl drops of uranyl formate stain on a piece of parafilm
Apply 2.5 μl of sample to a glow-discharged EM grid covered with a continuous carbon film and let the sample adsorb for 30 sec
Blot the grid from the side with a piece of filter paper, briefly touch the first drop of water with the grid, blot with filter paper, briefly touch the second drop of water, blot with filter paper, briefly touch the first drop of uranyl formate, blot with filter paper, touch the second drop of uranyl formate for 20 sec, and blot with filter paper (avoid complete drying of the grid in between the drops)
Completely dry the grid by vacuum aspiration touching only the rim of the grid

The particles on the carbon film should be well separated (to allow for their extraction into individual images for computational processing) but not too sparse (to avoid having to collect too many images). The particle concentration on the grid is best adjusted by dilution of the sample solution. It is also possible to vary the time for glow discharging and for sample adsorption.

Back to the Table of Contents

Useful modifications to the protocol

For sensitive specimens, distilled water can be replaced by buffer solution for the washing steps
The number of washing drops can be increased to remove detergent or omitted to induce immediate fixation of the specimen
Glycerol or glucose can be added to the sample solution or the staining solution to minimize specimen flattening (only recommended for calculating 3D reconstructions)

Carbon sandwich technique

Place two 50 μl drops of deionized water and one 50 μl drop of uranyl formate stain on a piece of parafilm
Float thin piece of carbon in a small container of uranyl formate stain
Apply 2.5 μl of sample to a glow-discharged EM grid covered with a continuous carbon film and let the sample adsorb for 30 sec
Blot the grid from the side with a piece of filter paper, briefly touch the first drop of water with the grid, blot with filter paper, briefly touch the second drop of water, blot with filter paper, briefly touch the first drop of uranyl formate, blot with filter paper, touch the second drop of uranyl formate for 20 sec
With the sample side facing up, plunge the grid into the container holding the uranyl formate with the floating piece of carbon.

Position the grid under the carbon and then lift the grid out of the container picking up the floating piece of carbon in the process. Gently blot the grid from the side using filter paper

Useful modifications to the protocol

Uranyl formate can be substituted with any other stain
Glycerol or glucose can be added to the sample solution and the grid can be frozen in liquid nitrogen to minimize specimen flattening

Plunge Freezing

Plunge freezing is a semi-automated method of preparing frozen-hydrated samples for cryo-EM. Sample is added to a plasma cleaned grid, blotted to a thin fluid layer (~100nm), and rapidly frozen in liquid ethane. After freezing the sample must be kept below -150 ºC in order to prevent formation of crystallized ice.

Cp3 Plunge Freezer:

Cp3 Plunge Freezer Protocol:

Setup:

Turn on power switch on right side.
Open nitrogen tank.
Test plunger without a sample first by making sure safety shield is in place and pressing ‘reset’, then ‘start’. If there are any problems please contact Ashleigh or another member of EMG.
Insert sponge wand into chamber after wetting with hot water. You may need to re-wet the sponge several times. Ideally you want the relative humidity to reach 80-90% so as not to dry out your sample.
Add fresh, uncontaminated liquid nitrogen to the workstation.
Replace filter paper before using plunger. You are not expected to replace after use so it is safe to assume the filter paper is contaminated with another sample.

Gently pull out blotters to remove.
Use tweezers to remove pin and release used filter paper and plastic disk. Take care not to lose the plastic disk! Place inverted pin in bottom of filter paper loading station followed by new filter paper and plastic disk. Push down gently with blotter to attach. If you need to make more filter paper disks there is a hole punch available for this purpose.

Condense 5mL ethane in a liquid nitrogen-cooled 50mL conical tube. Be sure to wear protective gear and make sure to close both valves on ethane when finished.
Set blotting time.
When set temperature (-174ºC) and relative humidity has been reached, insert cryo plunge freezer tweezers with plasma cleaned grid into plunge rod.
Add 2-3µl of sample to grid using pipette. Rotate plunge rod 90 degrees.
Close safety shield, press ‘reset’ then ‘start’ to blot sample and plunge into liquid ethane.
Open safety shield and raise plunge rod just enough to remove tweezers by pressing blue button but not remove grid from ethane pool. Quickly transfer grid into liquid nitrogen and into cryo grid box.

Shutdown Procedure:

Lift plunge rod before turning off Cp3!
Turn off nitrogen and ethane.
Make sure plasma cleaner gas tanks are also closed.
Remove sponge wand from chamber.
Return tools to where you found them and clean up any messes.

For more advanced instructions, you can find the Cp3 manual here: http://www.nysbc.net/twiki/pub/Main/PlungeFreezing/CP3PlungeFreezerManual.pdf

Vitrobot Plunge Freezer:

Vitrobot Plunge Freezer Protocol:

Setup:

· Open nitrogen tank.

· Turn on Vitrobot at back right of machine.

· Replace blotting paper.

· Fill humidity cup to minimum fill line with picotap water and attach to humidifier.

· Enable foot pedal use if desired under Options menu on computer.

· Set blot time, set/enable humidity, and adjust any other parameters on computer.

· Cool cryo workstation with liquid nitrogen and use the “spider” to cool the ethane cup. Add ethane and remove

spider from workstation when ready to freeze.

· Select “Place new grid” on menu or use foot pedal to attach Vitrobot tweezers with glow discharged grid.

· Place cryo workstation on Vitrobot without the spider.

· Re-enable humidity on computer when chamber is fully sealed.

· Add sample to grid at sampling loading position and close side opening.

· Plunge freeze.

· Transfer grid to grid box only after removing tweezers and cryo workstation from Vitrobot.

Shutdown Procedure:

· Raise plunge rod to uppermost position in chamber.

· Remove humidity cup, empty, and invert to dry.

· Remove blotting paper.

· Close nitrogen, ethane, and Solarus gas tanks.

· Shutdown Vitrobot computer.

· Turn off Vitrobot.

· Place cryo workstation in storage bin in lab.

· Return tools to where you found them and clean up any messes.

The EMG Electron Microscopy Facility at NYSBC

The NYSBC

The New York Structural Biology Center is a 501 (c)(3) corporation incorporated in the State of New York. The corporation is governed by a Board representing nine institutional members. The institutional members are:

Albert Einstein College of Medicine of Yeshiva University
City University of New York
Columbia University
Memorial Sloan-Kettering Cancer Center
Mount Sinai School of Medicine
New York University
Rockefeller University
Wadsworth Center of the Department of Health
Joan and Sanford Weill Medical College of Cornell University

Hours of Operation

Overview of the EM Facility

Instrumentation

NYSBC has five electron microscopes for use by affiliate members: A 120kV screening microscope (JEOL 1230); two 200kV microscopes (Tecnai F20 and JEOL 2100); a 300kV energy-filtered microscope (JEOL 3200); and a dual beam FIB/SEM (FEI Helios 650). The 200kV and 300kV instruments have field emission guns and CMOS or Direct Detectors for image acquisition. All microscopes have computer interfaces that communicate with specialized programs for automated acquisition of data, which is convenient for single particle analysis and crystallography and absolutely essential for tomography. In addition to the microscopes, we have all necessary equipment for sample preparation, such as two plungers to freeze crystals or macromolecular suspensions, two high pressure freezers for tissue, a freeze substitution machine, an ultramicrotome, a carbon evaporator for making sample support films and a wide variety of negative stains. Thus, affiliates simply need to provide suitable samples and all EM-specific sample preparation can be handled at NYSBC. For further details see equipment ( http://cryoem.nysbc.org/equipment.html ).

Affliation with NYSBC

Users are required to become officially affiliated with NYSBC, in order to be eligible to use the instruments and will make you eligible for an ID card, for computer accounts and for access to our equipment signups. Steps for affiliation are detailed on the following Intranet page: http://www.nysbc.net/twiki/bin/view/Main/AffiliationProcedure.

EM Operations committee

Each institution has a representative.

Institution	Representative	Email
Albert Einstein College of Medicine	Hernando Sosa	hsosa@aecom.yu.edu
City University of New York	Reza Khayat	rkhayat@ccny.cuny.edu
Columbia University	Bob Grassucci	rg2502@columbia.edu
Memorial Sloan Kettering Cancer Center	Chris Lima	limac@mskcc.org
Mount Sinai School of Medicine	Iban Ubarretxena	Iban.Ubarretxena@mssm.edu
New York University	David Stokes	stokes@nyu.edu
Rockefeller University	Seth Darst	darst@rockefeller.edu
Wadsworth Center	Michael Marko	mike.marko.em@gmail.com
Weill Cornell Medical College	David Eliezer	dae2005@med.cornell.edu

EM Facility User levels

We have 3 user levels with different responsibilities:

Independent
Supervised
Collaborating users

Independent users

Access: Free and independent use of the microscope and sample preparation equipment for your allotted institutional allocation.

Responsibilities: Complete microscope and safety training (2-3 sessions). After demonstrating your capability on the microscope you will be tested on the microscope and knowledge of our standard operating procedures. To access the facility during nights and weekend you must obtain a C-14 from the FDNY. You are responsible for all matters relating to your project, but staff will be on hand to assist you set up the microscope and answer general EM questions.

Acknowledgement: In publications or presentations please note the use of NYSBC, see Publication/Authorship for more details.

Supervised users

Access: Supervised use of the microscope and sample preparation equipment for your allotted institutional allocation during normal hours of operation, pending staff availability.

Responsibilities: Complete microscope and safety training (2-3 sessions). You are responsible for all matters relating to your project, but staff will be on hand to assist you set up the microscope and answer general EM questions. Staff will also be available to help with aligning the microscope, sample insertion and optimizing imaging during your session. Outside of your allocated time staff can give you general guidance on your EM project.

Acknowledgement: In publications or presentations please note the use of NYSBC and acknowledge the staff that has assisted you, see Publication/Authorship for more details.

Collaborating users

Access: Staff assisted use of the electron microscopy facility for your project.

Responsibilities: Staff will have integral involvement in the EM part of your project to give general guidance and assist in the project from beginning to end. Projects have the most chance of success if there is a dedicated member in the lab devoted to the project.

Acknowledgement: Authorship/co-authorship in publications or presentations commensurate to the contribution, see Publication/Authorship for more details.

Publication/Authorship

For independent users:

Staff will certainly appreciate your appropriate acknowledgment of their efforts. There is no general requirement for acknowledgment of staff for microscope training and microscope maintenance. However, you will need to acknowledge the EMG at NYSBC.

For supervised/collaborating users:

Staff should be acknowledged and/or included as an author commensurate with the level of involvement in the project..

Support Statements in papers:

Generally... Principal Investigators should acknowledge their use of Electron Microscopy Group resources and EM Facility at NYSBC in papers and presentations as below for specific instruments -- Some journals have restrictions on how individual grants may be acknowledged, and of course you may need to modify the statement for their restrictions.

EM in general (If you need to modify this statement, please retain grant # in the modification)

All publications that emerge from use of C06-supported facilities must include the following or similar statement: “The data collected at NYSBC was made possible by a grant from NYSTAR. The investigation was conducted in a facility constructed with support from Research facilities Improvement Program Grant number C06 RR017528-01-CEM from the National Center for Research Resources, National Institutes of Health.”

EM, JEOL 3200FSC microscope (300kV) (If you need to modify this statement, please retain grant # in the modification)

Please add "The 300 kV electron microscope was purchased with funds from NIH grant S10 RR17291" to the above.

Requesting instrument time

Rules for microscope requests:

Every institution has a set allocation of use per quarter. If your institution has reached its allocated limit your use will be restricted. If you require additional microscope time, time may be purchased at an hourly rate.
To schedule time on the microscope you need to reserve time on the microscope scheduler: http://emg.nysbc.org/booked
Requests should be made for morning (AM: 9-1pm), afternoon (PM: 1PM-5PM), or both. This flexibility has been introduced to potentially open up more time for more efficient usage of the instruments.
Brief rules for booking time:

Each user may submit up to 2 session requests for instrument time. One for their current session and if needed an additional session in the future to help plan future experiments.
Note that the JEOL 3200 and the Helios FIB/SEM requires staff assistance. Although you may request time on the machine, if staff is not available then there is no guarantee that you will be able to use or collect data on these instruments.
Requests for after-hour use of TF20, JEOL 2100, JEOL 3200, and Helios should be made only after you are certified as an all-hours (24 h) access user.
If you require staff assistance you must inquire 1 week in advance and receive confirmation of availability at least 48 hours in advance of your instrument session.
If you cancel your session do not remove it from the calendar. You must ask EMG staff to cancel your session at least 48 hours in advance. Allowances will be made for emergencies.

Additional Notes:

Always use the request page for signing up for time even if you contact us at emg@nysbc.org. Except for EMG personnel bookings, the EMG staff only updates the microscope calendar at set intervals throughout the month.

Other instrumentation requests:

Signups for other instrumentation should be made through http://emg.nysbc.org/booked Sample prep equipment calendars.

Signup is on a first come first served basis.
List the time period you want to use the equipment (usually a minimum of 2hrs).
Contact Staff (emg@nysbc.org) if you need to schedule a training session

Evening or weekend microscope requests:

Signups for evenings (after 6pm), weekends and/or holidays should also be made through the scheduler: http://emg.nysbc.org/booked.

Only 24-hour users are allowed to request off hours time
You must be a completely independent user -- staff help will not be available

• This means you can run and safely follow end-of-day procedures on your own

Send an email to emg@nysbc.org that you have signed up for off-hours time

Microscopes

Staff assistance

It is your responsibility to complete microscope training and have a working knowledge of the standard operating procedures of using the microscope. The staff will be available to assist in aligning and setting up the microscope as well as any questions that may arise during the session. Please check the main EM lab to find the personnel on call to assist the particular microscope you are using.

Microscope rooms

No food, drink, eating, or application of cosmetics is allowed.
Please keep the microscope rooms tidy and clean up after your session. There should be trash cans for regular waste disposal in each room.
Dress appropriately. The temperature in the EM rooms should be maintained at a constant temperature, but we suggest you wear layers to make your session more comfortable.

Liquid nitrogen policy

When dispensing liquid nitrogen use personal protective equipment (PPE).

Example of PPE: Gloves and faceshield:

At the end of the session if you used an LD4, pour the excess nitrogen into the larger LD10 dewar. Then invert the LD4 dewar to warm up and dry overnight. We have several cryoEM users and want to reduce the sources of water/ice contamination.

LD10 and LD4 dewars:

Empty LD4:

If you have excess liquid nitrogen please pour them into styrofoam containers in each room.

Please minimize liquid nitrogen spills on the TEM microscope tables and floor. Do not pour excess nitrogen on the floor because certain rooms are close to drains.

If a tank is empty notify EM Staff and place an "Empty tank" notification sign on the tank. Move the empty LN2 to the "Empty cylinder" area.

Use appropriate signage, we have 4 signs: "In use" "Use next" "Full tank" and "Empty tank"

LN2 main area:

LN2 main area signs:

location of TF20 LN2 tank:

Cryo holders

You must be trained on proper handling of the cold stages before unattended use is permitted.

Always carry the cryo holder in the transfer station.
Always leave enough time to warm up the holder at the end of your session. This will take approximately 30 minutes.
Do not fill the transfer station to the top with liquid nitrogen. Fill at or just a little above the level of the metal insert. The holder can freeze in the transfer station requiring the holder and transfer station to be warmed up for removal.
Be careful when inverting the transfer station. The metal insert can fall out.
Before cooling down the holder make sure the O-rings are intact, free of dust and greased with fomblin.
All insertions must be supervised until training has been completed.

Performance tests

Before using the cryoholders make sure they are pumped down and a vacuum integrity for the dewar is good. The holders require roughly 30 minutes to stabilize temperature before use.

NYSBC F20 cryoholder test:

Warm up procedure

Gatan Dry pumping station

Insert specimen rod into the pumping station

Open "V1" and "V2"
Make sure the screw valve on tee is closed
Turn on pump

Warm up

A warm-up cycle should be run with the "Gatan Cold Stage Control" after every use.

Invert the dewar during warm up to facilitate expulsion of condensation

Pump on the specimen rod only during warm-up.

Attach the plug on the back of the "Gatan Cold Stage Control" to the dewar
Turn on "Gatan Cold Stage Control"

Switch is in back of box

Turn knob until "Start Warm-up Cycle" is selected

Press any one of the three buttons to select
Press far left button to begin
This cycle will take around 30 min. You must wait for the cycle to finish before pumping on the dewar. This prevents contamination on the Zeolite in the dewar; one of the causes of instability.

Room Temperature

Once the frost is evacuated form the specimen rod and the vacuum reads in the 10neg4 range

Close the "V1" valve
Return dewar to the upright position
Connect tygon hose from tee to dewar
Unscrew valve at end of tee

Allow the tygon tube to evacuate for a minute before opeing dewar
This is to prevent contamination of the dewar's zeolite

Unscrew the valve on the dewar
leave the stage pumping overnight

Gatan pumping station:

End of Day Procedure

TF20

Return microscope to standard condition

Lower main viewing screen
Change to ~50kx-62kX magnification
Make sure beam is spread

Close column valves
Reset holder
Remove objective aperture
Replace cover on viewing chamber
Remove sample holder and store
Clean up materials used
Fill/top off cold trap dewar

Additional Procedures for end of cryo session/week shutdown

If you have completed a cryo session or it is the end of the week we run a cryo cycle.

Remove Dewar for cold trap and place the styrofoam box/cup under the coils.
Run cryo cycle (setup menu)
Warm up cryoholder if used

Additional information

Useful EM parameters

Wavelengths

80kV: 0.04176 Å
120kV: 0.03349 Å
200kV: 0.02508 Å
300kV: 0.01969 Å

Lens changes

TF20

Lens	Magnification range
LM	21X → 2100X
M	1700X → 3500kX
SA	65kX → 280kX
Mh	390kX → 700kX

Calibrations

TF20

wavelengths

200kV: 0.02508 A

objective lenses

Cs = 2.1 mm

Aperture Sizes

Condensor : 150 um, 10 um, 50 um, 50 um
Objective : 100 um, 50 um, 50 um, 40 um
Selected Area: 200 um, 100 um, 70 um, 40 um

TF20 CMOS information

Size of camera : 4096 x 4096 pixels
Pixel size: 15 .6um
Post-film mag : 1.84

Ideas, requests, problems regarding this handbook, Send feedback: emg@nysbc.org

EM Training Manual, Eng version 0.1