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T12 User Guide

Version 0.6

2016/02/16

Table of Contents

Table of Contents

Quick start guide

New user workflow

User Responsibilities

Entering the facility

Hours of Operation

Sign In and Out

Identification Badges

Visitors

Personal Property

Liquid nitrogen dispensing

Care of equipment

EM toolbox/kit

EMG User Toolkit Item List:

EM Toolkit Parts Links:

Plunge freezer tweezers

Examples of EM toolkits

EM Vendors

Examples of EM Grids

Choosing the right EM technique

How to prepare samples?

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General considerations

Negative Stain or Frozen-hydrated?

Negative staining

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Plunge freezing

Negative staining vs. Plunge freezing

Sectioning

Principle of negative stains:

Commonly used negative stains:

Negative stain protocols

Preparation of a 0.75% uranyl formate solution

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Conventional negative staining protocol

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Useful modifications to the protocol

Carbon sandwich technique

Useful modifications to the protocol

Plunge Freezing

Cp3 Plunge Freezer:

Cp3 Plunge Freezer Protocol:

Vitrobot Plunge Freezer:

Vitrobot Plunge Freezer Protocol:

The EMG Electron Microscopy Facility at NYSBC

The NYSBC

Hours of Operation

Overview of the EM Facility

Instrumentation

AffliationAffiliation with NYSBC

EM Operations committee

EM Facility User levels

Independent users

Supervised users

Collaborating users

Publication/Authorship

For independent users:

For supervised/collaborating users:

Support Statements in papers:

Requesting instrument time

Rules for microscope requests:

Other instrumentation requests:

Evening or weekend microscope requests:

Microscopes

Staff assistance

Microscope rooms

Liquid nitrogen policy

Cryo holders

Performance tests

Warm up procedure

Gatan Dry pumping station

Warm up

Room Temperature

End of Day Procedure

TF20

Additional Procedures for end of cryo session/week shutdown

Additional information

Useful EM parameters

Wavelengths

Lens changes

TF20

Calibrations

TF20

wavelengths

objective lenses

Aperture Sizes

TF20 CMOS information


Quick start guide

How do I start?

Note: The resources of the NYSBC are available for your use at no cost if you belong to a member institution.

1) Learn about the resources and capabilities of the NYSBC Electron Microscopy (EM) facility at our website: http://cryoem.nysbc.org.

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

Member institutions:

3) Create an account.

4) Register your project.

5) Review our information.

6) Schedule a meeting.

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

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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.

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User Responsibilities

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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. 


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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.

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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.


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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.


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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.

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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.


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Care of equipment

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

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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.

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Ed’s EM Toolkit

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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


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EM Toolkit Parts Links:

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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

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Examples of EM toolkits

Example1: Standard components of an EM toolkit.

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

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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:

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Examples of EM Grids

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EM grid: 3mm diameter metal mesh

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

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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:    

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How to prepare samples?

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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.

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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.

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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)

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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

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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

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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.

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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)

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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.

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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.

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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.

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Preparation of a 0.75% uranyl formate solution

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

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Conventional negative staining protocol

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.

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Useful modifications to the protocol

Carbon sandwich technique

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

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:

Shutdown Procedure:        

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

 

Vitrobot Plunge Freezer:

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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:

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.

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 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:

  1. 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.
  2. To schedule time on the microscope you need to reserve time on the microscope scheduler: http://emg.nysbc.org/booked
  3. 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.
  4. Brief rules for booking time:

Additional Notes:

Other instrumentation requests:

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

  1. Signup is on a first come first served basis.
  2. List the time period you want to use the equipment (usually a minimum of 2hrs).
  3. 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.

  1. Only 24-hour users are allowed to request off hours time
  2. 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

  1. 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

Liquid nitrogen policy

 

 

Cryo holders

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

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

Warm up 

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

 Room Temperature

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

 

End of Day Procedure

TF20

  1. Return microscope to standard condition
  1. Close column valves
  2. Reset holder
  3. Remove objective aperture
  4. Replace cover on viewing chamber
  5. Remove sample holder and store
  6. Clean up materials used
  7. 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.

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


Additional information

Useful EM parameters

Wavelengths

Lens changes

TF20

Lens

Magnification range

LM

21X → 2100X  

M

1700X → 3500kX

SA

65kX → 280kX

Mh

390kX → 700kX

Calibrations

TF20

wavelengths

objective lenses

Aperture Sizes

TF20 CMOS information

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EM Training Manual, Eng version 0.1