Electron Microscopy Facility at the New York Structural Biology Center
Electron Microscopy Group
Training Session Manual
Version 0.3
2015/04/30
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.
 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)
o 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: [email protected]) 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.
2
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.
3
Table of Contents
Quick start guide ............................................................................................................................................... 2
New user workflow............................................................................................................................................ 3
Table of Contents .............................................................................................................................................. 4
User Responsibilities ....................................................................................................................................... 6
Entering the facility ....................................................................................................................................................... 6
Hours of Operation...................................................................................................................................................................... 6
Sign In and Out ............................................................................................................................................................................. 6
Identification Badges.................................................................................................................................................................. 6
Visitors .............................................................................................................................................................................................. 6
Personal Property........................................................................................................................................................................ 6
Liquid nitrogen dispensing ...................................................................................................................................................... 6
Care of equipment ......................................................................................................................................................... 6
EM toolbox/kit.................................................................................................................................................................. 7
EMG User Toolkit Item List: ................................................................................................................................................... 7
EM Toolkit Parts Links: ............................................................................................................................................................. 8
Plunge freezer tweezers .......................................................................................................................................................... 8
Examples of EM toolkits ........................................................................................................................................................... 9
EM Vendors ....................................................................................................................................................................10
Examples of EM Grids ...............................................................................................................................................10
Choosing the right EM technique ............................................................................................................ 11
How to prepare samples? ........................................................................................................................................12
General considerations........................................................................................................................................................... 12
Negative Stain or Frozen-hydrated? ................................................................................................................................ 12
Negative staining ....................................................................................................................................................................... 12
Plunge freezing ........................................................................................................................................................................... 13
Negative staining vs. Plunge freezing ............................................................................................................................. 13
Sectioning ...................................................................................................................................................................................... 14
Principle of negative stains: ...................................................................................................................................15
Commonly used negative stains:....................................................................................................................................... 15
Sodium............................................................................................................................................................................................................... 15
Neutral Phosphotungstic Acid ................................................................................................................................................................ 15
Uranyl Acetate ............................................................................................................................................................................................... 15
Ammonium Molybdate .............................................................................................................................................................................. 16
Other Stains ..................................................................................................................................................................................................... 16
Negative stain protocols...........................................................................................................................................16
Preparation of a 0.75% uranyl formate solution .............................................................................................16
Conventional negative staining protocol ........................................................................................................................ 16
Useful modifications to the protocol ................................................................................................................................. 17
Carbon sandwich technique ................................................................................................................................................. 17
Useful modifications to the protocol.................................................................................................................................................... 17
Plunge Freezing .............................................................................................................................................................18
Cp3 Plunge Freezer: ................................................................................................................................................................ 18
Cp3 Plunge Freezer Protocol: ............................................................................................................................................. 19
The EMG Electron Microscopy Facility at NYSBC ........................................................................... 20
The NYSBC .....................................................................................................................................................................20
Hours of Operation......................................................................................................................................................20
Overview of the EM Facility .....................................................................................................................................20
Instrumentation ........................................................................................................................................................................... 20
4
Affiliation with NYSBC ............................................................................................................................................................. 20
EM Operations committee..................................................................................................................................................... 21
EM Facility User levels ..............................................................................................................................................21
Independent users .................................................................................................................................................................... 21
Supervised users ....................................................................................................................................................................... 21
Collaborating users ................................................................................................................................................................... 22
Publication/Authorship .............................................................................................................................................22
For independent users: ........................................................................................................................................................... 22
For supervised/collaborating users: ................................................................................................................................. 22
Support Statements in papers: ........................................................................................................................................... 22
Requesting instrument time....................................................................................................................................23
Rules for microscope requests: .......................................................................................................................................... 23
Other instrumentation requests: ......................................................................................................................................... 23
Evening or weekend microscope requests: ................................................................................................................. 23
Microscopes ..................................................................................................................................................... 24
Staff assistance ............................................................................................................................................................24
Microscope rooms.......................................................................................................................................................24
Liquid nitrogen policy ................................................................................................................................................24
Cryo holders................................................................................................................................... 27
Performance tests ........................................................................................................................ 27
Warm up procedure ..................................................................................................................... 28
Gatan Dry pumping station ......................................................................................................................... 28
Warm up ..................................................................................................................................................... 28
Room Temperature .................................................................................................................................... 28
Start of Session Procedure ........................................................................................................... 29
TF20 ............................................................................................................................................ 29
Holder Insertion .......................................................................................................................................... 30
Room Temperature Holder Insertion ...................................................................................................... 30
Cryo Holder Insertion ............................................................................................................................. 31
Holder Removal .......................................................................................................................................... 31
Room Temperature Holder Removal ..................................................................................................... 31
Cryo Holder Removal ............................................................................................................................. 32
End of Session Procedure ............................................................................................................ 32
TF20 ............................................................................................................................................ 32
Additional Procedures for end of cryo session/week shutdown ................................................................. 32
Additional information ................................................................................................................... 33
Useful EM parameters ................................................................................................................. 33
Wavelengths ............................................................................................................................................... 33
Lens changes ............................................................................................................................................. 33
TF20 ....................................................................................................................................................... 33
Calibrations .................................................................................................................................. 33
TF20 ........................................................................................................................................................... 33
wavelengths ........................................................................................................................................... 33
objective lenses ...................................................................................................................................... 33
Aperture Sizes ........................................................................................................................................ 33
TF20 CMOS information ................................................................................................................................................................... 33
5
User Responsibilities
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.
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.
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.
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.
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.
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.
Care of equipment
You are expected to demonstrate proper care when using NYSBC instrumentation.
6
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.
Ed’s EM Toolkit
EMG User Toolkit Item List:
Ted Pella Item #
13215
160
510-5
505
EMS Item #
72940
71150
0202-N5-PO
72700-D
160-40
5002-29
71166-10
77937-26
Name/Description
4 ½” SCISSORS
GRID BOX
DUMONT ANTI-CAP BIO TWEEZER
DUMONT STAINLESS STEEL
TWEEZER
CRYO GRID BOX WITH LID
DISSECTING FORCEPS
Qty
1
2
1
1
Price Estimate
$8.00ea
$5.00ea
$50.00ea
$40.00ea
2
1
$10.00ea
$20.00ea
x1
x2
x2
$140.00ea
$300.00/50
$230.00/25
Other items for cryo-EM (not included in standard toolkit):
CP3690 CP3 CRYOPLUNGE PLUNGING TWEEZER
Q250-CR2 QUANTIFOIL, 200#, 2um, Copper
CF222C-100 C-FLAT GRIDS, 2um/2um-2C, Copper
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EM Toolkit Parts Links:
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Forceps:
http://www.tedpella.com/twzr-mis_html/twzr-ac.htm
http://www.dumonttweezers.com/Tweezer/TweezerType/1
Empty grids:
http://www.tedpella.com/grids_html/Pelco-TEM-Grids.htm
http://www.emsdiasum.com/microscopy/products/grids/gilder.aspx
Grids with carbon on them:
http://www.tedpella.com/supflm_html/suptfilm.htm
http://www.emsdiasum.com/microscopy/products/grids/support.aspx
Grid boxes:
http://www.tedpella.com/grids_html/gridbox.htm#anchor1313234
http://www.emsdiasum.com/microscopy/products/grids/accessories.aspx
• 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 :
◦
http://www.emsdiasum.com/microscopy/products/grids/cflat.aspx
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http://www.protochips.com/products/holey-carbon-support-grids-for-cryo-tem.html
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http://www.emsdiasum.com/microscopy/products/grids/quantifoil.aspx
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http://www.tedpella.com/calibrat_html/656.htm
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http://www.2spi.com/catalog/grids/quantifoil-carbon-films.php
• Dissecting forceps:
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http://www.tedpella.com/dissect_html/forceps.htm
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http://www.emsdiasum.com/microscopy/products/tweezers/forceps.aspx
• Cryo grid boxes:
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http://www.tedpella.com/grids_html/gridbox.htm#_160_40
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http://www.emsdiasum.com/microscopy/products/grids/accessories.aspx
• Wiha hex screwdriver for NYSBC cryo grid boxes:
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http://www.wihatools.com/200seri/264serie.htm
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 two plunge freezers: a custom vitrobox and a GatanCp3
(http://www.gatan.com/products/specimen_prep/products/CryoPlunge3.php), and will have a FEI
Vitrobot up and running in the near future.
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
8
Examples of EM toolkits
Example1: Standard components of an EM toolkit.
Example2: What’s in Ed's EM toolbox (Negative-stain and Cryo).
9
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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Electron Microscopy Sciences - http://www.emsdiasum.com/microscopy/
TedPella - http://www.tedpella.com/
SPI - http://www.microscopy.cc
Protochips - http://www.protochips.com
Examples of EM Grids
EM grid: 3mm diameter metal mesh
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:
 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.
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How to prepare samples?
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.
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.
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.
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Advantages
Disadvantages
high contrast
good signal-to-noise ratio of molecules <100 kD
prone to structural collapse
simple to apply
high background from surrounding stain
resistant to radiation
flattening/squashing artifacts
Works well on heterogeneous preps
distortions due to ionic strength and pH
Can induce preferred orientation
limited resolution (~25Å)
3-D reconstruction is possible
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
No artifacts due to fixation, dehydration or
staining
preservation of native conformation
Random orientation
Works well on homogeneous sample
Good contrast at high defocus
Disadvantages
low contrast
low signal-to-noise-ratio
sensitive to radiation
technically challenging
Difficult to visualize smaller than 100 kD
Higher resolution info than negative stain
Difficult to distinguish between different
orientations vs. conformations
low background
Freezing artifacts
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.
13
Negative staining
Plunge freezing
sophisticated equipment required
quick and easy to learn
involves learning curve
low tech microscope
resistant to radiation damage
high-end microscope with cryo
accessories
high contrast and signal-to-noise ratio
image dose limited by radiation damage
samples as small as 100kDa
low signal-to-noise ratio
structural artifacts due to staining and
dehydration
sample size >300kDa
preservation of native structure
low resolution
preservation of high resolution
surface contour only
internal details revealed in reconstructions
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 extract 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.
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)
14
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.
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.
15
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.
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.
Preparation of a 0.75% uranyl formate solution
Attention: uranyl formate is radioactive, toxic and light-sensitive
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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
Conventional negative staining protocol
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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
16
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.
Useful modifications to the protocol
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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
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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
17
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:
18
Cp3 Plunge Freezer Protocol:
Setup:
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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.
o Gently pull out blotters to remove.
o 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
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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
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 ).
Affiliation 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.
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EM Operations committee
Each institution has a representative.
Institution
Representative
Email
Albert Einstein College of Medicine
City University of New York
Columbia University
Memorial Sloan Kettering Cancer Center
Mount Sinai School of Medicine
New York University
Rockefeller University
Wadsworth Center
Weill Cornell Medical College
Hernando Sosa
Reza Khayat
Bob Grassucci
Chris Lima
Iban Ubarretxena
David Stokes
Seth Darst
Michael Marko
David Eliezer
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
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.
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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.
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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:
◦ 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 [email protected].
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.
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 ([email protected]) 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
3. Send an email to [email protected] that you have signed up for off-hours time
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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.
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• 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"
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• LN2 main area:
• LN2 main area signs:
26
• 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. Use fomblin very sparingly.
• 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.
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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
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•
•
•
◦
◦
•
•
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:
Start of Session Procedure
TF20
1. Bring up Vacuum Overview and make sure IGP1 < 10. V4 and V7 should be closed.
29
2. Make sure Gun, Column, and Camera are highlighted in green and High Tension is yellow.
Normal vacuum reading:
3. Fill cryo dewar half way with LN2 and top off after inserting coils.
4. Load grid into holder.
Holder Insertion
Room Temperature Holder Insertion
1. Make sure IGP1 < 10 on Vacuum Overview and Gun = 1, Column < 10, and Camera in 30s.
2. Turn on turbo pump: “Turbo on.” Button will be orange while pumping and yellow when ready. Takes
a few minutes to spin up.
3. “Pre-pump airlock.” Takes 4 seconds.
4. Line up pin on holder by orienting white line on holder at the 12 o’clock position.
5. Insert holder to first stop. Wait for the specimen airlock pump down timer to go to zero on
Vacuum Overview.
6. Select ‘Single Tilt’ as specimen holder.
7. Turn holder counter-clockwise to second stop and apply resistance to slowly let the vacuum pull the
holder in. You do not want it to slam but you also do not want to prevent it from going in.
8. Watch IGP1/Column vacuum for spike and wait for it to get below 10. If vacuum spikes over 50 and
Column is now red, contact an EMG staff member immediately.
9. Turn off turbo pump.
10. Do not open the column valves until IGP1/Column < 10. If you are ever in doubt, close the
column valves and contact an EMG staff member immediately.
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Reading after a vacuum crash:
Cryo Holder Insertion
1. Make sure IGP1 < 10 on Vacuum Overview and Gun = 1, Column < 10, and Camera in 30s.
2. Position Styrofoam box below goniometer to catch LN2.
3. Turn on turbo pump: “Turbo on.” Button will be orange while pumping and yellow when ready. Takes
a few minutes to spin up.
4. “Pre-pump airlock.” Takes 4 seconds.
4. Set holder alpha value at -60, goniometer will rotate.
6. Insert holder to first stop. Wait for the specimen airlock pump down timer to go to zero on
Vacuum Overview.
7. Select ‘ST Cryo Holder’ as specimen holder and reset alpha value to 0.
8. Turn holder counter-clockwise to second stop and apply resistance to slowly let the vacuum pull the
holder in. You do not want it to slam but you also do not want to prevent it from going in.
9. Watch IGP1/Column vacuum for spike and wait for it to get below 10. If vacuum spikes over 50 and
Column is now red, contact an EMG staff member immediately.
10. Turn off turbo pump.
11. Do not open the column valves until IGP1/Column < 10. If you are ever in doubt, close the
column valves and contact an EMG staff member immediately.
Holder Removal
Room Temperature Holder Removal
1. Close column valves.
2. Under Search, reset ‘Holder.’
3. Brace against the column and pull back to break vacuum. Turn clockwise and pull all the way out of
the column. If you feel the microscope sway or hear the air table then you are using too much force.
4. Watch IGP1/Column vacuum for spike. If vacuum spikes over 50 and Column is now red,
contact an EMG staff member immediately.
31
Cryo Holder Removal
1. Close column valves.
2. Under Search, reset ‘Holder.’
3. Position Styrofoam box below holder to catch LN2.
4. Brace against the column and pull back to break vacuum. Turn clockwise and pull all the way out of
the column. If you feel the microscope sway or hear the air table then you are using too much force.
5. Watch IGP1/Column vacuum for spike. If vacuum spikes over 50 and Column is now red,
contact an EMG staff member immediately.
End of Session Procedure
TF20
1. Return microscope to standard condition
◦
Lower main viewing screen
◦
Change to ~50kx-62kX magnification
◦
Make sure beam is spread
2. Close column valves
3. Reset holder
4. Remove objective aperture
5. Replace cover on viewing chamber
6. Remove sample holder and store
7. Clean up materials used
8. 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
32
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
LM
M
SA
Mh
Magnification range
21X → 2100X
1700X → 3500kX
65kX → 280kX
390kX → 700kX
Calibrations
TF20
wavelengths
 200kV: 0.02508 A
objective lenses
• Cs = 2.1 mm
Aperture Sizes
• Condensor : 150 um, 100 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
33
Copyright © by NYSBC. All material on this collaboration platform is the property of the contributing
authors from the NYSBC EM Facility.
Ideas, requests, problems regarding this handbook, Send feedback: [email protected]
EM Training Manual, Eng version 0.1
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