- Click on /
to expand/contract the menu item or on the link to go directly to the page.
Procedures for Performing Cryoelectron Microscopy
on the FEI Sphera Microscope
The procedures given below were written specifically for the FEI
Tecnai G2 Sphera microscope. Modifications will need to be made
for other FEI, non-Tecnai microscopes and for microscopes from other
manufacturers. It will be assumed that the microscope has a
thermionic gun, a Gatan 626 cryotransfer holder, and an intensified
TV camera. Procedures for freezing the sample, transferring it into
the cold holder, and transferring the cold holder into the
microscope are discussed in other tutorials. Words in Bold are either
software buttons to be pushed or options to enter.
Before insertion of the cold holder the anticontaminator/cold finger should be
filled with nitrogen, the user should be logged onto the computer system, the
filament saturated and a beam obtained. Make sure that the column valves on
the microscope are closed before inserting the holder.
After inserting the holder wait a minimum of a half an hour for the
microscope vacuum to recover. Do not retract the cryoshields on the
holder. This assures that any vapor that is inserted into the column
with the holder will be removed and not become contamination on the sample.
Now perform the necessary gun alignments. These need to be done without a
sample in the beam. Open the column valves and pull the holder out until it
no longer obstructs the beam but not so far out that the holder is pulled into
the airlock. The holder may be maintained in this position by propping a pencil
between the holder dewar and the goniometer.
- Remove the objective aperture.
- Perform the necessary gun alignments.
- Read in all previously stored microscope alignments except for the gun alignments.
Set the low dose magnification and illumination conditions (Fig. 1).
Fig. 1. Example low dose settings. The Options
flap-out box shows the dose measuring feature in the lower
right of the image.
- Turn on Low Dose on the low dose panel. The Search mode may be set up either in low-magnification,
bright-field mode or in defocused diffraction mode.
Bright-field search mode
- Hit the Search button
- Set the magnification to between 1000X and 3000X. This should allow the user to
view an entire grid square.
- Set the illumination conditions (C2 aperture, spot size, beam diameter) to
make the beam as dim as possible yet still allow the user to judge the quality
of the sample on the large, fluorescent, viewing screen. These settings will,
obviously, need to be tweaked when the sample is put in the beam. The aperture
in position three of the C2 aperture rod is most often used.
- Example conditions: 100 μm C2 aperture, 2500X, spot size 4, 100 mm
beam diameter would give a dose rate of ~0.1 e-/Å2/sec.
- Defocused diffraction search mode
- Make sure Low Dose is in Search mode
- The advantage of this mode is that the objective lens current, which
changes at different magnifications, remains constant throughout the procedure.
This results in a more stable beam.
- Choose your preferred C2 aperture and center it. The one in position three is most often used.
- Put the Magnification into the M mode (1,500 to 3,100X).
- Hit the Diffraction button on the right-hand control pad.
- Turn the Camera Length knob (same knob as magnification in bright field) to
around 135mm and spread the beam with the C2 illumination knob. This should
give you a view of a majority of a grid square.
- Select a spot size that is as small as you can get and still be able to see the
holes in the carbon film. This will reduce the dose the sample gets while in
search mode. This beam diameter and beam settings should not be changed throughout
the rest of the low dose session.
- Set up the Exposure mode.
- Go to Exposure mode
- Set the desired final magnification. This decision in based on the size of the macromolecule,
the pixel size at the specimen magnification, the digitization step size of the scanner if the
film is to be scanned, and the expected resolution of the final three-dimensional reconstruction.
- Center and spread the beam until it reaches the edge of the view screen. On the Sphera
this diameter is 165 mm. This beam diameter and beam settings should not be changed
throughout the rest of the low dose session although they should occasionally be checked to
make sure nothing has not moved.
- Measure the electron dose in the Exposure mode (Fig. 1). Open the "flap-out" box and click on
the Options tab. Make sure that 165 mm is in the beam diameter box at the
bottom of the screen and hit the Measure button. The readout shows the
dose in electrons/nm2. Move the decimal point two positions to the left to
get e-/Å2. For 200 kV electrons the dose rate should be
between 20-25 e-/Å2, at 50,000X, and an exposure of 1
second when using film. It is best to keep the value as low as possible but the
density on the developed film will be too light if the exposure is much less. Change the C2 spot size
to change the beam brightness.
- If you are using film go to the Plate Camera (User) page and set the
film camera parameters (Fig. 2). The Hold box should be checked, the camera
should be set on Manual rather than on Automatic exposure, and the exposure
time should be set to obtain the desired dose based on the measurement. The
Film text box is for typing in any information you may want to see printed on the
bottom of the film. Make sure you hit the ↵ key to enter the information. Also
take note that this information will be printed on all of the films until you
erase or change it.
Fig. 2. Plate Camera (User) panel
set for low dose data collection on film
- On the Low Dose page hit the flap-out button and go to th
e Settings tab. Set the Use: option for
the Exposure mode to use Plate Camera
if you are using film or US1000 if you are using the CCD. The CCD camera will
have to be on in order to see
this option (Fig. 3). If you are using the CCD enter the time of your exposure in
the Exposure time (s) box in the Exposure tab on the
bottom half of the main Low Dose page (Fig. 1).
Fig. 3. Choosing the plate film camera in the settings flap-out box in low dose.
- Set up the Focus mode. It is best to have the magnification on the focus
and exposure modes the same to minimize changes in the objective lens currents. The decision to do this, however, is usually based on if it is possible to do focusing and astigmatism correction at the working magnification. Focus mode is sometimes
set as high as 150,000X to accurately visualize any image astigmatism that may be present. If a TV-rate or a CCD camera is present it is possible to use lower Focus mode magnifications to do the corrections.
- The diameter of the beam and spot size settings are not a factor here as long as the beam
is not spread so far as to dose the Exposure area. Some microscopists suggest that the spot sizes be set the
same in all three modes.
- Example conditions: At 50,000X a focus distance of 2 μm and angles set at 0 and 180
degrees are sufficient. These values are set with the Multifunction knobs and the settings put the focus
points and the exposure point along the tilt axis of the holder. If commercially-available
C-flats or Quantifoil films are used (Fig. 4), the angles may have to be changed if the focus points fall in adjacent holes
rather than on the carbon.
Fig. 4. Low magnification view of a grid square. A mixture of dry holes and holes covered with the proper
thickness of vitreous ice can be seen. This regular array of holes is a feature in the commercial
Quantifoil and C-Flat films.
- Put the holder back into the beam. Minimize potential drift by removing any ice
crystals in the cryoholder dewar that form nucleation points and cause bubbling. This is done by taking
a cotton-tipped applicator stick and rubbing along all of the inside surfaces of the dewar and the central
rod. The crystals
then may be blown out of the dewar with a device that is made of a rubber stopper through which a length of
plastic tubing has been inserted. Put the tubing in the dewar of the holder and hold the stopper against
the opening until enough pressure is formed that it blows out some of the liquid nitrogen and the ice
crystals. Look in the dewar with a flashlight to see if the bubbling in the nitrogen has stopped. It
is also best that the level of nitrogen not be high enough to touch the central rod.
- Go to Search mode. Pull back the cryoshields on the
holder and check to see if you have a beam. Find a grid square that is nearly dry or one that
can be sacrificed. Your remaining alignments will be done in this area.
- Adjust the holder Eucentric Height. During microscopy if you ever move a
distance of more than a few grid squares recheck this.
- Go to Direct Alignments and correct the Rotation Center.
- Place an objective aperture into the beam and center it. Most of the time the
aperture in position three is sufficient but if more contrast is desired the aperture in position two
may be used. When searching for holes of an appropriate ice thickness it is often useful to alternatively
put in and retract the aperture. Areas of extremely thick ice will be too thick to get a beam through,
however, if the objective aperture is removed there may be enough brightness to allow for navigation to a
thinner region of the grid.
- Align the Search and Exposure mode positions. This is necessary so that the
area that is chosen in Search mode will be the same as the one photographed in Exposure mode.
- Place the beam stop/pointer into the beam and center it on the small
circle in the middle of the large fluorescent screen.
- Find an object of interest that will be recognizable at the both the
Exposure mode and Search Mode magnifications. Many times this can be a rip in the film or a large
chunk of ice such as that in the upper right corner of the grid in Figure 4. Go to Exposure
mode and move the object with the goniometer specimen shifts until the object is centered under the
tip of the pointer.
- Go to Search Mode. Move the object of interest
with the Multifunction X and Y knobs (image shift) until it is under the tip of the pointer. If
the Exposure and Search Modes are closely aligned this should be easy otherwise this may
need to be repeated until the object is aligned in both modes.
- Find a new object of interest and check the positioning in both modes
again. Make adjustments with the specimen shift while in Exposure Mode and with the
Multifunction X and Y knobs while in Search Mode.
- If you are doing your searching in bright-field mode defocus the objective
lens several hundred microns and then, if the object moves, bring it back into position with the
Multifunction X and Y knobs. This increases the contrast in the search mode and makes it easier to
recognize the sample in the ice. This will not be necessary if the defocused diffraction mode is
chosen for searching.
- In Search mode look across the grid for suitable areas of ice thickness and sample
concentration for data collection (Fig. 4). Judging the appropriate thickness of ice will become more
accurate with experience.
- When a good area is located that has the appropriate ice thickness go to one of
the corners of the grid square and recheck the eucentric height adjustment.
- A few techniques may be used to determine if there is sample in the ice-covered
holes and if the sample is properly dispersed.
- If a bottom-mounted, intensified, TV-rate camera is available the extra
magnification obtained by the difference in the physical mounting height of the camera compared with
that of the fluorescent screen may be enough to judge the quality of the preparation.
- Another method is to temporarily increase the magnification of the Search
mode until beam damage causes bubbling in the sample. This usually occurs first in the biological
sample which makes it possible to distinguish the sample from the background. This, obviously,
destroys this area and makes it unsuitable for photography. Checking a few areas should
result in a general idea of the sample distribution.
- If a CCD camera is present a few images may be recorded to check the sample.
- When an appropriate hole is found in search mode, center it under the pointer and
go to Focus mode. If the first Focus mode area falls on an undesirable area such as a dry hole select the
second Focus mode area. Focus and stigmate the image. This may be done with the binoculars on the fluorescent
screen, with an intensified, TV-rate camera or by calculating live FFTs from images collected with the
CCD camera. In Focus mode it is permissible to change the size of the beam with the Intensity knob
without affecting the beam in the other modes. This makes it much easier to focus the image.
- Focus the image to true focus and hit the Reset Defocus button. This button is
usually one of the options of the “L” and “R” user buttons that are on the inside edge of the control pads. The
Reset Defocus button zeroes out the focus value but it does not affect the actual focus setting of the
objective lens. It is then possible to defocus the image to the desired amount and view the value of the
defocus in the display box at the bottom of the screen.
- Hit the Expose button on the left control pad (not the Exposure Mode in Low Dose).
If you are using film you will hear a film plate being put into position. The shutter will open for the
set amount of time and then the film plate will be placed into the take-up box. If you are using the CCD,
the software will put the CCD camera into the beam and continue similarly as that of film. If you would
like a preview of what you recorded on film, hit the beam Blank, then go
to Exposure mode. When the beam is in position quickly remove the beam Blank
and take a CCD exposure manually with the Digital Micrograph software. After the image has
been recorded put the beam Blank back on and return to Focus mode.
Obviously, this image will not be suitable for processing but it will help you to pre-judge the quality
of your recorded film image.
- After the exposure is complete it is a good idea to occasionally check the
Exposure mode to see if the beam conditions have remained constant and that the area that was chosen to be
photographed is still centered.
- Go back to Search Mode. Note the areas of the grid square that were burned
due to focusing and photographing. Note the proximity of the two (or three) burn spots to make sure that your focus points are not dosing the areas you are recording. It is a
good idea to always focus your image in an area where data has already been recorded to maximize the
amount of data that can be collected from one grid square. If you are using C-flat or Quantifoil
grids you may need to adjust the focus distance and angle in Focus Mode if your focus points end up
in adjacent, good-ice holes.
- Systematically move across the grid to make sure that you are always moving
onto a new area and not onto one that has already been damaged by the beam. The use of the
specimen position display in Stage2, and with Tracks turned on, should
greatly help you decide if you have been in an area before.
- If you are collecting data with the CCD camera it is a good practice to
save your images as you collect them and not have more than a few images open on the
screen at one time. The computer does not have the capacity to run the microscope and
keep many images open at the same time. Too many images may cause problems with the microscope
operating system. It is easiest to name your images with the automatic
functions in Digital Micrograph.
- Go to File/Global Info… and click on the option Save Numbered in the left hand panel (Fig. 5).
Fig. 5. The Save Numbered
option in the Global Info
in Digital Micrograph
- Create or pick a folder in the File Directory. One way to name folders and
files is to use a combination of the date and the sample name. For example: 080910-BobVirus.
The date, September 10, 2008 is written the way it is so that folders are organized, and can be
- Check the radio button Build Using String and then add some text to name your
files. A good practice here is to use the same file name that you used for your folder.
- Set Next Index to 1.
- Under the File Content and Format area click on the radio button
Save Image As and in the dropdown box choose Gatan Format. The reason for this is
explained in Finding Image Parameters in
Digital Micrograph DM3 Files .
- Hit OK.
- To save an image go to File/Save Numbered or just hit CTRL-Y. Close the
image by hitting CTRL-W.
- The DM3 format has its advantages but it can only be read by a few programs.
Most users convert their DM3s into Tiffs and this can be done in batch mode. Click
on File/Batch Convert… and pick the folder where you stored your images by
clicking on Browse.
- In the File Content and Format area choose the radio
button Save Display As and choose Tiff Format as the
option in the drop down box. Hit OK to continue. If any of your
images are still open they will not be converted.
- If you are using film and you have completed your data collection remove and
replace the film camera. Make sure you hit the film stock reset button so the next user knows
there is available film in the microscope. Since the film has been underexposed for low dose
conditions it must be developed under low-dose conditions. Check
out Darkroom Procedures for
specifics on developing the film.