Procedures for Performing Cryoelectron Microscopy on the FEI Sphera Microscope

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 box 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 sorted, chronologically.
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.