School of Life Sciences

The Sussex Centre for Advanced Microscopy

Acquiring Digital TEM Images with

the Gatan Ultrascan 1000 CCD Camera

Getting Started ; Acquiring and Saving Images ;

Other Important/Useful Information ; Useful Tips ; Troubleshooting

********* N.B. *********

There is an intermittent alignment fault with the TEM. If, as you are viewing your specimen, you find that the electron beam suddenly disappears, please do the following:

PLEASE DON'T try to twiddle lots of knobs to find the beam again. Instead, turn down the filament current and turn off the accelerating voltage

Now press the RESET button below the CRT screen on the TEM left upper console

Turn on the accelerating voltage and turn up the filament current and you should find that everything is fine again. If you still have a problem then, please ask me to take a look.

However, if you lose the beam whilst changing your grids or changing the objective lens aperture, for example, firstly make sure that it is simply not a problem with aperture alignment or grid positioning (over a grid bar or at the end of a specimen shift).

Getting Started

Run up the Gatan Digital Micrograph software
N.B. If the camera/image control windows do not appear, click on 'Windows'/ 'Floating Windows'/ 'Show All'
Click on the ‘hammer and spanner’ icon on the top menu
Go to ‘Session’/’Session Info’ and enter your Specimen and Operator details, plus any Notes you would like saved with your image. N.B. The specimen identifier will be on every image name until you change it
Go to ‘Saving’/’Save Numbered’ and set ‘Next Index’ to 1 (N.B. *** or an appropriate number if you’ve already saved images with the same name previously ***)
Click on ‘Browse’ and go to the folder where you want your images to be saved, then click ‘OK’

Acquiring and Saving Images

Make sure your image is focused and any astigmatism corrected
Make sure the electron beam is centred and spread at least a little
Press the ‘PHOTO’ button on the left upper console of the TEM on (it will illuminate) and wait for the screen to raise fully
Now press the white button to the right of the PHOTO button upwards
Go to the ‘Camera View’ window and tick the ‘Camera Inserted’ box to slide the camera under the electron beam
In the ‘Camera Acquire’ window, make sure the ‘Auto Exposure’ box is ticked and click on ‘Start Acquire’
Type in the magnification and click ‘OK’ (make sure this is correct and enters correctly as the scale bar depends on this info - if the 'Num Lock' button has been left on, this needs to be turned off)
An image should now appear. N.B.(1): If you see just a white image, go to the Dropdown menu ‘Display’/’Image Display’ and key in, say, 10000 and 35000 into the Display/Contrast/Contrast Limits/Low and High boxes, respectively and click on 'OK'. N.B. (2): If there are jagged edges to the image, click on the 'hammer and spanner' icon within the 'Camera Acquire' window, click on 'Advanced Settings' and enter a zero into the 'Shuttering'/ 'Settling(s)' box, then press 'OK' and 'OK' in both windows)
Go to the ‘Display Control’ window and adjust the Contrast/Brightness/Gamma as required
To save the image, click on the ‘123’ save icon on the top menu to add your image identifier information to your image
Now click on the save icon to the left of the 123 icon to save your image
To resume TEM observation, firstly press the white button to the right of the PHOTO button downwards and then press the PHOTO button to turn it off

Other Important/Useful Information

When you change your sample, remember to change the Specimen identifier via the ‘hammer and spanner’ icon on the top menu, then ‘Session’/’Session Info’ before you acquire an image
At the end of a session, you can batch convert the Gatan format images into a format you can read remotely as follows:
Go to 'File' dropdown menu 'Batch Convert' and 'Browse' to your folder
Change 'Save Display As' to 'BMP Format', 'JPEG/JFIF Format' or 'TIFF Format' as required and then click on 'OK'. N.B. The JPEG/JFIF Format gives the smallest file size and there is no reduction in image quality (it's just all the meta-info you lose)
Please check that the batch conversions are successful if you are thinking of deleting the Gatan format images afterwards, as they sometimes do not convert properly. If this latter occurs, move or delete the successfully converted Gatan format images to another folder and then re-convert the remainder and keep doing this until they are all converted successfully (this is a bit of a software 'quirk' that I do not have an answer to as yet!)
Please remind me to back-up your images at the end of a session. If I'm not there, please drop me an email reminder mailto:j.r.thorpe@sussex.ac.uk

Useful Tips

When imaging immunogold labelled sections and the contrast of the specimen is so high that the secondary gold particles are not readily visible, please follow the suggestions of Gatan (pdf). N.B. If you do this, can you please make a note of the settings before you change them and then reset them to the previous values when you've finished your session.

Troubleshooting

1. Camera does not respond when you try and acquire an image

Remedy: Re-starting the PC normally cures this problem

2. Image appears white

Remedy: Go to the Dropdown menu ‘Display’/’Image Display’ and key in, say, 10000 and 35000 into the Display/Contrast/Contrast Limits/Low and High boxes, respectively and click on 'OK'. Afterwards, adjust as necessary. This is a bit of a software 'quirk' that I do not have an answer to as yet! Unfortunately, if this happens, you have to feed in these contrast limits each time you acquire an image. Taking a fresh gain reference sometimes gets over this problem.

3. The acquired image does not look good (it appears blurred or there are striations running across the image)

There are various possible reasons for this:

(a) The image is not correctly focused and/or the astigmatism is not corrected properly

Remedy: Check the image on the fluorescent screen and adjust the focus and/or astigmatism controls accordingly.

(b) The specimen is drifting under the heat of the electron beam

Remedy: Check the image on the fluorescent screen under the binocular to check if the specimen is drifting. If it is, try a specimen pre-irradiation to negate any thermal drifting.

(c) A camera 'gain reference' needs to be acquired

Instructions: Take a 'gain reference' as follows:

Retract the specimen rod from the TEM column into the 'park' position (pull out and turn to the right and leave in this position)

In the 'zoom' mode (with the objective lens aperture in), obtain a magnification of 20,000X and make sure that the beam is centred

Now spread the beam so that it fills the entire fluorescent screen and a little beyond that

Press the 'Photo' button and flick the switch next to it upwards as though you were about to take a normal image

Go to the 'Camera' dropdown menu and select 'Prepare Gain Reference'

Click on each 'OK' or 'Yes' to everything as a series of prompts pop up and the gain reference will start to be acquired

N.B. At some point during this process a prompt may pop up saying that you should adjust the illumination. Just use the brightness control to adjust it so that the cursor is within the green part of the pop-up window display

Wait till the gain reference is acquired and a prompt pops up to say that a new gain reference has been successfully acquired

Now you can re-introduce your specimen into the column and continue viewing. Bear in mind that the specimen may drift a little at first, having been out of the beam for a while. Do another specimen pre-irradiation if needs be

(d) There is some dirt in that part of your grid that is affecting the electron beam deflectors that operate when the camera takes an image

Remedy: (Memorise the position if it's important, and then.....) Move to a different part of the grid and try taking an image. If this appears good then some localised contamination on the grid is the problem. If the image is still not good, try taking an image of the other grid position in the specimen rod. If this image is good then it may mean that there is some dirt on the specimen holder around the other grid position and you can then try swapping the grids around (and asking Jules to clean the specimen rod later).