Gain Reference Dialog
This dialog box allows you to acquire a gain reference for the current camera. References are needed for older Tietz cameras, DE cameras with an older server version, Falcon 3, EMSIS, and DECTRIS cameras, and are optional for other cameras (except K2/K3 and OneView/Rio, where they cannot be taken). With a Gatan camera, this reference is separate from the gain reference that DigitalMicrograph maintains, and once a reference has been obtained through SerialEM, SerialEM will use this reference instead of DigitalMicrograph's reference. There are two advantages to using a SerialEM gain reference:
1)If SerialEM's capability for removing X-rays from dark references is configured, the gain reference will be protected from the effects of X-rays in its dark reference (which produce hot pixels in the gain reference.)
2)If you have a 4K camera with a slow readout and only need to use it as a 2K camera (e.g., fiber optic camera at 300 KV), SerialEM allows you take just a gain reference binned by 2, which is much faster than taking a reference in DigitalMicrograph.
Normalization is always done in DM for images from a K2/K3 camera because of the complexity of the processing there. For a OneView or Rio camera, drift correction can be done only with normalization within DM, so there seems to be no point in having a separate reference within SerialEM for images where drift correction is not needed.
To acquire a gain reference, you want to have a fairly bright, uniform beam with no specimen. The beam should be spread larger than the field of the camera, and will be more uniform if it is spread as large as the whole viewing screen. A dimmer beam may be appropriate for binnigs higher than 2. Check the following parameters and press YES to proceed.
Target number of counts
SerialEM will find the exposure time that will give this number of counts in each image that it takes. Enter a value in the text box or use the spin buttons to change the value by 500 . A high value (e.g. 8000) is generally appropriate for a sensitive camera, even if you are going to take low-dose images. However, if the camera is less sensitive, it may be necessary to make the target about the same as the number of counts expected in a typical image with normal exposure.
Number of frames to average
Use this entry to set the number of images to acquire and average. Five to ten frames will usually give good results; the higher number may be needed to average out X-rays more.
Select 1 to make an unbinned gain reference, which can be used to normalize images with any binning but could take a long time to acquire; or 2 to make a gain reference binned by 2. The latter can be used only to normalize images with even binnings. The text on this radio button will say whether an image with odd binning will be normalized with an older unbinned reference from SerialEM or with a reference from DigitalMicrograph. If your properties file contains an entry for 'ExtraGainReferences', you will also see choices for up to four higher binnings. Note that the target number of counts will change by a factor of 4 when you switch between unbinned and binned gain references, but the target does not change when switching between different binned references.
Average dark reference
Select this option to have the dark reference used for the gain reference images averaged by the number of times indicated in the text box. This averaging will reduce the effects of X-rays in the dark reference if you do not have X-ray removal set up or if it is not working well with the particular kind of X-ray signals produced in the camera.
Calibrate dose with last image
If this option is selected, then for an unbinned or binned by 2 reference, the program will use the last reference image to calibrate electron dose for the current spot size (see Electron Dose Calibration ). On a Thermo/FEI scope, the spot size will be normalized after you press the Yes button, in order for this to be as accurate as possible. This may change the beam position and brightness, so there will then be a message box reminding you to check the size and centering of the beam. On some scopes, this is not necessary, and there is a property that can be set to suppress the message. Otherwise, if the message is too annoying and you really do not care about dose, then deselect this option.
Autosave raw frames
This option is available for Direct Electron cameras; when selected, raw frames will be saved by the server for each gain reference image that is taken.