Camera Setup Dialog
This dialog box allows you to specify exposure parameters for image acquisition. The controls for a CCD camera are described first. Options specific to the Gatan K2 Summit camera are described in Controls for Gatan K2 Camera, one for Direct Electron direct detector cameras in Controls for Direct Electron Cameras, and ones for Falcon cameras in Controls for FEI Falcon Camera. Controls for STEM acquisition, and modifications of the existing controls, are described in Controls in STEM Mode.
Camera radio buttons
If you have multiple cameras, each available camera will be listed here. Each camera has an independent set of parameters. The current camera will be the one selected. If you select a different camera, that one will become the current camera when you press OK in this dialog, but not if you press Cancel.
Match pixel size and/or intensity when changing camera
These buttons will appear if you have two or more CCD cameras that are not on a GIF. If you select pixel matching, the program will change magnification if necessary so that the pixel size matches as well as possible when you change from one camera to another. If you select intensity matching, then beam intensity will be adjusted so that the electrons per pixel per second will be the same after switchin between cameras. The pixel matching can be controlled by setting a property 'PixelMatchFactor' for one or more cameras; for example, setting this factor to 2 for one camera will make it match binned pixels for that camera with unbinned pixels for the other camera. This factor also adjusts the effective pixel size for intensity matching as well.
Parameter set radio buttons
There are five independent parameter sets for taking pictures. Use these buttons to select a set and view and adjust its parameters. Whenever you change from one set to another, the parameters are stored for the set that you are leaving.
Copy from other camera or from View
This button is available if there are multiple cameras, or if there are separate Search camera parameters. In the latter case, when Search is the selected parameter set, press the button to copy the View parameters for the current camera to Search. Otherwise, press the button to copy parameters for the current parameter set from the camera listed on the button. Acquisition area will be scaled appropriately if the two cameras are different sizes.
These buttons determine whether a single image will be acquired or whether acquisition will repeat automatically. Continuous acquisition is relatively slow in SerialEM for some cameras, but for newer Gatan cameras it accesses continuous camera modes and is fast. If you start a continuous acquisition, you can stop it by pressing the space bar or Esc up pushing the STOP button, and you can restart it by pressing the space bar again. Most automated operations (i.e., tasks and tilt series) will use single-image mode even when taking an image with a parameter set in which you selected continuous mode. Thus, you should be able to use those operations without having to turn off continuous mode in any of your parameter sets.
These buttons determine whether you will acquire an unprocessed, raw image from the camera, an image with the dark reference subtracted, or a gain-normalized image. An image is gain-normalized by subtracting a dark reference then dividing by the gain reference.
This checkbox will activate removal of X-rays from an image taken with this parameter set. It will appear only if the ShowRemoveXRaysBox property is set for the particular camera, or if one of your parameter sets already has the option checked. Use this option only if necessary to prevent bad tracking due to large X-rays, since the X-ray removal in SerialEM is not as effective or reliable as the removal with Ccderaser in IMOD. The parameters controlling the X ray removal are adjusted through the Set Image Criteria command in the Processing menu.
This checkbox is present for a OneView camera and turns on the real-time drift correction by that camera.
These radio buttons present the options for binning an image directly as it is read out of the CCD camera.
This text box allows you to enter the exposure time in seconds. Unlike in DM, exposure time will not change when you change binning.
This text box allows you to enter the amount of time that the specimen will be exposed to the beam before the picture begins being recorded on the CCD camera. This will minimize the effects of image drift when the beam first hits the specimen. For a OneView camera, drift settling is achieved by throwing away initial camera frames andit thus works with one shutter connected. However, if there are two shutters connected, the settling works only when the 'Film shutter' option described next is selected, so the box is disabled and the settling value there will have no effect if the 'Beam shutter' option is selected.
These radio buttons allow a choice of how shutters will be operated to expose the specimen and camera. The first two options correspond to the two shuttering modes available in DM when there are two shutters:
For a Tietz camera, only two shutter modes are shown. In the first mode, the specimen will be exposed only during the drift settling time, if this is possible, plus the exposure time. In the second mode, the beam will be left on the specimen continuously.
Force new dark reference next time only
If this option is selected, SerialEM will take a new dark reference itself, or force DM to take a new dark reference, the next time a picture is taken with this parameter set, and then the option will be cleared. There is no control over dark references with a K2 or OneView camera.
Take new dark reference each time
If this option is selected, SerialEM will take a new dark reference itself, or force DM to take a new dark reference, each time a picture is taken with this parameter set, which should be unnecessary unless DM's drift settling is used in an older version of DM.
Average dark reference
If this option is selected, then the dark reference will be averaged the number of times indicated in the text box. Averaging can be helpful for low-exposure images if the camera readout is particularly noisy. This selection, like everything else on the dialog, is specific to the camera and parameter set. Checking this box does not affect when a dark reference is taken; if you change the setting, use 'Force new dark reference next time' to get a new dark reference. When an averaged dark reference is taken, the status bar will display 'Dark Ref #n' instead of 'Getting Dark Reference. If you press 'Stop' before the dark reference is finished, a dark reference will be saved based on the average of the references already obtained, and will be used on the next acquisition.
Top - Left - Bottom - Right
These text boxes display and allow you to change the coordinates of the area to be acquired. As in DM, Y coordinates start with zero at the top. Unlike in DM, coordinates are always expressed in terms of the full, unbinned size of the CCD camera.
Up-down and Left-right Spin Buttons and Recenter Button
The two pairs of spin buttons allow you to shift the capture area in any desired direction without changing its size. The Recenter button will undo these shifts and center the area on the camera.
Swap X & Y
This button will transpose the X and Y coordinates to the extent possible. Even on a square camera, the X and Y sizes may not exchange exactly because of different constraints on sizes in the two directions.
Quarter - Half - Full - Wide Quarter - Wide Half
These buttons provide the most convenient way to select the area of the camera to be acquired. The quarter and half buttons refer to linear dimensions. The wide quarter is one quarter of the height and one-half of the width of the camera, while the wide half is one half the height and the full width of the camera. These options are provided because camera readout time is dominated by the number of rows being read out, and a rectangle gives more pixels of data per unit time than a square.
10% Less - 10% More - A Bit Less or Square
These buttons provide a convenient way to adjust the area being acquired to sizes other than the stock ones provided by the buttons above. Again, the changes are in linear dimensions, not areas. Because of rounding, the '10% Less' button will not exactly undo the change made by the '10% More' button, and vice versa. To get back to a regular size, just push one of the upper buttons.
The 'A Bit Less' button was useful for creating parameter sets with a slightly different sizes so that they would have independent dark references in DM. For older versions of DM, if you had two parameter sets with the same image size and binning but different exposures, DM would take a new dark reference every time you switched from one to the other. Making one set 'a bit less' in size would prevent this. However, as of at least DM version 3.8, DM would keep only one reference per binning regardless of size, making this trick ineffective. For a camera that is not within 10% of being square, this button is converted to 'Square', which will make a square area by trimming the current size in its long dimension to match the short dimension.
Dose readout and Update Dose button
If electron dose is calibrated for the current spot size and intensity, the estimated dose will be shown for the given exposure. This readout is refreshed whenever you change parameter sets or when you press the Update Dose button, so you can change brightness and use this button to see the effect of the change. For a direct detector camera, there is also a line expressing the dose rate as electrons per unbinned pixel per second, which is the value that affects camera performance. Initially, this will be a dose rate incident on the specimen; but once an image is taken with the camera, the program estimates the dose rate at the camera based on the apparent attenuation in the dose rate from specimen to camera in the last image that was taken.
OK - Acquire - Cancel
When you press the OK button, any changes in the current parameter set will be saved; if you press Cancel, such changes will be discarded. Note that changes are saved for a parameter set whenever you switch to another set. If you press Acquire, the dialog will go away, an image of the current type will be taken, the Min/Max/Mean command of the Process menu will be run, and the dialog will reappear. If you have hidden the Search parameters with the Use View for Search option in the Camera menu, then in Low Dose mode this button will be labeled either Acquire Search or Acquire View when the View parameters are being shown, depending on whether Low Dose is currently in the Search area or a different area.
Controls for Gatan K2 Camera
When a K2 camera is selected, the bottom section of this dialog provides options for accessing the special features of this camera. Some general features of the camera images and of SerialEM's interface to this camera are explained in Direct Electron Detectors, Especially the Gatan K2. The most salient points are these:
Linear - Counting - Super-resolution Mode
These radio buttons allow one to select one of the basic camera modes. The image size and binning selection are preserved when switching into super-resolution mode, or when switching out of it if the binning is above 0.5.
Always reduce to "Binned size" with anti-aliasing instead of binning
Select this option to have anti-aliasing applied to binned images in counting and linear modes as well as in super-resolution mode.
Dose Fractionation Mode
Use this checkbox to select Dose Fractionation mode, which enables a number of other options.
Enter the exposure time of each frame of the exposure in Dose Fractionation mode. The frame time is constrained to a minimum of 0.025 second.
Select this checkbox to have frames aligned in one of two ways (in DM, or by the SerialEM plugin to DM), or to have the plugin write a command file for aligning with the Alignframes program in IMOD. Press the Set Up button to open the Frame Alignment Parameters dialog and choose the method and parameters for aligning. When frame alignment is selected, an indicator below this checkbox will show which method is selected: 'Align in DM', 'Align in Plugin', and either 'Align in IMOD' or 'TS only in IMOD' when the third method is chosen. In the latter two cases, frame saving will be activated automatically; the 'Save Frames' checkbox will be disabled and shown as checked. When frame alignment is turned off or the option to align with IMOD is no longer selected, the 'Save Frames' checkbox will revert to its original setting.
Select this checkbox to have SerialEM's plugin to DM save each frame to disk. See above.
Set File Options
Press this button to open the Frame File Options dialog and make choices for whether to save frames in MRC or compressed TIFF format, whether to save frames in a stack or in separate files, whether to acquire frames without software gain normalization regardless of the processing option selected here, and whether to pack data acquired without normalization into half the space. The dialog also allows you to control the names of these files and to put subsets of them into different folders automatically. The choices set in the dialog are summarized in a line just above this button when saving is turned on. That line also shows the number of frames to be saved and whether they will be raw or normalized.
Press this button to select the directory where subframes will be saved as a stack, or where a container directory will be created for saving subframes in single files. If the SerialEM is running on the K2 computer, a directory browser will open. Otherwise, a small dialog will appear with a text box in which any directory name may be typed, even one not accessible to the computer running SerialEM. The dialog may also have a 'Browse' button that will open the directory browser. This browser is able to select only a valid existing directory in file systems accessible from the local computer, but it allows you to create a directory. The plugin is able to create a directory, but only if its parent already exists. This means that if you do not specify the use of folder names in the Frame File Options dialog, you can specify a folder here that does not exist, as long as its parent directory exists. However, if you do select options for automatic folder creation in that dialog, the directory you select here does need to exist before image acquisition. In this situation, if you enter into the text box a new directory that is accessible only from a separate computer hosting the K2, be sure to create this directory before trying to acquire.
Save variable frame sums
Select this option to save sums of camera frames of variable size, e.g., single camera frames at the beginning of the exposure, then pairs of camera frames, then larger sums at the end. The first time that you turn this option on, the Frame Summing Selector dialog will open to allow you to specify the variable summing of frames. Thereafter, this dialog can be opened with the Set Up button.
Controls for Direct Electron Cameras
When a Direct Electron direct detector or its survey sensor is selected, a section at the bottom opens up to allow control of what frames are saved by the Direct Electron server. The choices are to save Raw frames, images that are sums of a specified number of successive frames (Sum images with frame count), or the final summed image (Final image). All of these images are unprocessed and require dark-subtraction and gain normalization to be useful. The server will save some appropriate dark and gain images at various times in the autosave directory.
The summed image option does not produce useful output with the version of the server available in October 2013 but will be supported by a later version.
Set File Suffix
Press this button to open the Frame File Options dialog, which allows you add a suffix to the frame filenames based on your selection of components, such as SerialEM output filename or tilt angle.
Controls for FEI Falcon Camera
Some aspects of these controls differ for different versions of the Falcon camera and the software interface to them. Originally, Falcon 2 was controlled through the standard microscope scripting interface; this will be referred to as 'Falcon 2, old interface'. A new, advanced scripting interface was developed to allow control of the Falcon 3 camera, and this interface can be used with FEI software under Windows 7 to control the Falcon 2; this will be referred to as 'Falcon 2, new interface'.
Under the old interface, when a Falcon 2 camera is selected and the SerialEM property file contains an entry for the configuration file that FEI uses to specify intermediate frame-saving from the camera, a section at the bottom opens to allow control of frame-saving. With frame-saving selected, the FEI software writes the intermediate frames to a location specified in that file. SerialEM reads those files, converts them to a single MRC stack and/or aligns the frames, and deletes the original frame files. Although the number of frames that could be saved was originally quite limited, it is possible to use script commands to take multiple exposures whose frames are stacked into a single MRC file.
Under the new interface, frames are saved by FEI software into an MRC stack under a fixed location. If aligning of frames is selected to occur in SerialEM, the frames are read in by its microscope plugin or server, passed to SerialEM, and aligned there. If only aligning is selected without saving, SerialEM then deletes the frame stack. Some options will not be available if SerialEM does not have direct access to the frame storage location, however. See the help for the Frame Alignment Parameters dialog for details.
Intermediate frame-saving is ON in FEI dialog (old Falcon 2 only)
For Falcon 2, old interface, frame-saving is enabled by a separate dialog that SerialEM cannot communicate with; however, there is another Falcon configuration file that SerialEM can check to determine whether it is enabled, and on some systems, modifying this file will enable it. There are thus 3 cases:
None of the other options in this section are enabled unless SerialEM thinks that frame saving is either enabled or can be enabled when needed. Once frame-saving is enabled in scenario 1 or 2 above, the intermediate frame configuration file will be managed for every image acquisition, either to specify what frames to save or to prevent the saving of any frames. In scenario 3, either the Falcon configuration file or the intermediate frame file, or both, are always managed on every acquisition. Also, when the frame-saving is enabled, images will be gain-normalized in FEI software instead of in SerialEM, since problems have been seen in images gain-normalized in SerialEM when frame-saving is enabled.
Linear or Counting Mode (Falcon 3 only)
Use these radio buttons to select whether images are acquired from a Falcon 3 in linear (normal) mode or in electron counting mode. If frame alignment is being done with the Falcon processor, switching to counting mode may change the exposure time to satisfy the minimum requirements for counting mode.
Set Up Frames to Save (or Align)
Press this button to open the Frame Summing Selector dialog, which allows you to set how many summed (intermediate) frames are to be created from an exposure, as well as whether to skip some portion of camera frames at the beginning or end of the exposure. This dialog will open automatically when frame-saving or aligning is selected for a parameter set that has no previous information about how to compose frames. Once this information is set up, it may not be necessary to access this dialog often. When exposure time is changed, the program will adjust the number of camera frames summed into each frame so as to maintain the same number of frames saved, if possible.
When aligning frames from a Falcon 3 in the Falcon processor, the saved frames are constrained to be either single camera frames or multiples of 6 frames. Thus, switching to this alignment will cause the frames sums to be reorganized to meet these constraints; they will not be changed back when switching away from this alignment.
Select this checkbox to have summed frames aligned in SerialEM, or by the Falcon 3 processor, or to write a command file for aligning with the Alignframes program in IMOD. Press the Set Up button to open the Frame Alignment Parameters dialog and choose the method and parameters for aligning. When frame alignment is selected, an indicator below this checkbox will show which method is selected: 'Align in Falcon Processor', 'Align in SerialEM', and either 'Align in IMOD' or 'TS only in IMOD' when the third method is chosen. In the latter two cases, frame saving will be activated automatically; the 'Save Frames' checkbox will be disabled and shown as checked. When frame alignment is turned off or the option to align with IMOD is no longer selected, the 'Save Frames' checkbox will revert to its original setting.
For a Falcon 3, if SerialEM does not have direct access to the storage server, only alignment by the Falcon processor is possible, and saving of frames must be selected to enable this checkbox. Other than that, saving and aliging can be selected independently.
Check this box to save frames for images takes with this parameter set. The number of summed frames to be saved will appear in a text line.
Set File Options
Press this button to open the Frame File Options dialog, which allows you to control the names of the files in which frames are stacked and to put subsets of them into different folders automatically.
For Falcon 2, old interface, press this button to open a directory chooser that allows you to specify where the stacked frames should be saved by SerialEM. The frame files written by FEI software will go to this folder by default, but a property can be set to have them written to a standard location that is accessible to the FEI software. You can create a new directory in this chooser. If the directory specified here no longer exists when it is needed, SerialEM will create it, but only if its parent directory already exists.
Under the new interface, the frame stack can be put into a subdirectory directly under the defined storage location, but not into deeper levels of folders. Thus, the button opens a simple text entry box that allows you to enter the name of the folder, which will be created by the storage server if necessary. Since only one level is allowed, you can specify a folder either here or in the Frame File Options dialog, but not both. If you enter one here, the options to name subfolders will not be available in the other dialog.
Controls in STEM Mode
In STEM mode, there are some new controls, while other controls are omitted or disabled. The Processing and Shutter Mode radio buttons, and items related to dark references and dose are omitted. Positioning controls are disabled. Only the Quarter, Half, and Full area size buttons are shown, and they are disabled for DigiScan. Other new controls, or ones with changed meaning, are as follows:
With DigiScan, selecting Continuous acquisition starts a continuous scan, which SerialEM periodically reads. Although DigiScan stops the scan whenever the image is being read out, this mode still presents an advantage because SerialEM can process an image (e.g., for Live FFT) during the next scan. For FEI STEM, Continuous just acquires single frames one after another, the same as for a CCD camera.
IIn STEM, 'binning' decreases the number of pixels acquired on each line and the number of lines of data. If total exposure time is kept the same, the pixel dwell time is increased by the square of the change in binning, so there is an increase in signal-to-noise ratio comparable to that achieved by binning on a CCD camera.
Mag for Autofocusing
These control appear for the Focus parameter set with DigiScan and allow one to change the magnification used for autofocusing. The primary purpose for this is to compensate for the inability to acquire subareas with DigiScan; thus one can choose to increase magnification by 2, 3, or 4 times. In addition, one can reduce magnification by a factor of 2; this would be useful, and more convenient than using Low Dose mode, if autofocusing needs a larger field of view to work reliably while tracking does not. In any case, the nearest available magnification is chosen for the Focus pictures. To see what the Focus pictures look like, check Do for all Focus shots.
Exposure time and Pixel time
The duration of the exposure can be controlled by entering a number in either the Exposure time text box (in seconds) or the Pixel time text box (in microseconds). Entering a number in either box will change the value in the other box. There is only one underlying value that controls the acquisition, the pixel dwell time. The exposure time is defined as the product of the time per pixel and the number of pixels; the actual acquisition time will be greater because of time spent between lines of the scan. When a new exposure time is entered, it may be modified slightly because of constraints on the pixel time, or more extensively if Line Sync is checked with DigiScan.
Keep pixel time when binning changes
Ordinarily, the exposure time is kept the same when binning changes, and thus the pixel time changes. Use this option to keep the pixel time the same and change exposure instead.
Scan rate and Set Exposure for Max Scan Rate
The line labeled 'Scan rate' shows the speed at which the beam moves during the STEM scan, in microns per millisecond. Because of limitations on beam deflection, images are either shifted or distorted when the scan rate is above a certain level (roughly 0.5 micron/msec). As scan rate increases, at first an image will just be shifted, then it will appear stretched, then it may have serious curved distortions at the side of the image where the scan starts, especially on an FEI scope. The camera property 'MaximumScanRate' can be used to define both a maximum limit on scan rate, and a lower 'advisable' limit that it may be preferable not to exceed. If this property is defined, then the scan rate output shows three stars when the rate is above the maximum limit, and one star when it is between the advisable and maximum limit. Also, the Set Exposure for Max Scan Rate button will be enabled and can be used to set the exposure time so that the maximum limit is not exceeded. Another consequence of having this property defined is that tasks such as eucentricity will use an exposure time that is long enough to avoid exceeding the maximum scan rate.
Use this option to have defocus change progressively during image acquisition in order to keep a tilted specimen in focus. The option is enabled as long as the exposure time is long enough to allow at least five focus steps during the acquisition. Steps are taken every 40 msec on FEI scopes or 80 msec on JEOL scopes, unless set differently by the property 'DynamicFocusInterval'.
For FEI STEM, a line will appear above this checkbox showing what kind of timing information is available for the current acquisition parameters. An accurate estimate of flyback time is important for having the focus change at the right rate during the scan. The status line may show that a measured flyback time is available, which is very good; that it is being interpolated between measured times, which may be very good or bad depending on exposure time; that it is being extrapolated from measured times, which is more likely to be problematic; or that it is unavailable, which is bad.
Use this option with DigiScan to synchronize the start of each scan line with the cycles of the electrical supply. This can radically increase exposure time.
Channels to acquire:
If you have more than one STEM detector, the detector(s) to use are selected in one or more combo boxes. If you can acquire only one channel at a time, then there is just one combo box showing the available detectors. If you can acquire multiple channels simultaneously, then there will be one combo box per possible simultaneous channel. Any detector can be selected in any combo box. The order of selection determines the order in which images appear in SerialEM buffers; namely, the first channel will be in buffer A, the second in buffer B, etc. For FEI STEM, only the detectors selected in the microscope user interface when the dialog is opened are available. Unavailable detectors will be marked as 'NA' in the combo boxes. Such detectors can be left as the choice for particular channels; the program will simply skip them when acquiring an image, as long as there is at least one available detector to acquire from.