NOTE: The TEM Image mode acquires images from a CCD camera connected to the computer via a USB port. Before images can be acquired, the CCD camera must be mounted to the microscope and connected to computer via the USB cable. Also, the USB driver and hasp must be installed (review the Hardware and Software Installation links in the table of contents before proceeding).
TEM Image display and acquisition functions are controlled from the TEM Image Toolbar. If the toolbar is not visible at the top of the computer screen, selecting Mode → TEM Image from the top menu will make it appear:
The tool bar is divided into four sections:
Image Options. Used to set the image scaling; the menu selections are common to those for SEM Imaging. This section is also used to match Revolution's magnification to the magnification set at the microscope (the software cannot directly read this information). Refer to the Micron Marker Preferences for detailed information. This section is also used to specify the beam voltage (informational only, Revolution cannot control or read the beam voltage from the microscope).
Acquire Image. Used to set or define image resolution for both the acquire and preview modes (they can be set independently). The "camera" and "eye" buttons initiate acquisition and on-screen preview of the image, respectively.
Tools. Used to select tools for image manipulation. In order from left to right, these are:
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Switch to magnify-cursor. Used to zoom in images. Use the option key (Macintosh) or alt key (Windows) to temporarily zoom out. | |
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Switch to demagnify-cursor. Used to zoom out images. Use the option key (Macintosh) or alt key (Windows) to temporarily zoom in. | |
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Switch to hand-cursor. Used to drag image around in zoomed mode. | |
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Switch to pointer-cursor. Used for selecting any item with a valid handle in an image. | |
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Switch to text-cursor. Used to create or edit text annotations in an image. |
Status. Lists useful information about the current operational state.
Other TEM Image parameters can be controlled via the TEM Image Preferences.
| If no cameras is connected, the status will reflect this fact as shown in the toolbar at the top of the page. If the hardware (camera and hasp) has been properly installed and connected, the status will change to show that the camera is connected: |  |
| To launch a preview window, click on the preview button | → |  |
| A blank preview window appears on screen and the exposure time begins, with the corresponding status change.
The film number, beam voltage, and magnification are copied from the toolbar and inserted below the image, and the date and time are appended. Also appended are the facility name and user comments, if they have been entered in the TEM Image Preferences. |
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| When the exposure is done, the image is read from the CCD camera into the Revolution buffer...
... and then displayed on the computer screen (right). The image appears on the screen as the data are transferred if Live Update is turned on in the TEM Image Preferences. In preview mode, this process is repeated over and over, creating a live, albeit slowly updating, image. |
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When the preview is first displayed on the computer screen, some controls may not be set correctly. Before desirable images can be acquired, two procedures should be followed. These procedures can be repeated at any time.
Select Resolution
Adjust Brightness and Exposure Time
Most users will use the full resolution of the CCD camera (1 × 1 binning) for publication quality images. However, for setup purposes, 4pi supplies two smaller resolutions, corresponding to 2 × 2 and 4 × 4 binning of the CCD's pixels, and also "Selected Area" resolutions.
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Click the menu to the left of the preview button to see the available preview resolutions. 2 × 2 and 4 × 4 binning is controlled exclusively by the CCD camera and its on-board controller. Each factor of 2 decrease in resolution results in a proportional (factor of 4) decrease in both exposure time and data transfer time. SA = Selected Area. The CCD camera performs a 1 × 1 binning in the center of the field of view, using a reduced number of pixels. This procedure is controlled exclusively by the CCD camera and its on-board controller. Exposure time is not reduced because the binning is still 1 × 1; however, data transfer time is reduced proportionally. |
There are no "gain" or "offset" circuits in the CCD interface. Brightness is set by adjusting the TEM condenser lens settings. The exposure time is set in concert with the brightness. There are no specfic rules other than to make the image look as desired. Exposure times for the CCD camera may be more or less than the equivalent for film; however, they are unlikely to differ by an order of magnitude. Note: while making this adjustment, the display setting (toolbar Image Options) should be Full Range.
Select the desired acquisition resolution:
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Click the menu to the left of the acquire button to see the available acquisition resolutions. 2 × 2 and 4 × 4 binning is accessible as for the preview described above. Each factor of 2 decrease in resolution results in a proportional (factor of 4) decrease in both exposure time and data transfer time. Selected Area is not available in acquisition mode. |
| To start an acquisition, click on the acquire button | → |  |
The image will be captured in a separate window. The acquisition may be done while the Preview window is open, or not.
Images (including those that are only partially acquired) can be saved straight to disk at any time by selecting File→Save (keystroke clover-S on Mac, control-S on Windows). The same will work on a preview window.
The default format of a CCD image is selected in the TEM Image Preferences. The data from an acquired image or map can be saved to disk by keystroke (clover-S on Mac, control-S on Windows), selecting Save from the File menu, or simply selecting the close box in an acquired window (in a preview window, only File → Save is applicable). The following dialog appears:
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The selections available are:
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This correction (or "calibration") step accounts for the imperfections in the light sensitivity of the individual pixel elements within the CCD array. A full discussion is outside the scope of this document; only the general procedure and a description of the software calculation are outlined here.
The procedure requires that two frames be acquired aside from the desired image: a "dark" (sometimes referred to as "bias") frame, and a "bright" (sometimes referred to as "flat-field") frame.
The dark frame is taken with the shutter closed; it is a measure of the dark current in the CCD. The bright frame is taken with the shutter open; it is a measure of the uniformity of illumination. The bright frame should be taken with an illumination close to what will be used for the actual image acquisition.
| The calculation that Revolution performs is given by the equations: |  |
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where Ical is the corrected image, Iraw is the uncorrected image, Ibright is the bright-field image, and Idark is the dark-field image. M is the average pixel value of the dark-corrected bright field.
To properly perform the flat-field correction, follow these steps:
Remove the sample (recommended), or otherwise adjust the sample so that the TEM viewing screen is uniformly illuminated. To set this correctly, use the histogram tools (right-click on image, select Adjust Levels) to set the illumination just below saturation.
From the Acquire menu, select Acquire and Save Bright/Dark Fields. This can also be accomplished with the keyboard shortcut clover-D (Mac) or alt-D (Win). Note that this can be done only at full camera resolution. During the acquisition, This step will save two special files to disk: Bright Field.mbd and Dark Field.mbd. The files are saved to the top level of Revolution's directory, and are used to perform all flat-field corrections going forward. These files and should not be moved, nor should their names be changed. To create a new correction, repeat the acquisition of the two frames — the special files will be updated with the new information.
After the flat-field correction files have been created, the Flat Fielding checkbox will be available in the toolbar. When on, the flat-field correction will be applied to any image as it is being acquired. To temporarily circumvent this, turn the checkbox off.
The flat-field correction can be applied after the fact to any image of full resolution (for example, if the user forgets to turn the Flat Fielding checkbox back on). To execute this procedure, simply open the image in question and select Analyze → Post Flat Fielding. Once the Post Flat Fielding has been performed, it cannot be undone (in this case, the user can close the image without saving, and reopen it).
All images that are flat-field corrected will show a "(FF)" notation in the information field below the image.
