The ACT-1 software main window contains two display areas for live and captured images, as well as an area for display of thumbnail-size saved images. The window's menu bar and toolbars provide the capability to easily enlarge, reduce, crop, and position the images, to display them full-screen, and to interchange images in the large and small areas. The small subwindow also serves to display graphs for line profile and histogram analyses of image brightness. If images have been deleted from the thumbnail display area during file manipulation, a refresh feature makes it possible to re-create the thumbnail images corresponding to any saved image folder.

The dual image windows permit both a live image of a specimen currently being examined in the microscope and a previously captured image to be displayed simultaneously. The layout of the toolbars for the main (left-hand) and smaller (right-hand) windows are illustrated in Figures 1 and 2, respectively. The Switch Live Window button (Figure 1) causes the live and captured image windows to swap positions each time it is clicked with the mouse pointer. Selecting the Display Right option from the Live menu performs the same function. Two different methods of image enlargement, or zooming, are provided as toolbar functions, and image zooming may also be accessed from the windows menu bar. Zoom In buttons are provided on the toolbars for both the main left-hand image window and for the smaller right-hand subwindow. Clicking either button will cause the corresponding image to be enlarged at a specific magnification ratio, centered on the original image center. Each time the Zoom In button is clicked, an additional enlargement step occurs. In order to view the entire image after zooming, the horizontal and vertical scroll bars in the image window may be employed, or in the case of a captured image the Hand Pointer button (Pan Image, Figure 1) in the toolbar may be used to move the enlarged image. This feature is activated by clicking on the Hand Pointer icon, after which the cursor may be used to click and drag the image. Note that the hand pointer does not function on the live image, and scrolling the live image with the scroll bars is only possible when the image refresh is turned off (Live Window On/Off, Figure 1). Clicking on the Selection Pointer button switches from the hand pointer back to the standard arrow pointer. Enlargement of the live image cannot be performed when a line profile, histogram, or the focus mark is displayed.

The Zoom Out buttons in the toolbars for the left and right windows are utilized to reverse the enlargement function, applying an image reduction with each click, at the same predetermined ratios as applied by the Zoom In buttons. Both the zoom in and zoom out functions can also be accessed through the window menu bar by selecting Zoom + or Zoom - from the Live pull-down menu for the live image, and from the View pull-down menu for a captured image. The live image cannot be reduced if a line profile, histogram, or the focus mark are being employed.

Another method for enlarging a selected area of a captured image is enabled by clicking on the Magnifier Pointer button in the left-hand toolbar, which converts the standard pointer into the magnifier pointer. An image area to be enlarged can then be specified by positioning the pointer in the image window and dragging while holding the mouse button to outline a rectangular area for enlargement. When the mouse button is released, the specified area is enlarged to fill the display area. The magnifier pointer function may also be enabled by selecting the Zoom Rect option from the View menu (for a captured image). The magnifier pointer is returned to the standard arrow pointer by clicking on the Standard Pointer button.

Displayed images that have been enlarged or reduced may be resized to fit the display window, so that the entire image is visible, by clicking the Fit button in either the left-hand toolbar for the main image window or in the right-hand toolbar for the smaller subwindow. The Fit to Screen command in the View pull-down menu (for the captured image) or in the Live menu (for the live image) performs the same function. Immediately to the right of the Fit button on each toolbar is a Full Screen button that will cause the corresponding left-hand or right-hand image to be displayed full-screen surrounded by a black border (Figure 3). The Full Screen command in the View and Live menus, for the captured and live images respectively, functions in the same manner. To restore either image to its original displayed size in the main software window, the user should press the Esc, Space, or Enter key on the keyboard.

The usage of the computer CPU is reduced when the live image is not updated. A Start/Stop Live button is provided on the main window toolbar, which functions to start or stop updating the live image. Each time the button is clicked, the state of the live image is changed between on and off. The On option in the Live pull-down menu may also be utilized to start and stop live image refresh by clicking to place or remove a check mark adjacent to On.

Another image display feature accessed through the Digital Sight ACT-1 software main window toolbar is an image cropping function for application to captured images. In order to employ the cropping function, the captured image must be positioned in the main window by clicking the Change Live Window button, if required. When the mouse is clicked on the Crop Image button in the main display window toolbar, the mouse cursor changes to a cropping icon, which is utilized to delineate the image area to be cropped from the full image. After positioning the cursor in the image area, dragging with the mouse defines a rectangular area for cropping. To perform the crop after area selection, the Crop Image button on the toolbar must be clicked again.

The Thumbnail Display Area of the software window (illustrated separately in Figure 4) may be used to review captured images from a specified folder on the computer hard drive, in most cases the image folder being utilized for image storage during a microscopy session. Five image thumbnails are displayed at one time, and scroll arrows at each end of the area allow all images from the selected folder to be viewed. A thumbnail image is created as each captured image is saved, and is placed in a folder labeled s, immediately under the image folder in the file directory tree. If thumbnail images have been deleted during file manipulation, they may be recreated from the appropriate folder at a later time. This is done by first clicking on the File panel tab in the settings panel group to provide access to the image folder settings. The image folder for which thumbnails are to be recreated is then selected by bringing up the Browse for Folder dialog box or is entered directly into the Image Folder text field. After clicking on the OK button, Refresh Thumbnail should be selected from the Image pull-down menu to recreate the thumbnails from the selected folder.

Figure 4 illustrates the display and manipulation features of the thumbnail image area. The filename appears above, and the image size below, each displayed thumbnail. Arrows at the left and right ends of the display area allow scrolling backward and forward either one image or five images at a time within the currently selected image directory (set in the File panel). Clicking with the mouse on an image marks it as selected, and its border appears in red. When an image is right-clicked with the mouse, a Thumbnail Menu is displayed (see Figure 4), which provides a rapid means of selecting or unselecting all of the images (Select All, Unselect All), or jumping to the first or last thumbnail in the folder (Jump to TOP, Jump to END). An information field at the lower right edge of the thumbnail display area lists the images currently in the display window (numbered serially), the total number of images in the current image folder, and the number of images selected (labeled Check:).

Experimental information pertaining to an image can be displayed by right clicking on the desired image, and selecting Properties from the Thumbnail Menu. The File Information dialog box is displayed (Figure 5), which lists a number of default items specifying settings of the camera system for the selected image. The sequence of items in the list can be changed by clicking an item to be repositioned and using the up and down arrow buttons to move it to the desired location. Additional experimental information can be added by the user by clicking the Add button, after which a name for the item and comments can be typed directly in the appropriate fields. When the file information is printed, items that have nothing entered in the comment fields will be omitted. User-added information items can be deleted by selecting the item and clicking on the Delete button. The default entries in the information box can be repositioned in the list sequence, but cannot be deleted.

Assistance in focusing the microscope image is provided by the ACT-1 software through a feature that evaluates image sharpness at the position of a Focus Mark displayed in the live image window. Clicking the Focus checkbox in the image analysis panel (Figure 6) turns on the focus assistance feature, and displays both a numerical focus rating and a focus indicator in bar graph form. Higher numerical values of the focus rating, and further deflection of the indicator bar, are indicative of greater image sharpness (better focus). The microscope focus controls are adjusted to maximize the focus rating, which may provide more precise focusing than relying on visual evaluation alone. The bar graph focus indicator displays a peak value measured over multiple image frames. The focus rating is determined at the position of the Focus Mark in the image window, as illustrated in Figure 7. By clicking and dragging with the mouse, the mark can be moved to any desired position in the image. Clicking the Def button returns the Focus Mark to the default position at the image center. When the focus assistance feature is not turned on, or if the focus rating has a value of zero, N/A is displayed in the focus rating window.

The smaller subwindow located on the right-hand side of the main ACT-1 window serves to display either images or graphs that are generated for line profile and histogram analyses. Each of these analysis functions provides a method for evaluating image intensity on a more precise basis than afforded by averaged photometry measurements. Buttons for selecting the line profile and histogram analysis modes are located adjacent to the focus indicator in the analysis window (Figure 6). Clicking on the Line Profile button displays a graph of the red, green, and blue (RGB) signal levels of the image displayed in the main window. The form of the graph is an intensity profile across one line of image pixels, corresponding to the white horizontal line superimposed on the image. The position of the line may be changed by dragging it with the mouse, allowing analysis of the signal levels across any horizontal line of the image.

The focus rating bar graph is not functional when the graphical display is in use, and N/A is displayed in place of a numerical focus rating. The Graph Switching button allows the display to be switched from a single graph displaying the R, G, and B signal levels simultaneously (Figure 8(a)), to a set of three graphs illustrating the color channels separately (Figure 8(b)). The horizontal axis of the line profile graph displays image pixel count when the Use Unit check box is not selected, and is divided into absolute distance units when the box has been checked by clicking with the mouse. In order for the units to accurately represent distance in the specimen, the pixel resolution (the size of a single pixel) must have been previously determined either by specifying the total optical magnification of the microscope or by measurement of a known reference distance in the specimen utilizing the same microscope configuration as employed for the line profile analysis. Details of the procedure for determining pixel resolution with the ACT-1 software is described in the Annotation Tools section.

A histogram plot of intensity (brightness level) distribution of the image displayed in the main window is created when the Histogram analysis button is clicked with the mouse (Figure 6, Histogram Feature button). The plot represents the number of image pixels occurring at each of 256 intensity levels for the three color channels. In identical fashion to the line profile display, a Graph Switching feature allows the histogram to be changed from a combined display of three color channels in line graph form to one having separate plots for the red, green, and blue channels. Figures 9(a) and 9(b) illustrate the two histogram display types. An array of Color Channel Selection buttons are active in the combined RGB display mode, which provide the option to display any one of the red, green, or blue channels individually, or all three together. In Figure 9(a), the button designating the combined display of all three color channels is selected.