The Nikon DXM 1200 digital camera system software (ACT-1) has a live image control function (termed the Live settings panel, which is utilized to control the focus rating, white balance, exposure time, and camera sensitivity prior to capturing digital images. Each of these settings should be carefully scrutinized before selecting areas in the live window for photomicrography.

The Focus Rating is a relative value of the image sharpness represented in a normalized numerical form. Current focus conditions can be viewed in the Focus Rating frame located in the Live control panel, just beneath the image window. A green progress bar provides an indication of the current focus relative to the "best" focus position, which is represented by a red line positioned in the bar window. To the left of the progress bar is another window that presents a numerical value of the current focus conditions. Higher focus ratings indicate a better microscope focus for the specimen that is being viewed. In order to adjust and maximize the focus rating, the user must raise or lower the microscope stage.

Focus is measured at the position where the Focus Mark appears on the live image window. The user can relocate the focus position by moving the set of crosshairs to another position within the window. This is accomplished by clicking on the crosshairs (Focus Mark) and dragging it to a new location with the mouse. It should be noted that even when using an autofocus system, such as Nikon's V-IAF autofocus module or the Eclipse E1000, the focus conditions must be properly set in the ACT-1 software by maximizing the position (to the right) of the focus indicator progress bar. The Focus Mark feature can be deactivated by using the mouse cursor to place a check mark in the square radio box positioned in the Live control panel. Clicking the check mark to the on position (check mark present) activates the Focus Mark, whereas removing the check mark turns off the function.

The Live control settings panel also contains a Docking function selection. Turning the docking function on by clicking the radio box (placing a check mark) fixes the position of the Focus Mark in the center of the Exposure frame (Figure 1). When the Focus Mark is docked, translating the exposure frame with the mouse simultaneously moves both the frame corners and the set of crosshairs positioned in the center. To deactivate the Docking function, uncheck the square radio box. This allows independent movement of the Focus Mark and the Exposure frame. Note that when the Focus Mark is turned off, the focus rating does not appear in the control panel settings window and the green progress indicator bar is also absent. Instead, these symbols are replaced by the term N/A in the settings panel window.

The image can be evaluated for proper brightness based on the red, green, and blue (RGB) signal amplitude levels by examining these values with the Color Level monitor (see Figure 2). To activate the monitor, click the mouse on the Color Level monitor button that appears just beneath the Live image window, on the right-hand side of the ACT-1 software panel. When this button is activated, a line graph appears on the live image that plots each of the three RGB signal levels. The horizontal line of pixels at which the color level is monitored is located by a white bar (Pixel Line) that traverses the live image window. This bar can be translocated anywhere within the window by dragging with the mouse.

The Color Level monitor interprets individual pixel information for a selected horizontal row of pixels into a set of RGB values that are displayed on the screen in the form of a graph (Figure 2). For example, a pixel that has a RGB value of 30 (red), 238 (green), and 207 (blue) will appear teal when viewed alone. When displayed as a graph in the Color Level monitor, this pixel will be broken down into three individual RGB values that will each be represented as discrete points on the graph. A typical digital image having a single row of pixels displayed in RGB graphical format is illustrated in Figure 2. The information gathered in this format can be utilized to determine the relative brightness level of a digital image. If the graph displays a flat waveform on the top (Figure 3(a)), then the camera sensitivity is saturated and the gain (or microscope illumination) should be reduced to improve the image. Alternatively, when the graph shows a flat waveform positioned at the bottom of the live image window (Figure 3(b)), sensitivity is at a minimum (or zero) and either the camera gain or microscope illumination should be increased. Image gradation is determined by the waveform spread, with larger spreads leading to improved performance and a wider spectrum of color gradations.

A series of Color Level readings are presented in Figure 3. When the camera gain (or microscope illumination) is very high, as illustrated in Figure 3(a), all three RGB signal levels appear near the top of the window indicating a saturated image. In this case, the image appears drastically overexposed (white), which is indicative of total saturation. To remedy the situation, the camera gain or microscope illumination should be reduced. The negative slope of the red and green curves in Figure 3(a) is due to uneven illumination from the microscope. Utilizing the Color Level plots is a good mechanism to check for uniformity in microscope illumination when no specimen is present, provided the camera gain is not set too high or too low.

At very low illumination levels or when the electronic shutter speed is increased, a flat waveform appears in the Color Level window, as illustrated in Figure 3(b). In this figure, the DXM 1200 shutter speed was set to 1/4200 second with the gain (sensitivity) in Normal mode. Such a shutter speed is too fast for photomicrography under these conditions, and the situation is not improved even when the gain is increased (sensitivity set to High or Max) or the microscope lamp voltage is raised to 12 volts.

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Live Settings Panel Explore the various features available in the DXM 1200 ACT-1 control software Live settings panel, including exposure time, focus, color levels, and sensitivity.

Several additional examples of Color Level monitor utilization are presented in Figures 3(c) through 3(f). In Figure 3(c), a digital image illustrating scales on the edge of a butterfly wing also shows the dramatic changes that occur in the RGB levels when traversing from a clear area having a bright background (left-hand side of Figure 3(c)) to a region infested with yellowish-brown wing scales. Note how the spectrum spreads to produce a larger color gradation range on the right-hand side of the image.

When the color levels are measured on a specimen displaying a myriad of colors, such as the vitamin crystallites in Figure 3(d), the RGB signals levels oscillate in a sine wave-like fashion. If the specimen color is confined largely to a single hue (see the stained plant tissue in Figure 3(e)), the position of the resulting color signal levels will accurately reflect the percentage of each color present in the specimen. For example, the cyan-stained plant tissue illustrated in Figure 3(e) produces high signal levels of blue and green, which mix in equal proportions to produce cyan. The reduced amplitude of the red signal in Figure 3(e) is derived from darker shades of blue in the image.

Figure 3(f) illustrates the effects that occur when a single color overwhelms the image. In this case, a thin section of oak wood is heavily stained with hematoxylin and safranin, which bestows a deep red color to the specimen. Resulting color levels (Figure 3(f)) are split, with the red signal waveform (almost invisible over the dark red specimen background) being of much higher amplitude than the blue and green signals, which appear near the bottom of the window. Areas devoid of stain show peaks in the levels of all three color signals.

In conclusion, the Live Image window is equipped with a series of software features that allow the user to accurately monitor effects of both the camera sensitivity and exposure characteristics on the resulting digital image. This feature is very useful in producing sharp, crisp digital photomicrographs that display an excellent blend of color balance, saturation, and contrast.