To ensure that image analysis leads to accurate measurement and quantification data, the precise calibration data, normally dimensions in the x, y, and z directions, and crucial parameters, such as magnification and the corresponding scale bar, are recorded directly with each image.
Typically, users acquire images by using a specific objective lens and zoom lens setting to determine the overall magnification. For the zoom lens, Leica VZ series, there are click-stop positions which indicate a calibrated magnification value. If during operation users should happen to select a zoom setting that falls in between click-stop positions, then the scale bar in the image reflects this fact by displaying units of pixels rather than distance (mm or μm). The calibration data are then recorded in that way with the stored image. Plus, the Leica VZ zoom lenses have a light emitting diode (LED), which glows blue whenever the zoom is in a calibrated click-stop position. Figure 1 shows the Leica DVM2500 with the Leica VZ700 C zoom lens operated with a zoom setting both in and out of a click-stop position.
Fig. 1a–b: Leica DVM2500 operated with the Leica VZ700 C zoom lens:
1a) in a click-stop position, indicated by the LED glowing blue and showing an image with a calibrated scale bar (units of mm);
1b) in-between click-stop positions (LED off) and showing an image with an uncalibrated scale bar (units of px referring to pixels).
The original calibration data are stored with all images recorded with the Leica DVM2500 in either two dimensions (2D) or three dimensions (3D) and remain unaltered. Storage of the accurate calibration data with the saved images allows users to make reproducible measurements and analysis at any time.
Additionally, 3D images acquired with the Leica DVM2500 show a realistic representation of the sample surface and preserve fine details. Leica