Accurate and reliable analysis
To ensure that image analysis leads to accurate measurement and quantification data, the precise calibration data, normally dimensions in thex, 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 LAS software gives users full control over the amount of image processing, permitting a balance between nice visualization and realistic display of the 3D surface topography. There is no unwanted smoothing of topographic features. Examples of these advantages are shown below in Figure 2.
Work efficiently with the Leica DVM2500
To ensure ease of use, fast workflow, and high output, the Leica DVM2500 can be configured with the Leica VZ700 C zoom optics. These optics allow users to easily and quickly change the total magnification over a very large range from 35x to 2,500x with no need to swap out lenses or touch the sample. To achieve this rapid, seamless change in magnification, the Leica VZ700 C is equipped with a revolving objective nosepiece containing 3 objective lenses. This feature is shown in Figure 3.
Depth of field and digital microscopy
For microscopy using optical lenses, which includes both ocular and digital microscopes, the depth of field is determined by the relationships between the numerical aperture, resolution, and magnification (please refer to references [1] and [2] for more details). Digital microscopes, or ocular microscopes fitted with digital cameras, optimize the acquired image by finding a balance between the depth of field and resolution – parameters which are inversely correlated. Particularly at low magnifications, the depth of field can be significantly increased by reducing the numerical aperture, such as via a diaphragm [1, 2]. However, the smaller the numerical aperture, the lower the lateral resolution [1, 2]. Finding the optimal
balance between resolution and depth of field will depend upon the morphology of the sample being imaged.
Advantages of the Leica DVM2500
The Leica DVM2500 provides efficiency, ease of use, as well as highly reliable image data for analysis. It achieves these benefits for users via:
- Direct storage of accurate calibration data with the recorded images in both 2D and 3D permitting reliable measurements from the image data at any time
- Acquisition of 3D images with a realistic display of the sample surface and no unwanted smoothing due to the image processing capability of the Leica LAS software
- Real time feedback during operation to ensure that the selected magnification has valid calibration data, as indicated by a blue LED
- The ability to rapidly change magnification over a wide range from 35x up to 2,500x without having to touch the sample or change the lenses
A digital microscope offering reliable image data and highly efficient workflow makes it beneficial to use. The Leica DVM2500 has been purpose-designed to provide all of these benefits.
References / Additional reading
- Rottermann R, and Bauer P: How Sharp Images Are Formed: Depth of Field in Microscopy. Science Lab (2010).
- Goeggel D, and Schlaffer G: 3D Visualization of Surface Structures: Vertical Resolution – Small Steps, Big Effect. Science Lab (2012);
- PDF version for download: Leica DVM – 3D Visualisation: Vertical resolution in the balance between numerical aperture and depth of field.
