Imaging on Stereo Microscopes
Stereo microscopes (also called dissection microscopes) provide an intermediate range of magnification somewhere between a 1:1 macro lens on a consumer camera and a microscope (with overlap). Depending on lenses that are used on the stereo microscope and the projection lens on the camera, specimen widths from approximately 8 cm (3 inches) to 1 mm (~1/16 inch) can be imaged.
The denotation of "stereo" refers to separate and angled optical paths used for the right and left eyepiece to provide depth perception. For that reason, stereo microscopes are also used as a magnification tool for working with small objects.
For specimens with dimensions that exceed the field of view for a stereo microscope but are still small objects at less than 30 cm (~12 inches) or so, a copystand is often used. With additional diopters or a bellows, a copystand can duplicate magnifications of stereo microscopes, but the working distance between the object and lens can present problems with handling objects and with lighting.
Digital imaging choices when using image acquisition software is identical to what was covered earlier. Differences lie in the creation of flatfield images for front-illuminated specimens, where a white card is used as a reflective surface. The white card is put in place and imaged either before the specimen is put into place, or the specimen is removed (if practical) once the lighting decisions have been finalized.
On three-dimensional surfaces, flatfield images may be useless, depending on how the object is lit. Test the efficacy by comparing a flatfield corrected image of a specimen and an uncorrected image. If the lighting is focused more like a spotlight and the position of the lighting changes depending on the position of the specimen, or when uneven illumination is the objective of lighting, flatfield correction is counterproductive.
Controlling Glare and Lighting
The major disadvantage of macroscopy (small object photography) is that glare can be produced by specimens no matter what kind of lighting is used. For that reason, no stereo scope or copystand should be sold without a means to control glare. Often the thought is to place a polarizing filter in front of the lens, but that only controls glare when lighting is at a specific angle.
For effective control of glare, polarizing filters must be placed in front of lights and the lens (Figure 4.15). The polarizer in front of the lens turns so that the level of polarization can be controlled. The polarizers on the lights need to be fixed at a single angle of polarization (sometimes marked on filters). If the angle of polarization is not marked, a reflective object, such as crumpled tin foil, can be placed under the lens and the polarizer in front of one light can be turned until the glare disappears (or diminishes as much as possible). The disappearance of glare indicates "cross polarization." Then the same can be done for another light, one at a time. Do not turn the polarizer in front of the lens when going through this process, because the polarizers on the lights are being cross-polarized with the (stationary) polarizer on the lens.
Figure 4.15 Illustration showing polarizing filters in front of lens and light source (fiber-optic light guide).
Once the polarizers on the lights are set at the same angle of polarization, the polarizer on the lens can be turned to vary the level of polarizaton.
The advantage of stereo microscopes and copystands is in the multitude of ways to light specimens (when the lights are not fixed). Typically, lighting is done by using a fiber-optic lamp. Several ways of viewing a quartz rock with flecks of crystalline mica and a mouse embryo follow (Figures 4.16 through 4.18).
One fiber-optic light at 45 degrees, no polarization. This lighting shows both topographical information on the surface of the rock and specks of glare from the mica (Figure 4.16A). The bright specks help identify the mica as a crystalline structure.
Figure 4.16 Three methods for lighting a specimen.
- One fiber-optic light at 45 degrees, polarized. This lighting shows topographical information without glare from the mica by turning the polarizer in front of the lens to cross-polarize it with the polarizer at the end of the fiber optic (Figure 4.16B).
- Two line (or comb) lights pointed across the surface. Comb lights enhance the surface topography of the rock (Figure 4.16C). These lights are also effectively used to create darkfield images from unstained and near-transparent samples.
Backlit by a single fiber-optic light. This method of lighting eliminates surface features to reveal only veins and mica (Figure 4.17A and B). A specimen of a mouse embryo is also shown (Figure 4.17C): This image is backlit off-axis by a single fiber optic.
Figure 4.17 Backlit rock and mouse embryo.
On-axis lighting. Light can penetrate into crevices when using a 45-degree mirror in front of the lens (Figure 4.18). This mirror can be half-silvered (one half of the light is reflected, one half transmits through the mirror) or coated so that particular wavelengths are reflected (on the way to the specimen) and others pass through (emitted or reflected by the specimen to the camera). Ring lights can also be used for this purpose, but they are yet slightly off-axis from the lens, and some shadowing can result.
Figure 4.18 Portion of 96-well plate showing on-axis lighting and penetration of light into wells.
Tips for Imaging Challenging Specimens
Depending on materials or composition of specimens, some can be very challenging to position and light in order to produce accurate images. Their appearance can be visually confusing because of reflections or because textures and roundness do not translate well in images. Here are some suggestions:
- Specimens that reflect surrounding objects. A white tent can be created to surround highly reflective objects, such as highly polished metals. White paper can be made into a funnel. The camera is then focused through the opening in the funnel. Another alternative is to rub a thin layer of soft wax on reflective surfaces.
- Removing glare via setting specimen underwater. Glare can also be removed by setting the specimen underwater. Some elongation of the specimen will result because of the difference in the refractive index of air and water.
- Polymers and glass. Polymers and glass present difficult challenges for creating good representations of a specimen. Lighting is often pointed on the glass or the polymer itself. The better means for lighting may be to point a narrow beam of light on a part of the specimen that is not visible to the camera, and then to allow the light to "pipe" through the glass or polymer, or back light off-axis with a black background (as shown in Figure 4.18 with the embryo).