Microscope Requirements

The essential requirement for a microscope intended for image capture is a port for the electronic camera. This generally is referred to as a trinocular microscope, since the third port is also required for most photomicrographic systems, the old Zeiss Universal system incorporated the camera into the microscope, and therefore did not require a trinocular port. The camera must be matched to the microscope to insure optimal image quality. The microscope manufacturer will generally have available a standard mounting system for cameras, and there will most likely be different adapters depending on the type of mount on the camera, C mounts generally have shorter adapters than F mounts. Since C mounts are commonly associated with cameras that operate at video rates, C mounts are often referred to as Video mounts by microscope manufacturers. A microscope intended for electronic image capture must have an adequate illumination system. This does not necessarily mean that the light source must be very bright, but that the light source must be capable of fully illuminating the field of view, with as much uniformity of illumination as possible. This is not a new requirement, since the same capability is required for photomicrography. Since the field of view is larger at low magnifications, the best test of a microscope illumination system is at low magnifications.

If the light source is marginal, low magnification photographs will exhibit distinct variation in light intensity across the photograph, most commonly seen as dark corners in the photograph. The amount of light or the brightness of the light source is not critical for electronic image capture, since generally the camera sensitivity is sufficiently high that one or more neutral density filters will need to be inserted in the light path to reduce the total amount of light. Neutral density filters are filters that essentially reduce all wavelengths of light the same amount, so they simply reduce light intensity, and do not affect the color balance of the light. Color balance of light is very important in photomicrography with film. This is because film is manufactured to take advantage of specific types of light. A daylight film is rated for color balance of the sun, and sunlight is much bluer than a light bulb, which typically gives a red-yellow color. For this reason, photomicrography with film generally requires the microscope lamp to be operated as bright as possible, and to have a blue glass filter placed in the light path. Color balance is also important with electronic cameras, but electronic cameras are much more versatile than film. Either the camera control software, or the imaging software that collects the image will have capability of color correction.

This is generally done by setting different gain settings to the various color channels of the camera. For those systems that provide the user with direct feedback, one can instantly see that a standard microscope bulb is high in red and low in blue, since generally there will be no amplification of the red channel, and high amplification of the blue channel. The green will be between these two extremes. High quality camera software will perform these white balance steps automatically, with little user interaction. Generally this is accomplished by having the camera acquire an image at the illumination level that is going to be used for a given session at the microscope. The camera and software will then automatically adjust the picture for proper color balance, and then retain these values until instructed that the light has been changed. Even high-quality microscopes may have some difficulty with even field illumination, particularly at low magnifications. For this reason, many electronic cameras, and all image analysis software, have routines designed to produce flat-field images. These routines work by having the user set up the microscope with a slide in place, and align the light source. Then the user is instructed to move to a blank field, which is an area of the slide where there is no specimen, just the slide and the coverslip. An image of this “blank field” is grabbed, and is saved temporarily within the camera or the storage system. This blank field is then used as a mask to correct each subsequent field that is captured, typically by subtracting the blank field from the image, in bright field microscopes.