Shutters & Filters

• RS 232 Computer Interface
• GPIB 488 Computer Interface
• Parallel Mode (TTL compatible)

Filters

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1. Single or Multicolor Label: The researcher must decide if samples will be labeled with one fluorescently tagged target, or if there will be multiple targets and markers. Bandpass filters pass wavelengths in a certain range (passband) and absorb or reflect those outside the range. Researchers normally choose bandpass filters 20 to 35 nm wide, but a passband that is much wider (70nm) or much narrower (10nm) may be needed. A "long-pass" version of such a filter passes all wavelenths at or longer than a specific wavelength. Excitation filters are usually bandpass filters. Emission filters may be bandpass or long-pass filters. If you have only one target in your sample, choosing a filter set is easier. Long-pass filters maximize the signal passing to the detector, helping to reduce photobleaching, so these filters may be a good choice for single labeling research. More than one target in a sample complicates the choice of filters. Long-pass filters allow bleedthrough and have brighter backgrounds. For multiple labeling, you may choose a bandpass filter set, but be aware of the longer exposure times typical with bandpass filters. Images can be acquired with a series of individual bandpass filters, seeing only one color at a time. Multiband filter sets allow researchers to image all colors at the same time. It's also possible to combine techniques, using an excitation filter and imaging through a multiband dichroic mirror and emission filter. Viewing colors one at a time is the simplest, but requires multiple exposures. This can be turned into an advantage by choosing the detector (color or black/white, for example) that best suits each label. To see all the colors at once with this method requires using a computer and appropriate software. Changing filters and other imaging parameters can result in tiny shifts in registration, a problem that can be corrected with some imaging software packages. Multiband filters let the human eye or color CCD camera see all the colors in a sample at once, but in some cases the different labels may not all be in focus, and it is more difficult to keep one color from overwhelming other colors. Dim fluorescence may be masked completely. In the never-ending search for better images, researchers have teamed with hardware and software manufacturers to come up with filter wheels or sliders that a computer can change. The computer changes the excitation filer, but leaves the multiband dichroic mirror and emissiojn filter the same. Researchers must consider the cost of purchasing filter wheels or sliders and the software.
2. Light Source: epifluorescence microscopy usually involves a mercury or xenon arc lamp, note the lamp type and wattage. General fluorescence microscopes come with a mercury arc lamp marked "Hg" or "HBO", with wattage of 50 or 100 watts. In some microscopes, the type of lamp can be easily switched, in others this is a more difficult procedure.
3. Maximize the Light Source: Exploit the characteristics of the light source to enhance the image. Mercury arc lamps have an output that contains energy peaks at wavelengths the researcher can use to reduce photobleaching. An example: choosing an exitation filter that takes advantage of the 546nm spike in the mercury power spectrum, which results in the maximum signal when using rhodamine or CY3 markers. Xenon arc lamps and usually marked "Xe" and "SBO", with wattage of 75 or 150 watts. The power curve does not have sipikes like a mercury arc lamp, but is flat through visible light spectrum. However, Xenon power output increase at 750nm and remains strong to 1000nm--a good light source for ratio imaging or imaging in the far red. Confocal microscopes use lasers for illlumination, usually argon-ion or krypton-argon, but some may use helium-neon. A laser line excitation filter may enhance imaging characteristics. Doing without an excitation filter can be the best decision for some monochromatic sources as the narrowband filters used to isolate laser lines reduce laser transmission.
4. Detection Method: CCD cameras are sensitive to wavelengths of light farther into the red of the spectrum than the human eye sees, so filters must be chosen accordingly. What looks good to the eye may be overly saturated to the camera. An infrared blocking filter in front of the camera may be necessary.
5. Signal-to-Noise Ratio: Very bright samples against a very dark background have a high signal-to-noise ratio and may require a neutral density filter to dim the image so that it can be seen by the human eye. Imaging very dim specimens with low signal-to-noise ratios mandates a darker background and wider bandpass filter set.
6. Omega Filters: For more information, including graphs comparing filters, see http://www.omegafilters.com/