A majority of the common natural and artificial light sources emit a broad range of wavelengths that cover the entire visible light spectrum, with some extending into the ultraviolet and infrared regions as well. For simple lighting applications, such as interior room lights, flashlights, spot and automobile headlights, and a host of other consumer, business, and technical applications, the wide wavelength spectrum is acceptable and quite useful. However, in many cases it is desirable to narrow the wavelength range of light for specific applications that require a selected region of color or frequency. This task can be easily accomplished through the use of specialized filters that transmit some wavelengths and selectively absorb, reflect, refract, or diffract unwanted wavelengths. Filters are constructed in a wide variety of shapes and physical dimensions, and can be employed to remove or pass wavelength bands ranging in size from hundreds of nanometers down to a single wavelength. In other words, the amount of light excluded or limited by filters can be as narrow as a small band of wavelengths or as wide as the entire visible spectrum.
Basic Aspects of Light Filters - Many filters work by absorbing light, while others reflect unwanted light, but pass a selected region of wavelengths. The color temperature of light can be fine-tuned with filters to produce a spectrum of light having the characteristics of bright daylight, the evening sky, indoor tungsten illumination, or some variation in between. Filters are useful for adjusting the contrast of colored regions as they are represented in black and white photography or to add special effects in color photography. Specialized dichroic filters can be used to polarize light, while heat-absorbing filters can limit infrared wavelengths (and heat), allowing only visible light to pass through. Harmful ultraviolet rays can be exclusively removed from visible light by filters, or the intensity of all wavelengths (ultraviolet, visible, and infrared) can be reduced to specific ranges by neutral density filters. The most sophisticated filters operate by the principles of interference and can be adjusted to pass narrow bands (or even a single wavelength) of light while reflecting all others in a specific direction.
Filters for Color Photomicrography and Digital Imaging - Photography through the microscope is complicated by a wide spectrum of unexpected color shifts and changes that affect how the image is rendered on the film emulsion or electronic image capturing device. These unexpected imaging results are caused by a number of factors ranging from incorrect color balance between the light source and the film emulsion to optical artifacts such as aberration and lamp voltage fluctuations. A wide spectrum of filters are available to assist the microscopist in achieving the highest quality images in terms of color balance and saturation. These include color compensating and conversion filters, neutral density filters, didymium filters, filters to block ultraviolet light, and heat-absorbing filters.
Filters for Black & White Photomicrography and Digital Imaging - In black & white photography through the microscope, filters are used primarily to control contrast in the final image captured either on film or with a CCD digital camera system. Specimens that are highly differentiated with respect to colored elements from biological stains are translated into shades of gray on black & white film and will often appear to have equal brightness. When this occurs, important specimen details may be lost through a lack of contrast. Filtration techniques for black & white film are significantly different from those employed in color photomicrography.
Spectral Characteristics of Common Biological Stains - A wide variety of synthetic and naturally occurring biological dyes are available to the microscopist for selective staining of intracellular organelles in cells and tissues. Biological stains dramatically improve specimen contrast in brightfield illumination, and have been utilized for many years in histological preparations targeted at studies in anatomy, pathology, physiology, and similar disciplines. Compare the information contained in this section on the visible light absorption spectral data for common biological stains to determine suitability for use in black & white and color photomicrography and digital imaging.
Kodak Wratten Filters for Black & White Photomicrography - In black & white photography and digital imaging with the microscope, filters are employed primarily for control of image contrast. In many cases, filters are valuable in selectively imaging colored elements of stained specimens where contrast is obscured when the image is viewed in grayscale levels. Visible light transmission regions for Kodak Wratten color filters are presented in this section to aid microscopists in determining filter suitability for contrast and resolution control in black & white photomicrography.
Color Compensating Filters: Specifications and Spectral Data - Kodak color compensating filters are frequently used to fine-tune the color balance of tungsten-halogen microscope light sources for photomicrography with color films and digital imaging. These gelatin filters control color balance and contrast by absorbing differing amounts of the red, green, and blue portions of the visible light spectrum. This section is an index to pertinent information on color compensating filter specifications, including spectral data for each individual filter set.
Acousto-Optic Tunable Filters (AOTFs) - Several benefits of the AOTF combine to greatly enhance the versatility of the latest generation of confocal instruments, and these devices are becoming increasing popular for control of excitation wavelength ranges and intensity. The primary characteristic that facilitates nearly every advantage of the AOTF is its capability to allow the microscopist control of the intensity and/or illumination wavelength on a pixel-by-pixel basis while maintaining a high scan rate. This single feature translates into a wide variety of useful analytical microscopy tools, which are even further enhanced in flexibility when laser illumination is employed.
Interactive Java Tutorials
Absorption Filters - Absorption filters, commonly manufactured from dyed glass or pigmented gelatin resins, are one of the most widely used types of filter for brightfield and fluorescence microscopy. These filters operate by attenuation of light through absorption of specific wavelengths, so that spectral performance is a function of the physical thickness of the filter and the amount of dye present in the glass or gelatin matrix. This interactive tutorial explores how absorption filters pass certain wavelengths of light while blocking others.
Interference Filters - Recent technological achievements in bandpass filter design have led to the relatively inexpensive construction of thin-film interference filters featuring major improvements in wavelength selection and transmission performance. These filters operate by transmitting a selected wavelength region with high efficiency while rejecting, through reflection and destructive interference, all other wavelengths. Explore how interference filters operate by selectively transmitting constructively reinforced wavelengths while simultaneously eliminating unwanted light with this interactive tutorial.
Color Filters - Examine how color filters operate to change the color of objects visualized under filtered illumination. The tutorial enables visitors to drag and drop red, green, and blue virtual color filters over objects illuminated both with white light and also previously filtered with one of the primary additive colors.
Exposure and Color Balance in Photomicrography - Photography through the microscope is complicated by a wide spectrum of unexpected color shifts and changes that affect how the image is rendered on the film emulsion or digital electronic image capturing device. These unexpected imaging results are caused by a number of factors ranging from incorrect color balance between the light source and the film emulsion to optical artifacts such as aberration and lamp voltage fluctuations. This tutorial explores how exposure and color balance affect the qualities of color photographs using both everyday subjects and photomicrographs captured in the microscope.
Filters for Black & White Photomicrography - In black & white photography through the microscope, filters are used primarily to control contrast in the final image captured either on film or with a CCD digital camera system. Specimens that are highly differentiated with respect to colored elements from biological stains are translated into shades of gray on black & white film and will often appear to have equal brightness. When this occurs, important specimen details may be lost through a lack of contrast. Use this interactive Java tutorial to determine the appropriate starting point for contrast control in Black & White Photomicrography using Kodak Wratten filters.
Filter Control of Image Contrast in Black & White Photomicrography - As a general rule when employing color filters in black & white photomicrography, utilize filters that are complementary to specimen stain color (they absorb most of the predominant wavelengths transmitted by the stain) to maximize the amount of contrast in final images. To achieve a medium level of contrast, use filters that only partially absorb colors displayed by features of interest. Finally, to reduce contrast to a minimum, use filters that have colors identical to those of the specimen. A combination of filters can be used to enhance detail contrast in specimens stained with more than one color. This tutorial explores the use of Kodak Wratten color filters for contrast control in black & white photomicrography when using stained specimens.
Didymium Filters for Color Photomicrography - The intensities and hues generated by most biological tissue stains will reproduce very well on color film, but some stains tend to appear washed out or have their colors shifted, especially in multiple stain mixtures. In many instances, color compensating filters can help restore most or all of the lost color, but too much filtration can affect the color of neighboring stained features and the background. This problem occurs with the popular stains Eosin, Fuchsin, and Methylene Blue, which are not reproduced very well on most color films. Often, tissues stained with these dyes, either singly or in mixtures, appear muddy and lacking in color saturation. Explore, in this interactive tutorial, how didymium filters enhance intensities and hues of specimens stained with a variety of dyes for color photomicrography.
Acousto-Optic Tunable Filters - Wavelength selection is of fundamental importance in many arenas of the optical sciences, including fluorescence spectroscopy and microscopy. Electro-optic devices, such as the acousto-optic tunable filter (AOTF), are increasingly being employed to modulate the wavelength and amplitude of illuminating laser light in the latest generation of confocal microscopes. These filters do not suffer from the mechanical constraints, speed limitations, image shift, and vibration associated with rotating filter wheels, and can easily accommodate several laser systems tuned to different output wavelengths. In addition, acousto-optic filters do not deteriorate when exposed to heat and intense light as do fluorescence interference filters.
Liquid Crystal Tunable Filters - Liquid crystal tunable filters (LCTFs) use electrically controlled liquid crystal elements to select a specific visible wavelength of light for transmission through the filter at the exclusion of all others. This type of filter is ideal for use with electronic imaging devices, such as charge-coupled devices (CCDs), because it offers excellent imaging quality with a simple linear optical pathway.
Douglas B. Murphy - Department of Cell Biology and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Matthew J. Parry-Hill, Ian D. Johnson, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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