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. This interactive tutorial explores how absorption filters pass certain wavelengths of light while blocking others.
The tutorial initializes with a virtual absorption filter set to a wavelength pass band of 130 nanometers using the Bandwidth slider. The filter bandwidth size can be varied between 1 and 200 nanometers to demonstrate the wide spectrum of absorption filters commercially available for optical microscopy. Beneath the sliders is a graph of the filter characteristics that displays filter transmission percentage as a function of wavelength and calculates the center wavelength (CWL) and full width at half maximum (FWHM) for all of the bandpass filters in this tutorial. Filter neutral density can be adjusted from zero to 100 percent with the Transmission slider. As this slider is translated to higher densities (lower transmission values), the intensity of light passing through the filter diminishes and the bandpass peak on the graph is reduced in amplitude. The Filter Wavelength Maximum slider is utilized to adjust the bandpass center (center wavelength; CWL) of the virtual filter. As this slider is translated, the center wavelength can be varied between 350 and 750 nanometers, which is reflected by relocation of the curve in the graph presented in the lower portion of the tutorial window. The filter drawing in the upper portion of the tutorial simulates the spectrum of visible of light passing through the virtual absorption filter.
Absorption filters are the most widely utilized type of filter in fluorescence and other modes of optical microscopy, especially yellow and orange sharp-cutoff filters and black glasses that transmit ultraviolet and block most of the visible wavelengths. 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.
Among the advantages of absorption filters made from glass are the relatively low cost, high stability, and independence of spectral characteristics on the incident angle of light entering the filter. Chief disadvantages are the limited selection of glasses available for filter applications, poor slope performance and low peak transmittance of bandpass filters, a dependence of spectral performance on filter thickness, and autofluorescence of some glass formulations.