Page 54 - Fundamentals of Light Microscopy and Electronic Imaging

FILTERS FOR ADJUSTING THE INTENSITY AND WAVELENGTH OF ILLUMINATION 37

the lamp off, the reverse procedure should be used: First, turn off the accessory elec-
tronics, then turn off the arc lamp. It is advisable to post a simple warning on or near the
arc lamp power supply that states First on—Last off to act as a reminder to yourself and
other microscope users. Second, when an arc lamp fails, remember that some power
supplies try to continually reignite the lamp with a series of high-voltage pulses that can
be heard as a rapid series of clicks. When heard, the supply should be shut off immedi-
ately; otherwise, electromagnetic fields might damage peripheral equipment as well as
the power supply itself. There is always the risk that this may happen when you are out
of the room and away from the microscope. To protect against this, and especially if
time lapse studies are performed with no one at the microscope, be sure the power sup-
ply is protected with an automatic trigger override switch.

FILTERS FOR ADJUSTING THE INTENSITY
AND WAVELENGTH OF ILLUMINATION

Selecting and adjusting the lamp for a particular application is important, but the actual
control of the wavelength and intensity of illumination in a microscope requires the use
of filters, so it is important to understand the fundamentals of their action and perform-
ance. The microscopist needs to know how to interpret the transmission spectra of fil-
ters, select the best among several possible filters for a given application, and explain
differences in image quality, fluorescence quality, and cell behavior obtained with dif-
ferent filter combinations. This is particularly true in fluorescence microscopy, where
filters must match the excitation and emission requirements of fluorescent dyes. Fortu-
nately, the task is manageable, and filtering light to select a band of wavelengths from
the illuminating beam presents many challenges and rewards.
Neutral density filters regulate light intensity, whereas colored glass filters and
interference filters are used to isolate specific colors or bands of wavelengths. There are
two classes of filters that regulate the transmission wavelength: edge filters and band-
pass filters (Fig. 3-5). Edge filters are classified as being either long-pass (transmit long
wavelengths, block short ones) or short-pass (transmit short wavelengths, block long
ones), whereas bandpass filters transmit a band of wavelengths while blocking wave-
lengths above and below the specified range of transmission. Optical performance is
defined in terms of the efficiency of transmission and blockage (% transmission), and by
the steepness of the so-called cut-on and cut-off boundaries between adjacent domains
of blocked and transmitted wavelengths. Edge filters are described by referring to the
wavelength giving 50% of peak transmission; bandpass filters are described by citing
the full width at half maximum transmission (FWHM), and by specifying the peak and
central transmitted wavelengths. FWHM is the range of the transmitted band of wave-
lengths in nanometers and is measured as the distance between the edges of the band-
pass peak where the transmission is 50% of its maximum value. For high-performance
filters these boundaries are remarkably sharp, appearing on transmission spectra as
nearly vertical lines.
In part, the resurgence of light microscopy as an analytic tool in research has been
driven by the technologies used for depositing thin films of dielectric materials and met-
als on planar glass substrates. Companies now manufacture interference filters with
transmission and blocking efficiencies approaching 100% and with bandwidths as nar-
row as 1 nm anywhere along the UV-visible-IR spectrum—a truly remarkable accom-
plishment. This achievement has stimulated development of new research applications