Page 55 - Fundamentals of Light Microscopy and Electronic Imaging

38 ILLUMINATORS, FILTERS, AND THE ISOLATION OF SPECIFIC WAVELENGTHS

Short pass Band pass Long pass

100
Peak %T Peak CWL
%T

%T
50 50% 50%
cut-off cut-off
FWHM
(HBW)

Wavelength (nm)

Figure 3-5
Filters for isolating the wavelength of illumination. Short-pass and long-pass filters,
sometimes called edge filters, block or transmit wavelengths at specific cut-off wavelengths.
Bandpass filters exhibit broadband or shortband transmission centered on a particular band
of wavelengths. Filter performance is defined by the central wavelength (CWL) and by the full
width at half maximum (FWHM). Another term for FWHM is halfbandwidth (HBW). A
bandpass filter can also be created from two overlapping short-pass and long-pass filters.

involving laser-based illumination, new fluorescent dyes and molecules, and ratio imag-
ing at multiple specific wavelengths.

Neutral Density Filters

Neutral density filters are used in microscopy to attenuate uniformly the intensity of
light over the entire range of visible wavelengths. They are commonly employed in dif-
ferential interference contrast (DIC), polarization, and fluorescence microscopy with
high-intensity arc lamps that cannot be regulated with an adjustable power supply. In
these circumstances, neutral density filters must be used. As discussed in Chapter 6, it is
impermissible to reduce the intensity of illumination by closing down the condenser
diaphragm, as this action affects resolution and contrast. A light-absorbing filter is the
only solution.
Neutral density (ND) filters have a neutral gray color like smoked glass and are usu-
ally calibrated in units of absorbance or optical density (OD), where

OD log (1/T),
10
and T is the transmittance (intensity of transmitted light/intensity of incident light).
Thus, a 0.1 OD neutral density filter gives 79% transmission and blocks 21% of the inci-
dent light. Other manufacturers indicate the transmittance directly. ND filters can be
stacked in a series, in which case the total density of the combination is equal to the sum
of the individual filter densities.
ND filters are either absorbing or reflecting. Absorbing filters contain rare earth
elements throughout the glass, so there is no reflective coating that can be scratched
off, and their orientation in the beam is not critical. Reflecting ND filters contain an
evaporated coating of metal on one of the surfaces, so care must be taken not to scratch