Page 354 - Fundamentals of Light Microscopy and Electronic Imaging

GLOSSARY 337

small NA objective is used so that undeviated light does not enter the objective,
allowing only diffracted waves to enter the objective and form an image. 112
Dark frame. In image processing, a picture containing the dark count (thermal and bias
counts) and showing a featureless, low-amplitude background that is used to prepare
a flat-field-corrected image of the object. A dark frame is prepared using the same
exposure time as for the raw image but without opening the shutter. 292
Deconvolution microscopy. A method that applies computer deconvolution algorithms
to a through-focus stack of images along the z-axis to enhance photon signals spe-
cific for a given image plane or multiple focal planes in an image stack. A stepper
motor attached to the microscope focus drive guarantees image acquisition at regular
intervals through the specimen. In typical applications, deconvolution methods are
used to deblur and remove out-of-focus light from a particular focal plane of interest.
In more sophisticated applications, image frames of an entire stack can be decon-
volved to allow clear views of a specimen displayed in projection view or in 3D
viewing mode. 205
Depth of field. The thickness of the optical slice through a specimen that is in focus in
the real intermediate image. The thickness measurement is dependent on geometric
and wave-optical parameters. For a high NA objective, the thickness of the optical
2
slice in the specimen Z is given as Z nλ/NA , where n is the refractive index of the
medium between the lens and the object, λ is the wavelength of light in air, and NA
is the numerical aperture. 90
Depth of focus. The thickness of the image at the real intermediate image plane in the
microscope. Like depth of field, the focus thickness depends on geometric and wave-
optical parameters. The depth of focus is given approximately as [1000 M objective /(7
NA M total )] [ M objective 2 /2 NA objective 2 ], where M is magnification, is wavelength,
and NA is the numerical aperture. 90
Descanning. In confocal microscopy, the optical design of allowing the fluorescent
light emitted at the specimen upon excitation by the scanning laser spot to retrace its
path back through the objective lens and scanner mirrors to the dichroic mirror. With
descanning, the fluorescent image spot at the detector pinhole remains steady and
does not wobble. 210
De Sénarmont method of compensation. In polarization and DIC microscopy, the use
of a fixed quarter-waveplate retarder together with a rotating analyzer as a method for
measuring optical path differences (relative retardations) between O and E rays and
for introducing compensating retardation to adjust image contrast. Retardations of
/20 to 1 can be measured with an accuracy of 0.15 nm. 145
Destructive interference. In wave optics and image formation, the condition where the
summation of the E vectors of constituent waves results in an amplitude less than that
of the constituents. For interference to occur, a component of one wave must vibrate
in the plane of the other. 63
Dichroic mirror. In fluorescence microscopy, an interference filter that exhibits a
sharply defined transition between transmitted and reflected wavelengths. When
inclined at a 45° angle with respect to incident light beams, the mirror reflects short
excitation wavelengths through 90° onto the specimen and transmits long fluorescent
wavelengths to the real intermediate image plane. A dichroic mirror is one of the
three filters contained in a fluorescence filter set. 191
Dichroism. The property exhibited by linear polarizing films and certain naturally
occurring minerals, whereby incident wavelengths are differentially absorbed, caus-
ing the object to appear in two different colors depending on the angle of view and