Page 353 - Fundamentals of Light Microscopy and Electronic Imaging

336 GLOSSARY

Confocal laser scanning microscope (CLSM). A mode of light microscopy whereby a
focused laser beam scans the specimen in a raster and the emitted fluorescent light or
reflected light signal, sensed by a photomultiplier tube, is displayed in pixels on a
computer monitor. The dimensions of the pixel display depend on the sampling rate
of the electronics and the dimensions of the raster. A variable pinhole aperture,
located in a plane confocal with the specimen, rejects out-of-focus signals and allows
for optical sectioning. 208
Conjugate focal planes. In light microscopy, two sets of field and aperture planes
whose precise geometrical positioning in the microscope is assured by adjusting the
focus of the objective, condenser, and lamp collector lenses as required for Koehler
illumination. The two sets of focal planes are conjugate with each other but not with
the focal planes belonging to the other set; as a consequence, looking from one focal
plane along the optic axis simultaneously reveals the images of the other conjugate
focal planes. 4
Constructive interference. In wave optics and image formation, the condition where
the summation of the E vectors of the constituent waves results in an amplitude
greater than that of the constituents. For interference to occur, a component of one
wave must vibrate in the plane of the other. 63
Contrast. Optical contrast is the perceived difference in the brightness (intensity or irra-
diance) between an object and its surround, and is usually given as the ratio of the
light intensity of an object I to the light intensity of the object’s background I , thus:
o
b
C (I I )/I , or alternatively as C (I I )/(I I ). 22
o
b
b
b
o
o
b
Contrast threshold. The minimal contrast required for visual detection. The contrast
threshold is strongly dependent on the angular size, shape, and brightness of the
specimen, the brightness of the viewing environment, the region of the retina used for
detection, and other factors. For extended objects, the contrast threshold is usually
given as 2–3% in bright light and 30–100% or even greater in dim light. 22
Convolution. In image processing, the procedure of combining the values of image pix-
els with a 3 3, 5 5, etc., matrix of numbers through a defined function for pur-
poses of image sharpening and blurring. The processing values in the convolution
matrix or kernel are applied to each image pixel and to neighboring pixels covered by
the mask in order to calculate new pixel values in a processed image that takes into
account the values of neighboring pixels. 292
Curvature of field. An aberration of a lens that causes the focal plane to be curved
instead of flat. 52
Cyanine dyes. Fluorescent dyes produced by Amersham, Inc., that are known for their
photostability, solubility, and relatively high quantum efficiency. 185
Dark count and dark noise. In electronic cameras, the photon-independent signal in an
image. The major constituents of the dark count are the bias signal, thermal noise,
and camera read noises from the detector and processing circuits. The dark count
must be subtracted from a raw image in order to obtain a corrected image based
solely on photon-dependent counts. The dark noise is defined as the square root of
the dark count and remains a part of the corrected image. The dark count is deter-
mined by acquiring an image (a dark frame) without opening the camera shutter. 273
Dark-field microscopy. A mode of light microscopy in which 0th order undeviated
light is excluded from the objective, and image formation is based solely on the inter-
ference of waves of diffracted light. Typically, dark-field optics are obtained by illu-
minating the object with a steeply pitched cone of light produced with a transparent
annulus in an otherwise opaque mask at the condenser front aperture. A relatively