visualization of certain cells or cellular components under a microscope.
Because of this staining requirement, live cells usually cannot be viewed with
this technique.
(2) Reflected light microscopyis a popular illumination method used to examine
opaque specimens that are highly reflective and thus do not readily absorb or
transmit light. As the top lighting configuration in Fig.8.1shows, in this
technique the incident light originates from above the specimen and is often
referred to asepiscopic illumination,epi-illumination,orvertical illumination.
After the incident light has been specularly or diffusively reflected, it can be
viewed with the eye or a light-imaging system. An advantage of reflected light
microscopy compared to the transmitted light method is that the image created
through the reflected light can be viewed as a three-dimensional representa-
tion, provided that the microscope optics can distinguish between regions of
different heights in the specimen.
Many advanced modern optical microscopes have both illumination methods
built into the same instrument. This feature allows the viewer to examine a spec-
imen either by alternating between the two illumination modes independently or by
using both techniques simultaneously. In either situation, the same
specimen-holding platform and viewing optics are used. Recent instrument
enhancements include the use of energy efficient high-intensity light-emitting
diodes (LEDs) in place of the standard illumination lamps.
Three common microscope structures are the upright microscope, the inverted
microscope, and the stereomicroscope. In theupright microscopeshown in Fig.8.2
the viewer looks down at the stage and the specimen through a pair of eyepiece
lenses. The stage is a vertically and horizontally movable platform for mounting
specimen holders such as microscope slides. The translation mechanisms that
control the stage movements allow the viewer to accurately position, orient, and
focus the specimen to optimize visualization and recording of images. Above the
stage is a rotatablenosepieceorturretthat contains three or fourobjective lensesof
different magnifying strengths. Typically the lens magnifications are 4X, 10X, 40X,
and 100X, which are coupled with an eyepiece magnification of 10X to yield total
magnifications of 40X (4X times 10X), 100X, 400X, and 1000X. Light from the
optical source located at the bottom of the microscope travels horizontally toward a
mirror located below the stage and then is transmitted upward after being reflected
by the mirror. The function of thecondenser lensshown in Fig.8.2is to focus light
onto the specimen. The image then can be viewed directly by the eye or by means
of an image-capturing device, for example, a camera or an image-processing CCD
array. A variety of spectralfiltering, light intensity control, polarizing, and light
focusing optical elements (such as diaphragms, mirrors, prisms, beam splitters,
polarizers, and lenses) are located within the optical path from the light source to
the viewer. Many modern microscopes have motorized lens changeover,
motor-driven stages for precision positioning, and automatic adjustment of illu-
mination intensity capabilities.
236 8 Microscopy