Optical Systems

1. Stereomicroscope
The simplest optical microscope most frequently encoun-
tered in a laboratory is a stereobinocular microscope. This
is typically used to magnify an image from 5 to 60 times,
and the user sees a three-dimensional, noninverted im-
age. The three-dimensional, or stereo, image is formed by
two separate compound microscopes (one for each eye)
focused at the same point from two different angles, typi-
cally 10–15◦ apart. This results in the brain receiving two
slightly different images which, when combined, give the
third dimension to the object observed, just as in normal
everyday vision.
Most manufacturers offer very fine microscopes with
highly corrected objectives and methods to record an im-
age on film or videotape. Stereomicroscopes are used most
frequently as inspection devices; however, to the chemical
microscopist, the uses include preliminary sample obser-
vation or sample selection, segregation of materials, ma-
nipulation of a sample, and preparation such as picking a
particle or fiber from a bulk sample and mounting it in a
particular orientation on a microscope slide in preparation
for more detailed study under the higher magnification of
a compound microscope, most usually, a polarized light
microscope.
2. Compound Microscopes
The optical system of a simple compound microscope con-
sists of four basic elements: a light source, a condenser,
an objective lens, and an eyepiece or ocular.
The light source in present-day microscopes consists
of a tungsten filament light bulb or a higher brightness
quartz–halogen bulb. Some specialized techniques, how-
ever, require more sophisticated light sources. Fluores-
cence microscopy, for example, requires a lamp which
will emit light at a wavelength (365 nm) sufficient to cause
the specimen under observation to fluoresce in the visible
light range. The best sources for fluorescence are mercury
vapor arc lamps with a set of filters to remove unwanted
wavelengths and heat. The condenser is used primarily
to collect the light from the source and concentrate the
light upon the specimen plane in order to uniformly illu-
minate the sample. Condensers have other uses which are
discussed below.
The objective lens is the most important optical element
in the compound microscope; it gathers the light transmit-
ted through or reflected by the specimen and forms a pri-
mary image then further enlarged by the ocular. The objec-
tive is usually engraved on its barrel with various numbers
and letters; for example, 10 × POL, 0.25 or 40 Ph, 0.65,
0.17. The first, and usually largest number, is the magnifi-
cation; in the two examples above, this would be 10× and
40×. This is the nominal magnification of the intermedi-
ate image formed at the focal point of the eyepiece. The
letters signify the type of objective in use which, in the
examples, should be POL for polarizing and PH for phase
contrast. Others could be HI for homogeneous (oil) im-
mersion, EPI for episcopic, PLAN for flat field corrected,
APO for apochromatic, as well as combinations of these
and others.
The next set of numbers in the examples, 0.25 and 0.65,
respectively, is the numerical aperture (NA), a measure of
the light-gathering capability of the objective and, there-
fore, its resolution. Since the purpose of a microscope is
not solely to magnify an object but rather to resolve fine
details of the object, a higher NA objective is preferable
to a low-NA objective. The NA was stated by Ernst Abbe
to be related to Snell’s law as follows:
NA = n sin 1
2AA,
where n is the lowest refractive index of any element in
the lens/specimen system and AA is the angular aperture
(the angle between the two most divergent beams entering
the front element of the objective).
Diffraction theory states that if an object made up of
fine details is illuminated by a beam of light, diffraction
maxima will be formed on either side of the perpendicular
incident ray. The finer the detail, the larger the diffraction
angle; consequently, a wider AA (i.e., higher NA) would
be necessary to capture the diffracted rays. In addition to
the NA, microscope resolution depends on a number of
other factors, such as chromatic and spherical aberration,
coma, and astigmatism, any one of which can adversely
affect the image quality. For best results each objective
must be used with the proper thickness and refractive in-
dex of all materials between the object and the objective
front lens. Very important is the thickness of the coverslip,
especially for high-power dry objectives. The 0.17 on the
40× objective, for example, signifies the thickness of the
coverslip for which the objective has been corrected for
spherical and chromatic aberrations. Objectives are avail-
able with few or all of these problems corrected to varying
degrees and at appropriate costs.
The eyepieces, or oculars, are the final stage of mag-
nification. Oculars magnify the image formed by the ob-
jective and in some cases supplement the corrections of
the objective. Oculars range in power from 5× up to 30×;
however, increasing the magnification of the objective and
the ocular beyond a certain point does nothing to increase
resolution, rather it delivers “empty magnification” due
to the limit of resolution governed by the wavelength of
light used (usually white light). The generally accepted
rule for maximum useful magnification (MUM) is 1000×
the NA of the objective used. Thus, a 95 × 1.3NA ob-
jective would not be able to show any more detail with
the 25× ocular (total magnification = 2375×) than with a
15× ocular (1425×).
Numerous variations of the compound microscope are
available, the most basic being a standard biological mi-
croscope and the most complex being various types of
interference microscopes. Microscopists often will have
a universal or modular type of microscope which can be
altered by adding intermediate attachments or substage
components depending on the type of analysis to be per-
formed. The polarized light microscope is best for chem-
ical microscopy. Under some conditions, most frequently
in a crime lab where two similar samples must be com-
pared for possible common origin, two identical micro-
scopes can be optically connected to a single viewing
head. This is known as a comparison microscope; it is
used for fiber, hair, tool mark, bullet, and cartridge-case
comparisons.