Simple Microscopes

A magnifying glass, an ordinary double convex lenslens,

device for forming an image of an object by the refraction of light. In its simplest form it is a
disk of transparent substance, commonly glass, with its two surfaces curved or with one surface
plane and the other curved.
..... Click the link for more information. having a short focal length, is a simple microscope. The
reading lens and hand lens are instruments of this type. When an object is placed nearer such a
lens than its principal focus, i.e., within its focal length, an imageimage,
in optics, likeness or counterpart of an object produced when rays of light coming from that
object are reflected from a mirror or are refracted by a lens. An image of an object is also formed
when this light passes through a very small opening like that of a pinhole
..... Click the link for more information. is produced that is erect and larger than the original
object. The image is also virtual; i.e., it cannot be projected on a screen as can a real image.

Compound Microscopes

The compound microscope consists essentially of two or more double convex lenses fixed in the
two extremities of a hollow cylinder. The lower lens (nearest to the object) is called the
objective; the upper lens (nearest to the eye of the observer), the eyepiece. The cylinder is
mounted upright on a screw device, which permits it to be raised or lowered until the object is in
focus, i.e., until a clear image is formed. When an object is in focus, a real, inverted image is
formed by the lower lens at a point inside the principal focus of the upper lens. This image serves
as an "object" for the upper lens which produces another image larger still (but virtual) and
visible to the eye of the observer.

Computation of Magnifying Power

The magnifying power of a lens is commonly expressed in diameters. For example, if a lens
magnifies an object 5 times, the magnification is said to be 5 diameters, commonly written
simply "5x." The total magnification of a compound microscope is computed by multiplying the
magnifying power of the objective by the magnifying power of the eyepiece.

Development and Uses

The invention of the microscope is variously accredited to Zacharias Janssen, a Dutch

spectaclemaker, c.1590, and to Galileo, who announced his invention in 1610. Others are known
for their discoveries made by the use of the instrument and for their new designs and
improvements, among them G. B. Amici, Nehemiah Grew, Robert Hooke, Antony van
Leeuwenhoek, Marcello Malpighi, and Jan Swammerdam. The compound microscope is widely
used in bacteriology, biology, and medicine in the examination of such extremely minute objects
as bacteria, other unicellular organisms, and plant and animal cells and tissuefine optical
microscopes are capable of resolving objects as small as 5000 Angstroms. It has been extremely
important in the development of the biological sciences and of medicine.

Modified Compound Microscopes

The ultramicroscope is an apparatus consisting essentially of a compound microscope with an
arrangement by which the material to be viewed is illuminated by a point of light placed at right
angles to the plane of the objective and brought to a focus directly beneath it. This instrument is
used especially in the study of Brownian movement in colloidal solutions (see colloidcolloid
[Gr.,=gluelike], a mixture in which one substance is divided into minute particles (called
colloidal particles) and dispersed throughout a second substance. The mixture is also called a
colloidal system, colloidal solution, or colloidal dispersion.
..... Click the link for more information. ). The phase-contrast microscope, a modification of the
compound microscope, makes transparent objects visible; it is used to study living cells. The
television microscope uses ultraviolet light. Since this light is not visible, the apparatus is used
with a special camera and may be connected with a television receiver on which the objects (e.g.,
living microorganisms) may be observed in color.

Electron Microscopes

The electron microscope, which is not limited by the powers of optical lenses and light, permits
greater magnification and greater depth of focus than the optical microscope and reveals more
details of structure. Instead of light rays it employs a stream of electrons controlled by electric or
magnetic fields. The image may be thrown on a fluorescent screen or may be photographed. It
was first developed in Germany c.1932; James Hillier and Albert Prebus, of Canada, and V. K.
Zworykin, of the United States also made notable contributions to its development. The scanning
electron microscope, introduced in 1966, gains even greater resolution by reading the response of
the subject material rather than the direct reflection of its beam. Using a similar approach, optical
scanning microscopes achieve a resolution of 400 Angstroms, less than the wavelength of the
light being used. Finally, the scanning tunnelling microscope, invented in 1982, uses not a beam
but an electron wave field, which by interacting with a nearby specimen is capable of imaging
individual atoms; its resolution is an astounding one Angstrom.

Bibliography

See C. Marmasse, Microscopes and Their Uses (1980).

The Columbia Electronic Encyclopedia Copyright 2013, Columbia University Press. Licensed from
Columbia University Press. All rights reserved. www.cc.columbia.edu/cu/cup/

Microscope

An instrument used to obtain an enlarged image of a small object. The image may be seen,
photographed, or sensed by photocells or other receivers, depending upon the nature of the image
and the use to be made of the information of the image.

A simple microscope, hand lens, or magnifier usually is a round piece of transparent material,
ground thinner at the edge than at the center, which can form an enlarged image of a small
object. Commonly, simple microscopes are double convex or planoconvex lenses, or systems of
lenses acting together to form the image.
The compound microscope utilizes two lenses or lens systems. One lens system forms an
enlarged image of the object and the second magnifies the image formed by the first. The total
magnification is then the product of the magnifications of both lens systems (see illustration).

Compound microscope diagram

The typical compound microscope consists of a stand, a stage to hold the specimen, a movable
body-tube containing the two lens systems, and mechanical controls for easy movement of the
body and the specimen. The lens system nearest the specimen is called the objective; the one
nearest the eye is called the eyepiece or ocular. A mirror is placed under the stage to reflect light
into the instrument when the illumination is not built into the stand. For objectives of higher
numerical aperture than 0.4, a condenser is provided under the stage to increase the illumination
of the specimen. Various optical and mechanical attachments may be added to facilitate the
analysis of the information in the doubly enlarged image. See Lens (optics)

McGraw-Hill Concise Encyclopedia of Physics. 2002 by The McGraw-Hill Companies, Inc.

The following article is from The Great Soviet Encyclopedia (1979). It might be outdated or ideologically
biased.

Microscope

an optical instrument used to produce greatly magnified images of objects (or parts of objects)
that are invisible to the naked eye.

The human eye is a natural optical system characterized by a certain resolutionthat is, the
minimal distance between which elements of an observed object (perceived as points or lines)
can still be distinguished from each other. For the normal eye, the minimal resolution at the
minimal distance of distinct vision (D 250 mm) is approximately 0.08 mm (in many cases,
about 0.20 mm). Microorganisms, most plant and animal cells, small crystals, and the
microstructural elements of metals and alloys are much smaller. Various types of microscopes
are designed for the observation and study of such objects. The shape, size, structure, and many
other characteristics of microscopic objects are determined by microscope. The microscope
makes it possible to distinguish structures with only 0.20 micron () between elements.

Historical survey. In the Netherlands and northern Italy as early as the 16th century, craftsmen
making eyeglasses knew that a system of two lenses could be used to produce magnified images
of objects. Some sources claim that a microscope-like instrument was constructed by Z. Janssen
in the Netherlands in about 1590.

The rapid spread of microscopes and their improvement (chiefly by optical craftsmen) began in
160910, when Galileo, in the course of studying the optic tube he had designed, used it as a
microscope by changing the distance between the objective and the eyepiece.

The first brilliant advances in the application of the microscope to scientific research are
associated with the names of R. Hooke, who established the cellular structure of animal and plant
tissues (c. 1665), and especially A. van Leeuwenhoek, who discovered microorganisms (1673
77). Microscopes first appeared in Russia in the early 18th century, when L. Euler (1762;
Dioptrica, 177071) devised methods of computing their optical assemblies. G. B. Amici first
used an immersion objective in a microscope in 1827. In 1850, the English optician H. Sorby
built the first microscope for observing specimens in polarized light.

The scientific work of E. Abbe, who in 187273 developed the now classic theory of image
formation in the microscope with non-self-luminous objects, contributed greatly to the extensive
development of methods of microscopy and to the improvement of various types of microscopes.
The Austrian researchers R. Zsigmondy and H. Siedentopf developed the ultramicroscope in
1903. In 1935, F. Zernicke proposed the phase-contrast method for observing through the
microscope transparent objects that scatter light poorly. Soviet scientists, including L. I.
Mandelshtam, D. S. Rozhdestvenskii, A. A. Lebedev, and V. P. Linnik, have made a major
contribution to the theory and practice of microscopy.

Optical system operating principles, magnification, and resolving power. A diagram of a

typical microscope is presented in Figure 1. The specimen to be examined (7) is placed on a slide
(10). A condenser (6) concentrates a beam of light reflected from a mirror (4) onto the specimen.
A special illuminator, consisting of a lamp (1) and a collecting lens (2), generally serves as the
light source. (A mirror sometimes directs ordinary daylight onto the specimen.) A field
diaphragm (3) and an iris diaphragm (5) restrict the light beam and decrease the amount of
extraneous scattered light striking the specimen.
Figure 1. Diagram of typical microscope

The appearance of the image of a specimen in a microscope can be described in basic (although
extremely simplified) terms within the framework of geometric optics. Light beams originating
at the specimen (7) are refracted in the objective (8) and create an inverted and magnified real
optical image (7) of the specimen. The image is viewed through an eyepiece (9). The
microscope is focused so that the real optical image is located directly beyond the front focus of
the eyepiece (Foe). Under these conditions, the eyepiece functions as a magnifying glass: in
producing additional magnification, it forms a virtual image (7; inverted, as before). Upon
passing through the optical medium of the observers eye, the beams from the virtual image
create a real image of the specimen on the retina. The virtual image is usually located at the
minimal distance of distinct vision D from the eye. If the eyepiece is moved in such a way that
the real image is in front of Foc, then the image produced by the eyepiece becomes real and can
be produced on a screen or photographic film. Microscopic objects are photographed and filmed
in this manner.

The total magnification of a microscope is equal to the product of the linear magnification of the
objective and the angular magnification of the eyepiece: = . OC. The objective magnification
= /fob, where is the distance between the rear focus of the objective Fob and the front focus of
the eyepiece (the optical length of the body tube of the microscope) and fob is the focal length of
the objective. The eyepiece magnification, like that of a magnifying glass, is expressed by the
formula oc = 250/foc (foc given in millimeters). Objectives usually have magnifications from 6.3
to 100; eyepieces, from 7 to 15 (the values engraved on the mounting). Therefore, the total
magnification of the microscope ranges from 44 to 1,500.
Technically, of course, it is possible to use objectives and eyepieces that produce a total
magnification much greater than 1,500. Usually, however, this is inexpedient. High
magnification is not in itself a goal. The function of a microscope is to make it possible to
distinguish the smallest possible structural elements of a specimenthat is, to make maximum
use of the microscopes resolving power. However, resolving power is limited by the wave
properties of light. (Although these properties are ignored in geometric optics, within whose
framework image production in a microscope has so far been discussed, they are precisely what
determines a microscopes limitations.)

According to general principle, it is impossible when observing a specimen under illumination

with a given wavelength X to distinguish elements of the specimen separated by distances much
smaller than X. This principle is also manifested with the microscope, its quantitative expression
differing slightly for self-luminous and non-self-luminous objects. Even when produced by an
ideal (distortionless) objective, the image of a point emitting monochromatic light is not
perceived by the eye as a point: because of light diffraction, it is actually a round spot of light of
finite diameter d surrounded by several alternately dark and light rings. This image is called a
diffraction spot, Airy spot, or Airy disk; d = 1.22/A, where is the wavelength. (When the
specimen is illuminated in nonmonochromatic light, is usually the shortest wavelength of the
light, or the wavelength at which the intensity of the irradiation is maximal.) In the same formula
for the finite diameter d, A is the numerical aperture of the objective (A = n . sin um, where n is
the refractive index of the medium separating the luminous point and the objective and um is half
the aperture angle of the light beam emanating from the point and striking the objective).

When two luminous points are located close to each other, their diffraction patterns are
superimposed, producing in the image plane a complex illumination distribution (see Figure 2).
The least relative difference in illumination visible to the eye is 4 percent. This corresponds to
the shortest distance between points at which the images of the points can be discerned (the
maximum resolution of the microscope max = 0.42d = 0.51 /A). As was shown in Abbes
classic microscope theory, for non-self-luminous objects the maximum resolution max = /(A +
A) where A and A are the numerical apertures of the objective and the condenser (the aperture
values being indicated on the mounting).

Figure 2. Distribution of illumi-nation intensity in image of two proximate points in limiting case of
visual resolution
The image of any specimen consists of the sum of the images of its structural elements. The
smallest of these are perceived as points, the limitations resulting from light diffraction in micro-
scopes being completely applicable here. When distances between points are less than the
maximum resolution of the microscope, the points merge and cannot be seen separately. The
resolving power of a microscope can be increased significantly only by increasing A. In turn, A
can be increased only by increasing the refractive index n of the medium between the specimen
and the objective (since sin um 1). This has been accomplished in immersion systems, whose
numerical apertures reach A = 1.3 (in ordinary dry objectives, the maximum A 0.9).

The existence of a resolving power limit has an effect on the selection of magnifications. The
range from 500 A to 1,000 A is considered effective magnification, since at these powers the
observers eye can distinguish all of the structural elements of a specimen that are resolvable by
the microscope. Beyond this point, the capabilities of the microscope with respect to resolving
power are exhausted. No new structural details are revealed at magnifications above 1,000 A,
although such magnifications are sometimes used in microphotography, image projection onto
screens, and certain other cases. The electron microscope has a significantly greater resolving
power and, consequently, a significantly higher effective magnification than has the ordinary
microscope.

Methods of illumination and observation (microscopy). The structure of a specimen can be

discerned only when its particles absorb or reflect light differently or differ in refractive index
from each other or from the surrounding medium. These properties account for the amplitude
and phase differences of light waves that have passed through different parts of a specimen (the
determinant, then, of image contrast). Therefore, methods of observation with a microscope are
designed and selected according to the character and properties of the specimens to be studied.

LIGHT FIELD IN TRANSMITTED LIGHT. The method of a light field in transmitted light is used in
studies of transparent specimens with incorporated light-absorbing particles and components.
Examples of such specimens include thin stained slices of animal and plant tissues and thin
sections of minerals. In the absence of a specimen, the light beam from the condenser (6, in
Figure 1) passes through the objective (8) to produce a uniformly illuminated field near the focal
plane of the eyepiece (9). If an absorptive element is present in the specimen (7), it partially
absorbs and partially scatters the incident light (broken line) and results in an image. This
method can also be useful in observing nonabsorptive specimens, but only if they scatter the
illuminating beam so strongly that a large part of it fails to strike the objective.

OBLIQUE ILLUMINATION. The method of oblique illumination is a variation of the light field in
the transmitted light method, differing in that the light is directed onto the specimen at a large
angle to the direction of observation. In many cases, this makes it possible to reveal the relief of
the specimen, a result of shadow formation.

LIGHT FIELD IN REFLECTED LIGHT. A light field in reflected light is used to observe
nontransparent light-reflecting specimens, such as thin sections of metals or ores (see Figure 3).
Illumination of the specimen (4) from an illuminator (1) and a semitransparent mirror (2) is from
above, through the objective (3), which simultaneously functions as a condenser. The structure of
the specimen can be seen in the image created in the image plane (6) by the objective and the
tube lens (5) because of the difference in the reflecting power of its elements. Irregulari-ties that
scatter the incident light are also revealed in a light field.

DARK FIELD IN TRANSMITTED LIGHT. A dark field in transmitted light is used to produce images
of transparent nonabsorptive specimens that are invisible when illuminated by the light-field
method (see Figure 4). Biological specimens are frequently of this type. Light from an
illuminator (1) is directed by a mirror (2) onto the specimen by a specially designed dark-field
condenser (3). Emerging from the condenser, most of the light rays, which have not changed
direction in passing through the transparent specimen, form a beam in the shape of a hollow cone
and fail to strike the objective (5; located within the cone). The image in the microscope is
created by only a small portion of the

Figure 3. Light field in reflected light

rays: those that have been scattered by the microparticles of the specimen on the slide (4) into the
cone and have passed through the objective. Light images of the structural elements of the
specimen that differ from the surroundings in refractive index can be seen in the field of view (6)
against a dark background. For large particles, only the light edges, which scatter the light rays,
can be seen. The dark field in transmitted light cannot be used to determine whether particles are
transparent or opaque or whether their refractive index is larger or smaller than that of the
surroundings.
Figure 4. Dark field in transmitted light

ULTRAMICROSCOPY. The principle of the dark field in transmitted light is also the basis for the
method of ultramicroscopy. Specimens in ultramicroscopes are illuminated perpendicularly to
the direction of observation. The method makes it possible to detect (but not to see, in the literal
sense) extremely small particles whose dimensions are far less than the maximum resolving
power of the strongest microscopes. The presence of particles as small as 2 X 10-9 m can be
detected by means of immersion ultramicroscopes. It is impossible, however, to determine the
shape and exact size of such particles by this method. The observer sees their images in the form
of diffraction spots whose dimensions depend not on the size and shape of the particles
themselves but on the aperture of the objective and the magnification of the microscope. Since
such particles scatter very little light, extremely strong light sources (for example, carbon arcs)
are required to illuminate them. Ultramicroscopes are used chiefly in colloid chemistry.

DARK FIELD IN REFLECTED LIGHT. A dark field in reflected light is used for opaque specimens,
such as sections of metals. The specimen is illuminated from above, through a special annular
system (an epicondenser) located around the objective.

POLARIZED LIGHT METHOD. Polarizing microscopy is used for the study of specimens containing
or consisting entirely of optically anisotropic elements (many minerals, grains in sections of
alloys, and certain animal and plant tissues). The optical properties of anisotropic objects are
different in different directions and manifested in a variety of ways, depending on the orientation
of the specimen with respect to the direction of observation and the plane of polarization of the
incident light. The specimens can be observed in either transmitted or reflected light. The light
projected by the illuminator passes through a polarizer. The polarization imparted to the light
changes as the light passes through or is reflected from the specimen; these changes are studied
with an analyzer and various optical compensators. From such changes, the basic optical
characteristics of anisotropic microobjects can be assessed (the degree of birefringence, the
number and orientation of optical axes, the rotation of the plane of polarization, and dichroism).

PHASE-CONTRAST METHOD. The phase-contrast method and a variation of it called anoptral

contrast are used to obtain images of transparent and colorless specimens, such as unstained live
animal tissues, which are invisible when observed by the light-field method. The basis for the
method is that even when the difference in the refractive indexes of different elements of the
specimen is very small, a light wave transmitted through these elements undergoes different
phase changes (that is, acquires phase relief). These phase changes, which can be directly
perceived neither by the eye nor on a photographic plate, are transformed by a special optical
device into changes in the amplitude of the light wavethat is, into changes in brightness (the
amplitude relief), which are discernible to the eye and can be recorded on a photosensitive film.
In other words, the brightness (amplitude) distribution in the resultant visible image duplicates
the phase relief. Such an image is called a phase-contrast image.

In a typical diagram of this method (Figure 5), an iris diaphragm (2) with an annular opening is
installed at the front focus of the condenser (3). The image of the iris diaphragm appears near the
rear focus of the objective (5), where a phase plate (6) whose surface has an annular ridge or
groove (a phase ring) is installed. The phase plate may or may not be placed in the focal plane of
the objective (the phase ring often being made on the surface of one of the objective lenses). In
either case, rays from the illuminator (1) that are undeflected in the specimen (4) and produce the
image of the iris diaphragm (2) must pass completely through the phase ring, which (being
absorptive) greatly attenuates them and changes their phase by A/4 (where X is the wavelength).
At the same time, rays (even those that are only slightly deflected or scattered in the specimen)
pass through the phase plate and bypass the phase ring (broken lines) without undergoing an
additional phase shift. Taking into account the phase shift in the specimen, the total phase
difference between deflected and undeflected rays proves to be near 0 or X/2; as a result of light
interference in the image plane (4) of the specimen (4), the rays noticeably amplify or attenuate
each other and
Figure 5. Diagram of phase-contrast microscope

produce a contrast image of the structure of the specimen. The deflected rays have a considerably
lower amplitude than do the undeflected rays; therefore, by making the amplitude values closer,
the attenuation of the primary beam in the phase ring also leads to greater image contrast.

The phase-contrast method makes it possible to distinguish small structural elements that show
exceedingly little contrast in the light-field method. Comparatively large transparent particles
scatter light rays at such small angles that deflected rays pass through the phase ring together
with undeflected rays. For such particles, there is a phase-contrast effect only near the contours,
where significant scattering takes place.

INTERFERENCE-CONTRAST METHOD. In interference microscopy, every beam entering the

microscope is bisected. One of the resultant beams is directed through the observed particle; the
second bypasses the particle along the same or a supplementary optical path of the microscope.
The beams are recombined in the eyepiece area and interfere with each other. The result of this
interference is determined by the path difference of the beams 8, which is expressed by the
formula = N = (n0nm)d, where n0 and nm are the refractive indexes of the particle and the
surrounding medium, respectively; d is the particle thickness; TV is the order of interference;
and is the wavelength.

A diagram of one of the methods of interference contrast is presented in Figure 6. The condehser
(1) and the objective (4) are fitted with double-refracting plates (marked with diagonal arrows in
the figure), the first splitting the original light beam into two; the second, recombining the
beams. One of the beams, upon passing through the specimen (3), is phase delayedthat is, it
acquires a path difference relative to the second beam. The extent of the delay is measured by the
compensator (5).

Figure 6. Interference-contrast method

The interference-contrast method is similar in some respects to the phase-contrast method: both
are based on the interference of beams that have passed through and bypassed the microparticle.
Like the phase-contrast method, interference-contrast microscopy makes it possible to observe
transparent and colorless objects, although the images may also be multicolored (interference
colors). Both methods are suitable for the study of living tissues and cells and are frequently used
for this purpose. The main distinguishing feature of the interference method is that, using
compensators, it is possible to measure with high accuracy (to 1/300X) the path differences
introduced by the microobjects. This presents broad opportunities for quantitative studiesfor
example, for calculation of the total mass and concentration of dry matter in a microscopic
specimen, such as a plant or animal cell; of the refractive index; and of the size of the specimen
(see Figure 7).

The interference-contrast method is frequently combined with other methodsin particular, with
polarizing microscopy. Its use in conjunction with ultraviolet microscopy makes it possible to
determine the nucleic acid content of the total dry mass of a specimen. Methods involving
microinterferometers are also usually classified under interference microscopy.

Figure 7. Micrograph of human erythrocyte in monochromatic light ( 0.546). Bend in interference

band reproduces the thickness of the erythrocyte to scale

LUMINESCENCE METHOD. Luminescence, or fluorescence, microscopy consists in observing

microscopic specimens under the green-orange luminescence produced when the specimens are
illuminated with blue-violet or invisible ultraviolet light. There are two light filters in the optical
diagram of such microscopes. The first is placed in front of the condenser and allows only those
wavelengths to pass that stimulate the luminescence of either the specimen itself (natural
luminescence) or of special dyes introduced into the specimen and absorbed by its particles
(secondary luminescence). The second light filter, which is mounted behind the objective,
transmits only the luminescent light to the observers eye or the photosensitive film. Specimens
can be illuminated either from above, through the objective (which, in this case, also acts as a
condenser), or from below, through an ordinary condenser. Illumination from above is
sometimes called reflected light luminescence microscopy (an arbitrary term, since the excitation
of the fluorescence of the specimen is not a simple reflection of the light). The method is
frequently combined with phase-contrast observation in transmitted light.

Luminescence microscopy is used extensively in microbiology, virology, histology, cytology, the

food industry, soil studies, microchemical analysis, and flaw detection. The abundance and
diversity of its uses are associated with the extraordinarily high color sensitivity of the eye, the
high image contrast of self-luminous specimens against dark, nonluminescent backgrounds, and
the great value of the data that can be obtained concerning the composition and properties of
substances, when the intensity and spectral composition of their luminescent radiation are
known.

ULTRAVIOLET METHOD. Ultraviolet microscopy makes it possible to increase the maximum

resolving power of a microscopethat is, to lower its minimal discernible value, which, as
already mentioned, depends on the wavelength of the radiation used (for the ultraviolet rays
used with a microscope, = 400250 nanometers [nm]; for visible light, = 700400 nm). This
method expands the possibilities of microscopic research, since the particles of many substances
that are transparent in visible light nevertheless strongly absorb ultraviolet radiation of certain
wavelengths and are consequently easily distinguishable in ultraviolet images. A number of
substances present in plant and animal cells (for example, purine and pyrimidine bases, most
vitamins, aromatic amino acids, certain lipids, and thyroxine) have characteristic absorption
spectra in the ultraviolet region. This accounts for the extensive use of ultraviolet microscopy in
cytochemical analysis.

Ultraviolet rays are invisible to the human eye. Therefore, the images in ultraviolet microscopy
are recorded either photographically or with an image converter or luminescent screen.

The following method for the color presentation of such images is widely used. The preparation
is photographed at three wavelengths in the ultraviolet region of the spectrum. Each of the
resultant negatives is illuminated with visible light of a particular color (blue, green, or red), and
all three are projected simultaneously onto a single screen. A color image of the specimen in
standard colors (depending on the absorptivity of the specimen in ultraviolet light) is created on
the screen.

INFRARED METHOD. Like the ultraviolet method, infrared microscopy calls for the conversion of
an invisible image into a visible one by photography or with an image converter. Infrared
microscopy makes it possible to study the internal structure of specimens that are opaque in
visible light, such as dark glass and certain crystals and minerals.
MICROPHOTOGRAPHY AND CINEMICROGRAPHY. The production with a microscope of images on
light-sensitive films is used widely in conjunction with all other methods of microscopic
research. In microphotography and cinemicrography, the optical system of the microscope
requires some adjustmentan eyepiece focus, with respect to the objective image, that differs
from the focus used for visual observation. Many modern microscopes have permanent
(mounted) devices for microphotography that make it possible to make such adjustments and to
project the images onto a photographic plate or film. Most microscopes can also be fitted with
accessories for this purpose. Microphotography is indispensable for substantiating research,
studying specimens in invisible ultraviolet and infrared rays, and studying specimens with weak
luminescence intensity. Cinemicrography is important in studies of processes that occur over a
period of time, such as the vital activities of tissue cells and microorganisms, the growth of
crystals, and the course of simple chemical reactions.

Principal assemblies. In most microscopes (with the exception of the inverted type, to be
discussed), a device for securing the objective is placed above, and a condenser below, the stage.
All microscopes have a body tube in which the eyepieces are mounted. Coarse and fine focus
mechanisms (changing the relative positions of specimen, objective, and eyepiece) are also
essential parts of any microscope. All of these assemblies are attached to the microscope stand or
frame.

The type of condenser used depends on the method of observation selected. Light-field
condensers and phase-contrast and interference-contrast condensers are two- or three-lens
systems that are very different from each other. Light-field condensers, whose numerical
aperture may reach 1.4, contain an iris diaphragm that can be moved aside to allow oblique
illumination of the specimen. Phase-contrast condensers are equipped with annular diaphragms.
Dark-field condensers are complex systems of lenses and mirrors. Epicondenserssystems of
annular lenses and mirrors mounted around the objective for dark-field observation in reflected
lightconstitute a special group. Special mirror-lens (catadioptric) and lens condensers that are
transparent for ultraviolet rays are used in ultraviolet microscopy.

In most modern microscopes, the objectives are detachable and are selected according to the
particular observation conditions. Often, there are several objectives in a single rotating (turret)
head; the objective can be changed simply by turning this head.

A distinction is made between achromatic and apochromatic objectives, according to the degree
of correction for chromatic aberration. Achromatic objectives are simpler in design. They correct
for chromatic aberration in only two wavelengths, so that when the specimen is illuminated in
white light the image remains slightly colored. Apochromatic lenses correct for three
wavelengths and produce achromatic images. The image plane with achromatic and
apochromatic lenses is somewhat curved. With visual observation, the accommodation of the eye
and the possibility of seeing the entire field by readjusting the microscope compensate somewhat
for this shortcoming. The phenomenon has a strong effect, however, in microphotography, in
which the edges of the image are insufficiently sharp. Plane-achromats and plane-apochromats,
objectives that make additional corrections for field curvature, are therefore widely used. Special
projection systems that are inserted instead of the eyepiece to correct for curvature of the image
surface are used in combination with ordinary objectives. These systems are unsuitable for visual
observation.

Objectives are also classified according to (1) their spectral characteristics (those used for the
visible region of the spectrum and those for ultraviolet and infrared microscopy [lens or mirror-
lens objectives]); (2) the length of the body tube for which they are designed (those for a 160-
mm tube; those for a 190-mm tube; and those for an infinite length tube, which create an
image at infinity and are used together with an additional body-tube lens, which transfers the
image to the focal plane of the eyepiece); (3) the medium between the objective and the
specimen (dry and immersion types); (4) the method of observation (for example, normal, phase-
contrast, or interference); and (5) the type of specimen (those for use with a cover slip or
without). A special type is the epiobjective, a combination of a normal objective and an
epicondenser.

The diversity of objectives has to do with the diversity of methods of microscopic observation,
the variety of microscope design, and differences in the need for aberration correction under
various operating conditions. Each objective is used only under those conditions for which it is
designed. For example, an objective designed for a 160-mm body tube cannot be used in a
microscope with a 190-mm body tube, and specimens without a cover slip cannot be observed
with an objective designed to be used with a cover slip. It is especially important to adhere to the
design conditions when working with dry objectives of large apertures (A > 0.6), which are very
sensitive to any deviations from normal. Where cover slips are used, the cover-slip thickness
should be 0.17 mm. An immersion objective may be used only with the immersion for which it is
designed.

The type of eyepiece used with a given method of observation is governed by the choice of
objective. A Huygens eyepiece is used with achromatic lenses of low and moderate
magnification; a compensation eyepiece, which is designed so that its residual chromatic
aberration is of a sign opposite that of the objective (to improve image quality), is used with
apochromatic and achromatic lenses of high magnification. There are also special eyepieces used
for photography and projection, which project the image onto a screen or photographic plate.
Quartz eyepieces that are transparent for ultraviolet rays constitute a separate group.

The diverse accessories available for microscopes make it possible to improve the observation
conditions and expand the possibilities for research. Illuminators of various types are designed to
create optimal lighting conditions, ocular micrometers are used to measure the size of the
specimens, and binocular tubes make it possible to observe a specimen with both eyes
simultaneously. Photographic attachments and apparatus are used in microphotography, and
drawing units make it possible to sketch the images. Special devices, such as
microspectrophotometric attachments, are used for quantitative studies.

Types. The design of a microscope and its attachments and the characteristics of its main
assemblies are determined either by the field for which the microscope is used, the range of
problems, and the kind of specimens studied or by the method or methods of observation, or by
both. Various types of specialized microscopes have therefore been developed to make it
possible to study, with high precision, strictly defined classes of objects (or even certain of their
properties). On the other hand, there are universal microscopes with which a variety of objects
can be observed by a variety of methods.

BIOLOGICAL MICROSCOPES. Among the most widely used microscopes are those developed for
botanical, histological, cytological, microbiological, and medical research and for observing
transparent specimens in chemistry, physics, and other nonbio-logical fields. There are many
models of biological microscopes, of differing design and accessories, which greatly expand the
range of specimens that can be studied. The accessories include detachable illuminators
providing transmitted and reflected light, detachable condensers for working with light and dark
fields, phase-contrast devices, ocular micrometers, photographic attachments, and sets of light
filters and polarizing devices that make it possible to use luminescence and polarization
techniques with ordinary (nonspecialized) microscopes. The devices of microscopic technology
used for readying specimens and conducting various operations on them (including work during
observation) are especially important among the auxiliary equipment for biological microscopes.

Biological research microscopes are equipped with a set of interchangeable objectives, including
epiobjectives for reflected light and, frequently, phase-contrast objectives, for various conditions
and methods of observation and various types of specimens. There is a separate set of eyepieces
(for visual observation and microphotography) suitable for each set of objectives. Biological
research microscopes usually have binocular body tubes for observation with both eyes.

In addition to general-purpose microscopes, extensive use is also made in biology of various

microscopes that are specialized according to the method of observation.

INVERTED MICROSCOPES. Inverted microscopes are distinctive in that the objective is located
below the specimen, and the condenser, above. The path direction of the rays, transmitted from
above downward through the objective, is altered by a system of mirrors such that they usually
reach the eye from below (see Figure 8). Microscopes of this type are designed for the study of
cumbersome specimens that are difficult or impossible to place on the stage of an ordinary
microscope. Examples include tissue cultures in nutrient medium that are kept in a thermostatic
chamber to maintain a preset temperature. Inverted microscopes are also used to study chemical
reactions and to determine melting points. They are used when cumbersome auxiliary equipment
is required for the observation of various processes. Inverted microscopes for microphotography
and cinemicrography are fitted with special devices and cameras.
Figure 8. Diagram of the optical system of an inverted microscope

The inverted microscope is especially convenient for the observation of structures of various
surfaces in reflected light. It is therefore used in most metallographic microscopes: the specimen
(a section of a metal, alloy, or mineral) is mounted on the stage, polished surface downward; the
nature of the rest of the sample is arbitrary and does not require treatment. Metallographic
microscopes are also made in which the specimen is inserted from below, secured to a special
plate. The assemblies in such microscopes are in the same relative positions as in ordinary
(uninverted) microscopes. The surface under study is often etched beforehand, so that the grains
of its structure become sharply distinguishable. Metallographic microscopes can be used with the
light-field method, direct and oblique lighting, the dark-field method, and polarization. With
work in a light field, the objective also acts as a condenser. Parabolic mirror epicondensers are
used for dark-field illumination. A special attachment makes it possible to achieve phase contrast
in a metallographic microscope with an ordinary objective.

LUMINESCENCE MICROSCOPES. Luminescence microscopes are equipped with a set of

detachable light filters. By choosing the proper filter, it is possible to single out the part of the
spectrum in the illuminators radiation that excites the luminescence of the specimen under
study. A light filter can also be selected that only lets through luminescent light from the
specimen. The luminescence of many objects is excited by ultraviolet radiation or the shortwave
part of the visible spectum. Superhigh-pressure mercury vapor lamps produce precisely this type
of radiation (and very brightly) and are therefore used as light sources. In addition to special
luminescence microscopes, there are luminescence devices that are used in conjunction with
ordinary microscopes. These contain an illuminator with a mercury vapor lamp, a set of light
filters, and an opaque illuminator (for lighting specimens from above).

ULTRAVIOLET AND INFRARED MICROSCOPES. Ultraviolet and infrared microscopes are used for
research in the invisible regions of the spectrum. Their optical diagrams are analogous to those of
ordinary microscopes. Since making aberration corrections in the ultraviolet and infrared regions
is very complicated, the condenser and objective of such microscopes are often catadioptric
systems in which chromatic aberration is significantly reduced or eliminated altogether. The
lenses are made of materials that are transparent to ultraviolet (quartz, fluorite) or infrared
(silicon, germanium, fluorite, lithium fluoride). The microscopes are equipped with cameras in
which the invisible images are fixed. Visual observation through the eyepiece in normal (visible)
light is used where possible only for prefocusing and preadjusting the specimen in the
microscopes field of view. As a rule, ultraviolet and infrared microscopes have image
converters to make invisible images visible.

POLARIZING MICROSCOPES. Polarizing microscopes are designed for the study (with optical
compensators) of changes in the polarization of the light transmitted through or reflected from a
specimen. This opens up opportunities for the quantitative and semiquantitative determination of
various characteristics of optically active specimens. The assemblies of such microscopes are
usually made so as to facilitate precise measurements: the eyepieces are equipped with cross
hairs and a micrometric scale or reticule; the rotating stage has an angularly graduated dial for
measuring the angle of rotation; and a Fedorov stage is often secured to the main stage, making it
possible to rotate and tilt the specimen at will in order to find its crystallographic and
crystallooptic axes. The objectives of polarizing microscopes are specially chosen for an absence
of internal stresses, which lead to depolarization. Such microscopes usually contain a detachable
auxiliary (Bertrand) lens to be used for observation in transmitted light. The Bertrand lens makes
it possible to view the interference figures formed in the objectives rear focal plane by the light,
after it passes through the crystal under study.

INTERFERENCE MICROSCOPES. Transparent specimens are observed by the interference-contrast

method with interference microscopes. Many interference microscopes are analogous in design
to ordinary microscopes, differing only in the presence of a special condenser, objective, and
measuring unit. If the specimens are observed in polarized light, the microscopes are equipped
with a polarizer and an analyzer. Since they are used chiefly for biological research, interference
microscopes may be classified as specialized biological microscopes. Microinterferometers
(microscopes of a special type that are used to study the microrelief of surfaces of finished metal
parts) are often classified as interference microscopes.

STEREOSCOPIC MICROSCOPES. Although they make possible the convenience of observation

with both eyes, the binocular tubes used in ordinary microscopes do not produce a stereoscopic
effect: the same rays enter both eyes at the same angles, only separated into two beams by a
prism system. Stereoscopic microscopes, which make for a truly three-dimensional perception of
the microscopic specimen, are actually two microscopes in a single unit, made in such a way that
the right and left eyes see the specimen at different angles (see Figure 9). Such microscopes are
used most widely where stereoscopic perception facilitates work that must be done on a
specimen during observation (biological research, surgical operations on the blood vessels or
brain, micrurgy in the eye, and the assembly of miniature devices, such as transistors). The
inclusion in the optical diagram of prisms, which act as erecting systems, also serves to facilitate
orientation in the field of view. In microscopes of this type the image is not inverted but is erect.
Since in stereoscopic microscopes the angle between the optical axes of the objectives is usually
12, the numerical aperture does not generally exceed 0.12. The effective magnification of
such microscopes is therefore no greater than 120.
Figure 9. Diagram of stereoscopic microscope, which provides three-dimensional perception of
observed objects

COMPARISON MICROSCOPES. Comparison microscopes consist of two structurally joined

ordinary microscopes with a common eyepiece system. The observer sees the images of two
specimens at once in the two halves of the field of view; this enables him to compare the images
directly with respect to color, structure, distribution of elements, and other characteristics.
Comparison microscopes are widely used in evaluating the quality of treated surfaces and in
grading work (comparing with a standard sample). Special microscopes of this type are used in
criminologyin particular, to identify the weapon from which a bullet was fired.

TELEVISION MICROSCOPES. Television microscopes operate with a microprojection circuit. The

image of the specimen is converted into a sequence of electric signals, which reproduce the
image on a magnified scale on the screen of a cathode ray tube (kinescope). Image contrast can
be altered and image brightness controlled by purely electronic meansby altering the
parameters of the electric circuit through which the signals pass. Electric amplification of the
signals makes it possible to project the image on a large screen, whereas ordinary
microprojection requires extremely strong illumination, often harmful to the microscopic
specimens. The great advantage of television microscopy is that it can be used for studying
specimens at a distance, where proximity to the object (radioactive specimens, for example)
would be dangerous to the observer.

In many studies, it is important to make a count of the microscopic particles in a specimen (for
example, bacteria in colonies, aerosols, particles in colloidal solutions, blood cells) or to
determine the areas occupied by the grains of a particular type in sections of an alloy. The
conversion of an image into a series of electric signals (impulses) in television microscopes
makes it possible to construct automatic microparticle counters that record particles by number
of impulses.
MEASURING MICROSCOPES. The function of measuring microscopes is to measure precisely the
linear and angular dimensions of the specimens (which are often not at all small). There are two
types of measuring microscopes, depending on the method of measurement. The first type is
used only when the distance to be measured does not exceed the linear dimensions of the
microscopes field of view. In such microscopes, direct measurement is made not of the object
itself but of its image at the focal plane of the eyepiece (by using a scale or a screw-type ocular
micrometer). The distance measured on the specimen is then calculated from the known
magnification of the objective. The images of specimens are often compared with sample
profiles marked on plates of the detachable heads of the eyepieces.

In measuring microscopes of the second type, the stage bearing the specimen and the frame of
the microscope can be shifted with respect to each other (most often the stage being shifted with
respect to the frame) by using precise adjustment mechanisms. Measuring this shift with a
micrometer screw or scale (securely attached to the stage) makes it possible to determine the
distance between elements of the specimen.

Measuring microscopes also exist in which measurement is made in only one direction (single-
coordinate microscopes). Much more widely used are microscopes in which the stage is moved
in two perpendicular directions (the limits being as great as 200 X 500 mm). For special cases,
there are microscopes in which measurements (and, consequently, relative stageframe shifts)
are possible in three directions, corresponding to the three axes of rectangular coordinates.
Measurements can be made in polar coordinates with some microscopes: the stage can be rotated
and is equipped with a scale and vernier for reading the angles of rotation. Glass scales are used
in the finest measuring microscopes of the second type; the readings are made with an auxiliary
(reading) microscope.

The accuracy of the measurements in microscopes of the second type is much greater than in
those of the first type. In the best models, the linear accuracy is usually on the order of 0.001
mm, and the angular accuracy, on the order of 1. Measuring microscopes of the second type are
used extensively in industry (especially in machine building) to measure and monitor the size of
machine parts and tools.

In devices designed for especially precise measurements (for example, geodesic and
astronomical), readings on the linear scales and graduated dials of goniometric instruments are
made with special reading microscopesscale microscopes and micrometric microscopes. Scale
microscopes have an auxiliary glass scale; by adjusting the magnification of the objective, the
image of this scale is equalized with the intervals observed between markings on the primary
scale (or dial). The position of the division between the marks on the auxiliary scale can then be
directly determined with an accuracy of about 0.01 interval between primary scale divisions. The
accuracy of the readings in micrometric microscopes, whose eyepieces contain a crosshair or
spiral micrometer, is still higher (on the order of 0.0001 mm). The magnification of the objective
is controlled so that an integral number of turns (or half-turns) of the micrometer screw
corresponds to the shift of the crosshair between the images of the markings on the scale
measured.
In addition to the microscopes described, there are a number of more narrowly specialized types
of microscopes. Examples include microscopes used for counting and analyzing the tracks of
elementary particles and the fragments of nuclear fission in nuclear photographic emulsions;
high-temperature microscopes, used to study specimens heated to temperatures on the order of
2000C; and contact microscopes, used to study the surfaces of living human and animal organs
(the objective being pressed against the surface to be studied and the microscope focused by a
special built-in system).

In conjunction with other instruments, microscopes are frequently an important component of

compound units. Examples include microspectrophotometric units, in which the microscopes are
coupled to special monochromators and to devices that measure luminous fluxes; a number of
the instruments used in ophthalmology; comparators; and microphotometers.