19a.

Optical microscope
The optical microscope (light microscope) is widely used in the diagnostic work of
clinical pathologists, biologists, microbiologists and other specialists in the field of medical
practice and research. The optical microscope commonly uses visible light and a system of
lenses to generate magnified images of small objects.
The optical microscopes designed for viewing samples by transmitted light share the
same basic components of the light path. In addition, the vast majority of microscopes have the
same structural components:

• Eyepiece (ocular lens/es);

• Objective turret, revolver,
or revolving nose piece (to
hold multiple objective
lenses);
• Objective lenses;
• Focus knobs (to move the
stage);
o Coarse adjustment;
o Fine adjustment;
• Stage (to hold the
specimen);
• Light source (a light or a
mirror);
• Diaphragm and condenser.

The main components of the microscope's

optical system are the objective and the eyepiece.
The objective and eyepiece are complex systems of
high magnification lenses centered and connected
in a tube.
The closest to the object, located on a table,
is the front lens of the objective – a condenser lens,
from which the image of the object is formed. The
remaining lenses in the objective are used to
correct the defects of the front lens. The objective
makes a major contribution to microscope
magnification.
The eyepiece, or ocular lens, is a cylinder
containing two or more lenses. It magnifies the
image obtained by the objective. Its function is to
bring the image into focus for the eye. The
eyepiece is inserted into the top end of the body
tube. Eyepieces are interchangeable and many
different eyepieces can be inserted with different
degrees of magnification. Typical magnification
Figure 1 values for eyepieces include 5×, 10× (the most
common), 15× and 20×.
 The scheme for obtaining the image in the microscope is presented in Fig.1 as the
objective and the eyepiece are presented for simplicity as two converging lenses.
The object is placed on a stage and may be directly viewed through one or two eyepieces
on the microscope.
The subject 𝑨𝑩 is placed in front of the objective at a distance slightly greater than the
focal length (shortly before the front focus of the objective 𝑭′′𝒐 ). The resulting image 𝑨′ 𝑩′ is
inverted, real and magnified. This image falls between the focus and the eyepiece lens. The
eyepiece creates an image 𝑨″ 𝑩″ of the actual image obtained from the objective, i.e. on 𝑨′ 𝑩′ .
𝑨″ 𝑩″ is the final image from the microscope. With respect to the object, it is inverted, virtual
and enlarged and is obtained at the distance of the clearest vision 𝑫 (for a normal eye 𝑫 = 25
cm) from the eye (located in the posterior focal plane of the eyepiece). The distance between
the rear focus of the objective 𝑭′𝒐 and the front focus of the 𝑭′𝒆 eyepiece. is called the optical
length of the L tube.
The magnification of the objective is:
𝑨′ 𝑩′
𝑾о𝒃𝒋. = ,
𝑨𝑩
and of the eyepiece:
𝑨″ 𝑩″
𝑾ок. = ′ ′ .
𝑨𝑩
The total magnification of the microscope is:
𝑨″ 𝑩″
𝑾= = 𝑾об. 𝑾ок.
𝑨𝑩
Through some geometric examinations and approximations, the magnification of the
microscope gives the expression:
𝑳𝑫
𝑾= ,
𝒇о 𝒇𝒆
where 𝒇о and 𝒇𝒆 are the focal lengths of the objective and eyepiece, respectively. This formula
gives the magnification of the microscope a good approximation at high magnifications when
the focal lengths of the objective and eyepiece are small.
In addition to magnification, a very important feature of the microscope is its resolution.
Let 𝜹𝒐𝒎 denote the smallest distance between two points of the object, the images of which are
𝟏
seen separately in the microscope. The resolution of a microscope is called the magnitude: 𝜹 .
𝒎
It is limited by the phenomenon of light diffraction. For the resolution, the dependence is valid:

𝟏 𝒏 𝐬𝐢𝐧 𝜸
= ,
𝜹𝒐𝒎 𝝀
where 𝒏 is the refractive index of the medium between the object and the front lens of the
objective, 𝜸 is called the aperture angle and is equal to half the angle between the end rays
entering the lens, and 𝝀 is the length of the light wave.
When illuminating the object obliquely (at an angle), the resolution of the microscope
is:
𝟏 𝟐𝒏 𝐬𝐢𝐧 𝜸
= .
𝜹𝒎 𝝀
The quantity 𝑨 = 𝒏 𝐬𝐢𝐧 𝜸 is called the numerical aperture and is a characteristic
constant for each objective. The formula for the resolution shows what are the possibilities for
increasing the resolution, namely:
• Increasing 𝒏 – instead of air (dry lens, 𝒏 = 𝟏), between the objective and the object is
placed a medium with a higher refractive index – a liquid called immersion. Such objectives
are called immersion objectives. In addition to increasing the resolution of the microscope,
immersion lenses achieve greater image brightness, as light passes through an optically uniform
medium without reflection from the front of the front lens.
 • Increasing the aperture angle – by bringing the subject closer to the lens, but this
approach is limited by a certain distance specific to each lens at which the subject is located.
The largest aperture angle reached in modern microscopes is 70°.
• Reducing the wavelength, which is not justified within the visible range, but is used in
electron microscopes.
In order for the images of two points of the object, located at a distance 𝜹𝒐𝒎 to differ as
separate from the eye, the distance between these images must be at least equal to the distance
𝜹𝒆 between two points, which the eye sees as separate. Therefore, the magnification of the
microscope must fulfill the condition:
𝜹𝒆
𝑾 ≤ 𝑾𝒎 = .
𝜹𝒎
The magnification 𝑾𝒎 is called the useful magnification of the microscope. It is
between 500 A and 1000 A, in which the eye distinguishes all elements of the structure of the
object allowed by the resolution of the microscope. At magnifications above 1000 A, no new
details of the object structure are visible. Such magnifications are used in microphotography.

Optical microscopy methods

The sample can be lit in a variety of ways.

Transparent objects can be lit from below and solid objects can be lit with light coming
through (bright field) or around (dark field) the objective lens.
Polarised light may be used to determine crystal orientation of metallic objects.
Phase-contrast imaging can be used to increase image contrast by highlighting small
details of differing refractive index.
Epifluorescence microscope, designed for analysis of samples which include
fluorophores.
Confocal microscope, a widely used variant of epifluorescent illumination which uses a
scanning laser to illuminate a sample for fluorescence.
Two-photon microscope, used to image fluorescence deeper in scattering media and
reduce photobleaching, especially in living samples.