MICROSCOPY: TYPES Light Field Microscope Compound microscopes Bright-Field Microscope Phase-contrast microscope Fluorescence microscope

1. MICROSCOPY: TYPES
Dr. Saraswathi, M.S.c, Ph.D
Assistant Professor,
Dept. of Life Sciences
Kristu Jayanti College, Autonomous, Bengaluru

2. MICROSCOPY
• Microscopy is defined as the use of a microscope to magnify and study the
small objects that are too small to be visualized with the naked eye.
• Naked eye ~ 0.1 mm, Light microscope ~ 0.1 μm • Electron microscope ~
2.5 nm
• Magnification on a microscope refers to the amount or degree of visual
enlargement of an observed object.
• Magnification is measured by multiples, such as 2x, 4x and 10x, indicating
that the object is enlarged to twice as big, four times as big or 10 times as big,
respectively.

3. Resolution: Resolving power is the ability of a lens to separate or
distinguish small objects that are close together.
When a ray of light passes from one medium to another, refraction
occurs that is, the ray is bent at the interface.
- Depends on the quality of lens and the wavelength of illuminating
light.
• Shorter wavelength having greater resolution.
• Standard light microscopes : resolution 0.5 micrometers (mm).
• Electron microscopes : resolution of up to 1 nanometer (nm).

4. • Resolution is described mathematically by an equation developed in the 1870s by
Ernst Abbe, a German physicist.
Abbé equation states that the minimal distance (d) between two objects that reveals
them as separate entities depends on the wavelength of light (λ ) used to illuminate the
specimen.
• Numerical aperture of the lens (n sin θ), which is the ability of the lens to gather
light.

5. n= refractive index
λ= wavelength
sinθ= angle of light
d= distance
d= λ/NA
d= λ.0.5/n sinθ

6. BASIC QUALITY PARAMETERS OF MICROSCOPIC IMAGES
• Focus: It refers whether the image is well defined or blurry (out
of focus).
• The focus can be adjusted through course and fine adjustment
knobs of the microscope.
• Brightness: It refers how light or the dark the image is.
• depends on the illumination system and can be adjusted by
changing the voltage of the lamp and by condenser diaphragm.
• Contrast: It refers how best the specimen is differentiated from
the background or the adjacent area of microscopic field.
• More the contrast will give good images, phase contrast
microscopes are designed
• Resolution and Magnification

7. • Lenses act like a collection of
prisms operating as a unit.
• When the light source is distant
so that parallel rays of light strike
the lens, a convex lens will focus
these rays at a specific point, the
focal point.
• The distance between the center
of the lens and the focal point is
called the focal length .

8. Contrast:
• Reflects the number of visible shades in the specimen.
• Is needed to make objects stand out from the background.
• Achieved through various staining techniques.
• Microorganisms are essentially transparent and must be stained for bright-
field microscopy

9. TYPES

10. Basic components of the light microscope
• The simplest form of light microscope consists of a single glass lens
mounted in a metal frame – a magnifying glass.
• Here the specimen requires very little preparation, and is usually held
close to the eye in the hand.
• Focussing of the region of interest is achieved by moving the lens and
the specimen relative to one another.
• The source of light is usually the Sun or ambient indoor light. The
detector is the human eye. The recording device is a hand drawing.
THE LIGHT MICROSCOPE

11. Light microscopes
Eyepiece Lens - This is the lens that you
look down.
Coarse Focussing Wheel - This moves the
stage by a large amount to bring the image
into focus (make the image clear and not
blurry).
Fine Focussing Wheel - This moves the
stage by a small amount to focus the image
carefully (make the image clear and not
blurry).
Objective Lens - This is the lens next to the
specimen that magnifies the image.
Stage - This is where the sample is placed.
Light Source - This can be a lamp or a
mirror used to shine light through the
specimen

12. 1. Light microscope
2. Electron microscope.
• Light microscopes use a series of glass lenses to focus light in order to
form an image whereas electron microscopes use electromagnetic lenses to
focus a beam of electrons.
• Light microscopes are able to magnify to a maximum of approximately
1500 times
• Whereas electron microscopes are capable of magnifying to a maximum of
approximately 200000 times.
There are two fundamentally different types of microscope:

13. Compound microscopes
• All modern light microscopes are made up of more than one glass lens in
combination.
• The major components are the
and, such instruments are therefore called compound microscopes.
• The main components of the compound light microscope include a light source that is
focussed at the specimen by a condenser lens.
• Light that either passes through the specimen (transmitted light) or is reflected back
from the specimen (reflected light) is focussed by the objective lens into the eyepiece
lens.

14. • The image is either viewed directly by eye in the eyepiece or it is most often
projected onto a detector, for example photographic film or, more likely, a
digital camera.
• The images are displayed on the screen of a computer imaging system, stored
in a digital format and reproduced using digital methods.

15. COMPOUND LIGHT MICROSCOPY

16. Two basic types of compound light microscope. An upright light microscope (a) and an inverted light
microscope (b). Note how there is more room available on the stage of the inverted microscope (b). This
instrument is set up for microinjection with a needle holder to the left of the stage.
(a) (b)

17. Two basic types of compound light microscope
• The Upright microscope: The light source is below the
condenser lens in the upright microscope and the objectives
are above the specimen stage. This is the most commonly used
format for viewing specimens.
• The inverted microscope : The light source and the
condenser lens are above the specimen stage, and the
objective lenses are beneath it. Moreover, the condenser and
light source can often be swung out of the light path.

18. • A compound microscope is used for viewing small specimens.
• Used to visualize different samples at high magnification (40 – 1000x).
• Used to observe both prokaryotic and eukaryotic cells.
• To study cells and cell structures.
• Plays a pivotal role in the Clinical laboratory.
• To detect the microorganisms directly in clinical specimens.
• To characterize the growth of the organism in culture
APPLICATIONS

19. The Bright-Field Microscope
• The ordinary microscope is called a bright-field microscope because it
forms a dark image against a brighter background.
• The microscope consists of a metal body or stand composed of a base
and an arm to which the remaining parts are attached.
• A light source, either a mirror or an electric illuminator, is located in the
base.
• Two focusing knobs, the fine and coarse adjustment knobs, are located
on the arm and can move either the stage.

21. • This light is gathered in the condenser, then shaped into a
cone where the apex is focused on the plane of the specimen.
APPLICATIONS:
• Used for observing stained or naturally pigmented or highly
contrasted specimens.
• Used for visualizing different types of bacteria and cell
structures.

22. • Understanding cell structures in cell Biology, Microbiology,
Bacteriology to visualizing parasitic organisms in
Parasitology.
• Most of the specimens to be viewed are stained using special
staining to enable visualization.
• Negative staining and Gram staining.
• Used to visualize and study the animal cells
• Used to visualize and study plant cells.
• visualize and study the morphologies of bacterial cells
• Used to identify parasitic protozoans such as Paramecium.

23. Disadvantages of Brightfield microscope
• The aperture diaphragm may cause great contrast which may distort the
outcome of the image, therefore iris diaphragm is preferred.
• It can not be used to view live specimens such as bacterial cells.
• Only fixed specimens can be viewed under the brightfield microscope.
• The maximum magnification of the brightfield microscope is 100x but
modification can readjust the magnification to 1000x which is the
optimum magnification of bacterial cells.
• It has low contrast hence most specimens must be stained for them to be
visualized.
• The use of oil immersion may distort the image
• The use of a coverslip may damage the specimen

24. • Staining may introduce extraneously unwanted details into the
specimen or contaminate the specimen.
• It is tedious to stain the specimen before visualizing it under the
brightfield microscope.
• The microscope needs a strong light source for magnification
and sometimes the light source may produce a lot of heat which may
damage or kill the specimen

26. • The dark-field microscope allows a viewer to observe living, unstained cells and organisms
by simply changing the way in which they are illuminated.
• A hollow cone of light is focused on the specimen in such a way that unreflected and
unrefracted rays do not enter the objective.
• Only light that has been reflected or refracted by the specimen forms an image
• The field surrounding a specimen appears black, while the object itself is brightly
illuminated.
• The dark-field microscope can reveal considerable internal structure in larger eucaryotic
microorganisms. It also is used to identify certain bacteria like the thin and distinctively
shaped

27. Principle
• In dark field microscopy uses an extra opaque disc
underneath the condenser lens.
• Having a central blacked-out area, due to which the light
coming from the source cannot directly enter into the objective.
• The path of the light is directed in such a way that it can pass
through the outer edge of the condenser at a wide-angle and
strike the sample at an oblique angle.
• light scattered by the sample reaches the objective lens for
visualization.

28. • All other light that passes through the specimen will miss the
objective, thus the specimen is brightly illuminated on a dark
background.
• Uses of Dark-Field Microscopy
• Treponema pallidum (syphilis), Borrelia burgdorferi (lyme
borreliosis) and Leptospira interrogans (leptospirosis) in clinical
samples.
• Microbial motility; tufts of bacterial flagella can often be seen
in unstained cells by dark-field.
• Internal structure in larger eukaryotic microorganisms such as
algae, yeasts, etc.

29. Advantages of Dark-Field Microscopy
• Resolution by dark-field microscopy is better than bright-field
microscopy.
• It improves image contrast without the use of stain, and thus do
not kill cells.
• Direct detection of non-culturable bacteria present in patient
samples.
• No sample preparation is required.
• It requires no special setup, even a light microscope can be
converted to dark field.

30. Limitations of Dark-Field Microscopy
• Necessity to examine wet, moist specimens containing living
organisms very quickly, because visualization of the moving
bacteria is essential to detection.
• The sample must be very strongly illuminated, which can cause
damage to the sample.
• Besides the sample, dust particles also scatter the light and appear
bright.
• Sample material needs to be spread thinly, dense preparations can
affect the contrast and accuracy of the dark field’s image.

31. (a) T. pallidum: dark-field microscopy (b) Volvox and Spirogyra: dark-field microscopy

36. Phase-contrast microscope
• A phase-contrast microscope converts slight differences in
refractive index and cell density into easily detected variations in
light intensity and is an excellent way to observe living cells.
• Phase contrast is used for viewing unstained cells growing in tissue
culture and for testing cell and organelle preparations for lysis.
• The method images differences in the refractive index of cellular
structures.

37. • The background, formed by undeviated light, is bright, while the
unstained object appears dark and well-defined. This type of microscopy
is called
• Phase-contrast microscopy is especially useful for studying microbial
motility, determining the shape of living cells, and detecting bacterial
components such as endospores and inclusion bodies.
• These are clearly visible because they have refractive indices

41. (c) Pseudomonas: phase-contrast microscopy
(d) Desulfotomaculum: phase-contrast microscopy

42. • All Living cells can be observed in their natural state without previous
fixation or labeling process. The images are all label-free.
• No special preparation of fixation or staining is needed to observe a
biological sample under a phase-contrast microscope.
• It saves a lot of time for researchers and scientists.
• intracellular components of living cells at relatively high resolution.
mitochondria of cells, mitotic chromosomes, vacuoles, etc.
• To study living cells and how they multiply through cell division at their
respective stages of life.
• Phase-contrast microscope components can be added to virtually any
standard light microscope, provided the specialized phase objectives
conform to the tube length parameters, and the condenser will accept an
annular phase ring of the correct size.

43. Applications
• Phase contrast microscope enables the visualization of
unstained living cells.
• It makes highly transparent objects more visible
• It is used to examine various intracellular components
of living cells at relatively high resolution.
• It helps in studying cellular events such as cell division.
• It is used to visualize all types of cellular movements
such as chromosomal and flagellar movements..

44. Limitations of Phase contrast Microscopy
• Phase-contrast condensers and objective lenses add considerable cost to a
microscope,
• Phase contrast is often not used in teaching labs except perhaps in
classes in the health professions.
• To use phase-contrast the light path must be aligned.
• More light is needed for phase contrast than for corresponding bright-
field viewing,
• Technique is based on the diminishment of the brightness of most
objects.

45. Fluorescence microscope
• The fluorescence microscope exposes a specimen to ultraviolet, violet, or blue
light and forms an image of the object with the resulting fluorescent light.
• The most commonly used fluorescence microscopy is epifluorescence
microscopy, also called incident light or reflected light fluorescence
microscopy.
• Epifluorescence microscopes employ an objective lens that also acts as a
condenser
• A mercury vapor arc lamp or other source produces an intense beam of light
that passes through an exciter filter.

46. • The exciter filter transmits only the desired wavelength of
excitation light. The excitation light is directed down the
microscope by a special mirror called the dichromatic mirror.
This mirror reflects light of shorter wavelengths (i.e., the
excitation light).
• The excitation light continues down, passing through the
objective lens to the specimen, which is usually stained with
special dye molecules called fluorochromes.

47. • The fluorochrome absorbs light energy from the excitation light and
fluoresces brightly. The emitted fluorescent light travels up through the
objective lens into the microscope.
• Emitted fluorescent light has a longer wavelength, it passes through the
dichromatic mirror to a barrier filter, which blocks out any residual
excitation light.
• Finally, the emitted light passes through the barrier filter to the
eyepieces.

49. Application
• The fluorescence microscope has become an essential tool in medical
microbiology and microbial ecology.
• Bacterial pathogens (e.g., Mycobacterium tuberculosis, the cause of
tuberculosis) can be identified after staining them with fluorochromes or
specifically labeling them with fluorescent antibodies using
immunofluorescence procedures.
• In ecological studies the fluorescence microscope is used to observe micro-
organisms stained with fluorochrome-labeled probes or fluorochromes that
bind specific cell constituent

50. • In addition, microbial ecologists use epifluorescence microscopy to
visualize photosynthetic microbes, as their pigments naturally fluoresce
when excited by light of specific wavelengths.
• It is even possible to distinguish live bacteria from dead bacteria by the
color they fluoresce after treatment with a special mixture of stains.
• Thus the microorganisms can be viewed and directly counted in a
relatively undisturbed ecological niche.
• Imaging the genetic material within a cell (DNA and RNA).
• Viewing specific cells within a larger population with techniques such as
FISH.

51. Photon
electron