Microscope and Microscopy Principal , Function & Difference of various types of Light & Electron microscope.Microscopy is the technical field of using microscopes to view samples & objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye). Microscopists explore the relationships between structures & properties for a very wide variety of materials ranging from soft to very hard, from inanimate materials to living organisms, in order to better understand it. Zachariaz Janssen 1585 Robert Hooks 1665 Joseph Jackson Lister1830

1. MICROSCOPE &
MICROSCOPY
Principal , Function & Difference of various
types of Light & Electron microscope

2. MICROSCOPY
⦿Microscopy is the technical field of using
microscopes to view samples & objects that
cannot be seen with the unaided eye (objects that
are not within the resolution range of the normal
eye).
⦿Microscopists explore the relationships between
structures & properties for a very wide variety of
materials ranging from soft to very hard, from
inanimate materials to living organisms, in order
to better understand their
behaviour.https://www.digistore24.com/redir/35
1558/Mariamkareem/

3. INTRODUCTION
⦿in microbiology, the common unit for
measuring length is the micrometer(µm),
which is equivalent to a millionth (10–6) of a
meter. To appreciate how small a micrometer
is, consider this: Comparing a micrometer to
an inch is like comparing a housefly to New
York City’s Empire State Building, 1,472 feet
high. Microbial agents range in size from the
relatively large, almost visible protozoa (100
µm) down to the incredibly tiny viruses (0.02
µm)
⦿Most bacterial and archaeal cells are about 1
µm to 5 µm in length, although notable
exceptions have been discovered recently

4. HISTORICAL BACKGROUND
Zacharias Janssen 1585 Robert Hooks 1665

5. HISTORICAL BACKGROUND
Joseph Jackson Lister1830
https://www.digistore24.com/redir/351558/Maria
mkaree
Antoine van Leeuwenhoek 1677

6. INTRODUCTION
Achaea & Bacterial cells Empire state building `
1,472 feet high

8. MAGNIFICATION
Most microscopes used in microbiology have
several objective lenses, including 10* (low
power), 40* (high power), and 100* (oil
immersion). Most ocular lenses magnify
specimens by a factor of 10. Multiplying the
magnification of a specific objective lens with
that of the ocular, we see that the total
magnifications would be 100* for low power,
400* for high power, and 1000* for oil
immersion. Some compound light
microscopes can achieve a total magnification
of 2000* with the oil immersion lens.

9. RESOLUTION
Resolution (also called resolving power) is the ability of the lenses to
distinguish fine detail and structure. Specifically, it refers to the ability of
the lenses to distinguish two points that are a specified distance apart.
if a microscope has a resolving power of 0.4 nm, it can distinguish two
points if they are at least 0.4 nm apart
shorter the wavelength of light used in the instrument, the greater the
resolution.
white light used in a compound light microscope has a relatively long
wavelength and cannot resolve structures smaller than about 0.2 μm.
Light micrscope resolutionm 1500x
van Leeuwenhoek’s tiny spherical lenses had a resolution of 1 μm.
human eye are measured at a lateral resolution of 8.55 µm, 576 pixel

10. TYPES OF MICROSCOPES
⦿ Microscopy Categories
1: Light (optical) microscopes 2: Electron microscopes
Light or optical microscopes further categorized as
1. Polarizing Microscope,
2. Reflected Light Microscope,
3. Bright field microscopy
4. Dark field (Fig. 5a),
5. Phase contrast microscopy
6. Fluorescence microscopy
7. Confocal Microscope
8. SAM
9. Super light resolution microscopy
10. - Multiphoton Microscope –
11. Three-Dimensional Optical Microscopy
Electron microcopy is of two types
1. Transmission microscopy and 2. Scanning Electron microscopy

11. LIGHT (OPTICAL) MICROSCOPE
⦿ Light travels as wave in crests & troughs.
⦿ The amplitude of the crests & troughs determine the
brightness of the light.
⦿ The number of time complete wave occur per unit time is
called as frequency and the distance between two
consecutive crests is called wavelength (λ) of the light.
⦿ Light microscope wavelength in the range 400-700 nm
make up visible spectrum.
⦿ While the UV region consists of wavelengths ranging from
100-385 nm.
⦿ Visualizing any object directly by human eye involves
incidence & reflection of light in the visual range.
⦿ Microscopes use day light or light emitted by incandescent
bulb.
⦿ Fluorescent & UV microscope employ UV radiations

12. LIGHT MICROSCOPE
❖ A modern compound light microscope (LM) has a series of lenses and
uses visible light as its source of illumination
❖ We can calculate the total magnification of a specimen by multiplying
the objective lens magnification (power) by the ocular lens
magnification (power)
❖ Most microscopes used in microbiology have several objective lenses,
including 10x (low power), 40x (high power), and 100x (oil immersion)
❖ Most ocular lenses magnify specimens by a factor of 10. Multiplying the
magnification of a specific objective lens with that of the ocular, we see
that the total magnifications would be 100x for low power, 400x for high
power, and 1000x for oil immersion

13. CHANGING REFRACTIVE INDEX
⦿ To obtain a clear, finely detailed image under a compound light
microscope, specimens must contrast sharply with their medium
(the substance in which they are suspended)
⦿ refractive index is a measure of the light-bending ability of a
medium.
⦿ To attain such contrast, we must change the refractive index of
specimens from that of their medium
⦿ To achieve high magnification (1000*) with good resolution, the
objective lens must be small
⦿ To preserve the direction of light rays at the highest
magnification, immersion oil is placed between the glass slide
and the oil immersion objective lens
⦿ field of vision in a compound light microscope is brightly
illuminated. By focusing the light, the condenser produces a
brightfield illumination

14. HISTORICAL BACKGROUND
⦿Antonie van Leeuwenhoek used first single
lens which magnify 300x times in 17th
centuary
⦿Robert Hooke, built compound microscopes,
which have multiple lenses.
⦿Zaccharias Janssen, is credited with making
the first compound microscope around 1600.
However, these early compound microscopes
were of poor quality and could not be used to
see bacteria.
⦿until about 1830 that a significantly better
microscope was developed by Joseph
Jackson Lister (the father of Joseph
Lister).

15. HOW TO MAKE IMAGE QUALITY BETTER
⦿ Change contrast of image by changing refractive
index in compound microscope.
⦿ Change refractive index of medium and specimen
by staining specimen.
⦿ Both have different refractive index so light bend
different
⦿ As the light rays travel away from the specimen,
they spread out and enter the objective lens, and
the image is thereby magnified.
⦿ shorter the wavelength of light used in the
instrument, the greater the resolution
⦿ Immersion oil has an index of refraction of 1.5,
which is almost identical to the index of refraction
of glass

20. DARK FIELD MICROSCOPY
darkfield microscope is used to examine live microorganisms that
either are invisible in the ordinary light microscope, cannot be
stained by standard methods, or are so distorted by staining that
their characteristics
A darkfield microscope uses a dark field condenser that contains an
opaque disc are obscured
This technique is frequently used to examine unstained
microorganisms suspended in liquid
One use for darkfield microscopy is the examination of very thin
spirochetes, such as Treponema pallidum (trep-o¯ -NE¯-mah PAL-
li-dum), the causative agent of syphilis.

22. PHASE-CONTRAST MICROSCOPY
⦿ Phase-contrast microscopy is especially useful
because the internal structures of a cell become
more sharply defined, permitting detailed
examination of living microorganisms
⦿it isn’t necessary to fix (attach the microbes to the
microscope slide or stain the specimen—
procedures that could distort or kill the
microorganisms
⦿One light source and one diffracted source
⦿When brought together make in phase and out-
phase ocular image

25. DIFFERENTIAL INTERFERENCE
CONTRAST (DIC) MICROSCOPY
⦿Differential interference contrast (DIC)
microscopy is similar to phase-contrast
microscopy in that it uses differences in refractive
indexes.
⦿DIC microscope uses two beams of light instead of
one.
⦿prisms split each light beam, adding contrasting
colors to the specimen. Therefore, the resolution
of a DIC microscope is higher than that of a
standard phase-contrast microscope
⦿he image is brightly colored and appears nearly
three-dimensional

27. FLUORESCENCE MICROSCOPY
⦿ Fluorescence microscopy takes advantage of fluorescence, the
ability of substances to absorb short wavelengths of light
⦿ fluorochrome auramine O, which glows yellow when exposed to
ultraviolet light, is strongly absorbed by Mycobacterium
tuberculosis, the bacterium that causes tuberculosis
⦿ . Bacillus anthracis, the causative agent of anthrax, appears apple
green when stained with another fluorochrome, fluorescein
isothiocyanate (FITC)
⦿ The principal use of fluorescence microscopy is a diagnostic
technique called the fluorescent-antibody (FA) technique, or
immunofluorescence
⦿ Antibodies and antigen relation
⦿ This technique can detect bacteria or other pathogenic
microorganisms, even within cells, tissues, or other clinical
specimens

29. CONFOCAL MICROSCOPY
⦿ Confocal microscopy is a technique in light microscopy used to
reconstruct three-dimensional images. Like fluorescent
microscopy, specimens are stained with fluorochromes so they
will emit, or return, light. But instead of illuminating the entire
field, one plane of a small region of a specimen is illuminated
with a short-wavelength (blue) light which passes the returned
light through an aperture aligned with the illuminated regi
⦿ two-dimensional images can be obtained, with improved
resolution of up to 40% over that of other microscopes.
⦿ The scanned planes of a specimen, which resemble a stack of
images, are converted to a digital form that can be used by a
computer to construct a three-dimensional representation.
⦿ This procedure makes it possible to image cells up to 1 mm deep
in detail

32. TWO PHOTONS MICROSCOPY
⦿ Two-photon microscopy uses long-wavelength (red) light, and
therefore two photons, instead of one, are needed to excite the
fluorochrome to emit light
⦿ longer wavelength allows imaging of living cells in tissues up to 1
mm (1000 μm) deep
⦿ Confocal microscopy can image cells in detail only to a depth of
less than 100 mm
⦿ Additionally, the longer wavelength is less likely to generate
singlet oxygen, which damages cells
⦿ advantage of TPM is that it can track the activity of cells in real
time. For example, cells of the immune system have been
observed responding to an antigen.

34. SUPER RESOLUTION LIGHT
MICROSCOPY
⦿ Maximum resolution for light microscopes was 0.2 μm. However, in
2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan
Hell, and William Moerner for the development of a microscope that
uses two laser beams
⦿ super-resolution light microscopy, one wavelength stimulates
fluorescent molecules to glow, and another wavelength cancels out all
fluorescence except for that in one nanometer.
⦿ Cells can be stained with fluorescent dyes that are specific for certain
molecules such as DNA or protein, allowing even a single molecule to be
tracked in a cell
⦿ . A computer tells the microscope to scan the specimen nanometer by
nanometer and then puts the images together

36. SCANNING ACOUSTIC
MICROSCOPY
⦿Scanning acoustic microscopy (SAM) basically
consists of interpreting the action of a sound
wave sent through a specimen. A sound wave of a
specific frequency travels through the specimen,
and a portion of it is reflected back every time it
hits an interface within the material. The
resolution is about 1 μm. SAM is used to study
living cells attached to another surface, such as
cancer cells, artery plaque, and bacterial biofilms
that foul
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