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MICROSCOPES

Compiled by
Dr.CH.Aruna kumari PG

Guided by
Dr. K.R.L Surya Kirani MD Prof&HOD
Dr. K. Rajasekhar MD Asst.Prof
Dr. S.S.Sarma, MD Asst.Prof
History
The term microscope was coined by Faber in 1623.

In 1672 Leeuwenhoek developed a simple microscope with


a magnification of 200x – 300x.

He is called as Father of microscopy.

1590 - Zaccharias Janssen and his son Hans Janssen


observed enlarged images with multiple lenses placed in a
tube.

In 1665 Robert Hooke developed a first laboratory


compound microscope.

Later, Kepler and Galileo developed a modern class room


microscope.
MICROSCOPE :

A microscope is an optical instrument that uses a


lens or a combination of lenses to magnify and resolve
the fine details of an object.

MICROSCOPY :

The science of investigating small objects using a


microscope is called microscopy.

Applications:

 Preliminary and final identification of organisms &


micrometry.
 Evaluation of phagocytes or epithelial cells.
 Determination of organism’s clinical significance.
 To provide pre-culture information.
TYPES OF MICROSCOPES
1.Simple microscope.

2.Compound microscope.

Depending upon magnification involved these are of 2 types.

a. Light microscope : use sunlight or artificial light.

b.Electron microscope : use of electron.

1.Simple microscope

•Contains a single lens.

•Similar to magnifying glass.

•Leeuwenhoek used simple microscope


to observe microorganisms
Compound
Microscopes

Light Electron
Microscopes Microscopes

Phase Transmission
Bright Field Dark Field Flourescent Scanning EM
Contrast EM
COMPOUND MICROSCOPE
• This is an optical instrument containing one or
more lenses producing an enlarged image
WORKING OF COMPOUND MICROSCOPE

 Light is transmitted and focussed by mirror and condenser.

 Focussed light illuminate the object or specimen.

 The refracted light is collected by an objective where


primary image of the object is formed, it is real, inverted
enlarged image of the object.

 The eyepiece further magnifies this primary image into


virtual, erect enlarged image, this is the final image that lies
above the stage.
Principles of Light Microscopy

1. Magnification

2. Resolution

3. Contrast
Magnification:

• It is the ratio of the size of an object seen under microscope to


the actual size observed with unaided eye.

• Objective lens operates at a distance roughly equivalent to its


focal length, forming an enlarged, real, inverted image.

• Field lens collect them to pass through eye lens that magnifies it
further.

• Virtual image reaches the eye of the observer.


Magnification = Optical tube length/ focal length of objective .

Ex : 16 mm objective = 160/16 = 10 = low power


4 mm objective = 160/ 4 = 40 = high power
2mm objective = 160/ 2 = 80 = oil immersion

Total magnification = objective magnification X eye lens


magnification.

It implies that, for oil immersion, total magnification is


100x X 10x = 1000x

for high power, 40x X 10x = 400x


for low power, 10x X 10x = 100x
Resolution :

The extent to which details in the magnified object are


maintained.
Resolving Power (RP) :

• It is the ability to differentiate two close points as separate.

• Wavelength of light used is major factor in resolution. “Shorter


the wavelength, greater the resolution” .

•The resolving power of human eye is 0.25 mm

Light microscope - 0.25µm.

Electron microscope - 0.5nm.


Numerical aperture(NA)

• The numerical aperture of a lens is the ratio of the


diameter of the lens to its focal length.

• NA = n Sin θ
n = refractive index,
θ = angle of aperture

• NA of a lens is an index of the resolving power.


• NA can be decreased by decreasing the amount
of light that passes through a lens

10X objective NA 0.25

20X objective 0.45

40X objective 0.65

100X(oil) objective 1.25

• As NA value is directly proportional to n ( refractive


index), oil immersion objective will be having higher NA
and therefore higher resolving power.
Immersion oil or Mounting fluid

To achieve resolution, immersion oil with same refractive


index as glass must be used.

Examples :

1. Cedar wood oil


2. Liquid paraffin
3. Sandal wood oil
4. Xylol-balsam
5. Dried Canada balsam
6. Olive oil
7. Glycerol
8. Euparal
Contrast
 Makes objects stand out from the background.

 Owing to high water content and their microscopic


dimensions, bacteria are essentially transparent.

 Achieved by staining.
Uses

1.To study structural details and to measure approx. size of


bacteria, fungi, protozoa.

2.To examine wet films or hanging drop for demonstration


of motility.
3. Wet mount of clinical specimens like urine, CSF.

Candida Cysts of Entamoeba coli


Carrying a Microscope
Dark-field Microscope
DARK FIELD MICROSCOPE

PRINCIPLE

1.A special condenser is used, which prevents the


transmitted light from directly illuminating the specimen.

2.Only oblique scattered light reaches the specimen and


passes onto the lens system causing the object to appear
bright against a dark background.
An ordinary light microscope may be converted to a dark
ground microscope by using:

1. Dark-ground Condenser : It has a central circular stop


which illuminates object with a cone of light

2. The Illuminant : high-intensity low-voltage lamps

3. The Funnel Stop : to reduce the NA to < 1. this consists


of small funnel shaped piece of metal which fits into the
objective.
• Abbe condensor is fitted with a dark field stop—
eliminating all light from central portion of
condensor.

• A thin cone of light reaches the specimen, which is


focused in such a way that unreflected and
unrefracted rays do not enter the objective.
APPLICATIONS

For detecting:

Motile Treponema pallidum in chancre fluid.

Motile borreliae in blood.

Motile leptospires in urine.

Pathogenic microfilariae in blood. The sheath and nuclei


can be clearly seen.

Cryptococci in cerebrospinal fluid. The capsule


surrounding the cells can be seen.

Vibrios in specimens and cultures.


DEMERITS

1.Slides and coverslips should be free from dirt dust and


scratches.

2.Not possible to distinguish internal structure of bacteria.

3.Objects look bigger than they actually are.

TREPONEMA UNDER DARK LEPTOSPIRA UNDER DARK FIELD


FIELD MICROSCOPY MICROSCOPY
FLUOROSCENT MICROSCOPE

HISTORY

1.The fluoroscent microscope was first designed by August


Kohler, Carl Reichert & Heinrich Lehmann in 1911.

2.Initially used to detect primary or autofluorescence.

3.In 1930, Max Hatinger developed the technique of secondary


fluorescence which uses fluorochromes to stain tissues and
bacteria.
Principle:

Depends on emission of light by the specimen.

Release
Absorb
light of
Molecules radiant Excited
longer
energy
wavelength
Tissues,cells,bacteria

Stained with
fluoroscent dye

Examined under
microscope with UV
light

Luminous objects
around dark
background
FLUOROCHROMES

FLUOROCHROME EXCITATION EMISSION


Acridine Orange 490 nm 640nm Nucleic acids
Auromine 450-490 nm 515nm Mycolic acids
Calcofluor 355-425nm 460nm Cellulose
Acriflavin 430nm 515nm DNA
Acridine Yellow 470nm 550nm Nucleic acids,
acid fast cells
Fluorescein 490nm 520nm Antibody labelling
isothiocynate
Texas Red 596nm 620nm Antibody labelling

Rhodamine B 570nm 595nm Antibody labelling


isothiocynate
WORKING
ADVANTAGES

1.Its sensitivity, specificity, rapid testing, and easy use.

2.The sensitivity is high enough to detect as few as 50 molecules


per cubic micrometer.

3.Different molecules can now be stained with different colors,


allowing multiple types of molecule to be tracked simultaneously.
Limitations

FADING
 Reduction of intensity of fluorescence.

 Subdivided into quenching and bleaching.

 Bleaching is the irreversible decomposition of the fluorescent


molecules in the excited state because of their interaction with
molecular oxygen.

 Quenching reduces fluorescence intensity by mechanisms


involving nonradiative energy.

 result of oxidizing agents or the presence of salts of heavy


metals or halogen compounds.

 change the field frequently


 Fluorescence microscopy only allows observation of the
specific structures which have been labeled for fluorescence.

 For example, observing a tissue sample prepared with a


fluorescent DNA stain by fluorescent microscopy only reveals
the organisation of the DNA within the cells and reveals nothing
else about the cell morphologies.
APPLICATIONS

1)The Auramine-rhodamine dye to visualize


Mycobacterium tuberculosis.
-More sensitive than the Ziehl-Neelson and more cost
effective.

2) Examination of specimens for Fungal Diseases-


Calcofluor.
3)Demonstrating Cryptosporidium spp.oocysts

4) The QBC MalariaTest

•QBC capillary tube is coated with acridine orange


and centrifuged.

•The fluorescing parasites can then be observed


under ultraviolet light at the interface between red
blood cells and buffy coat.
5) Blood smears for the diagnosis of bacterial septicaemia.

6) Cerebrospinal fluid specimens for the diagnosis of


bacterial meningitis.

7) Urine for the diagnosis of bacteriuria.

8) Early detection of microorganisms in blood cultures.

9) Diagnosis of leptospirosis in blood, urine, or cultures..


IMMUNOFLUORESCENCE

Principle:

It involves labeling of antibody with fluorescent dye


followed by its use in detection or identification of
antigen.
In Immunofluorescence, following methods are used:

1) Direct Method (one stage method) - Coons and Kaplan


1950

2) Indirect Method(Two Stage Method) -Weller and


Coons 1954

3)Indirect compliment amplified Fluorescent antibody


technique -Boldwasser & Shepard 1958
ImmunoFluorescent Technique

•Fluorescent tag (fluorescein) conjugated to


antibodies.

•Antibodies bind to surface of bacteria.

•Illuminate with specific wavelength of light


(FITC- excite with blue, fluoresces green)

•Observe with fluorescence microscope.

•Used with syphilis spirochete and other


organisms. (FTA-ABS test).
Low Light Fluorescent Microscope
 1- inch SIT camera is used.

 Increased sensitivity.

 Provides images with good spatial resolution.

 Uses weakly fluorescent dyes that attach to specific


parts of cell.

 Computer enhances fluorescent signals given off as


biochemical processes occur in cell.
Uses of low light fluorescent microscope :

1. To measure

Local pH and ca+2 levels

Redox potentials

Fluidity of membranes

Turnover of molecules in living cells

2. To visualize individual threads of DNA and F-actin


molecules
Light sheet Fluorescence Microscopy

 An intermediate optical resolution, but good optical


sectioning capabilities and high speed.

 Only a thin slice of the sample is illuminated perpendicular to


the direction of observation.
Principle of Light sheet fluorescence microscope
As only the actually observed section is illuminated, this
method reduces the photodamage and stress induced on a
living sample.

Also the good optical sectioning capability, reduces the


background signal and thus creates images with high
contrast.

As LSFM uses a plane of light instead of a point, it can


acquire images at speeds 100 to 1000 times faster than
those offered by point- scanning methods.
•Starting in 1994, LSFM was developed as orthogonal plane
fluorescence optical sectioning microscopy or tomography
(OPFOS) mainly for large samples and later as
the selective/single plane illumination microscopy (SPIM) also
with sub-cellular resolution

•Since 2012, projects have been started to build up the


construction plans for LSFM.
LED Fluorescent Microscopy

Advantage of LED Fluorescent Microscopy

• Highly sensitive compared to ZN microscopy and


conventional mercury vapor lamp fluorescent microscopy
(Additional yield of 5-18% over ZN microscopy has been
documented ) and equally specific.

• Examination of the slide in less time.

• Less electricity consumption and cheaper maintenance.

• Non hazardous.
LED FM ZN

3+ 3+

2+ 2+
1+ 1+

Negative Negative
PHASE CONTRAST MICROSCOPE

• First described in 1934 by Dutch physicist Frits Zernike

• Produces high-contrast images of transparent specimens.

• Advantage - Living cells can be examined in their natural


state.
PRINCIPLE

-Unstained bacteria have constituents


of different refractive index .

-Differences in refractive index and


cell density are converted into easily
detected variations in light intensity.

-Light passing through a cell of high


refractive index is slowed down
relative to light that passes through
less dense medium.
ANNULAR DIAPHRAGM
-It is opaque flat-black (light absorbing) plate
with a transparent annular ring.

-Produces hollow cone of light.

PHASE PLATE
Placed in back focal plane of objective.

FUNCTION :

1. Enhances phase difference by


retarding diffracted wave front by one
quarter of wavelength .
2. Reduces intensity of direct rays and
equalizes it with diffracted rays
intensity.
APPLICATIONS

1.To estimate concentration of substance in


a cell.

2.Examining internal structure of living


cells.

3.To detect bacterial components like


-endospores
-inclusion bodies

4. To identify medically important fungi.


.

Budding yeast cells


6.To identify medically important protozoa

Trophozoites of Naegleria fowleri


from CSF of a patient with PAM

7.To examine cytopathic effect of viruses tissues and


cell cultures.

8.To examine growth and subdivision of bacteria.

9.To see flagellar movement.

10.Viable organisms can be seen.


11. To examine protozoa like Trichomonas, Entamoeba
etc.

TRICHOMONAS ENTAMOEBA
Differential Interference Contrast Microscopy

• DIC microscopy is similar to phase-contrast microscopy


as it uses differences in refractive indices.

• In this, 2 beams of light used instead of one. Prisms split


each light beam adding contrasting colours.

• So, its resolution is more than standard phase contrast


microscope. Image is brightly coloured and is a 3D image.
DIC Microscope
Advantages

• Useful for observing unstained cells because of its


ability to generate images that reveal internal cell
structures.

• Spores, vacuoles can be seen in 3D.

Embryo from single celled Giardia intestinalis


stage to two-fold stage
• It enables quantitative measurement of chemical
constituents of cells such as lipids ,proteins and
nucleic acids.

Budding Yeast Cells


Electron microscope

Invented in 1931 by Knoll and Ruska

Use electrons instead of photons (light) to illuminate a


specimen.

Electron: charged, has rest mass, not visible.

Photon: neutral, has no rest mass, visible at the wavelength


~ 400 nm-760 nm.

Wavelength of electron beam is much shorter than light,


resulting in much higher resolution.
Types

Transmission Electron Microscope (TEM)

Scanning Electron Microscope (SEM)

Reflection Electron Microscope

Scanning Transmission Electron Microscope (STEM)

Low-voltage Electron Microscope


Transmission electron microscope

PRINCIPLE

When a beam of electrons is passed through a


specimen, a part of it is transmitted and this part
when projected on fluorescent screen, its image
can be seen by the observer.
TEM

1. ELECTRON GUN
2. ELECTROMAGNE
TIC LENSES
3. VACUUM PUMPS
4. OPENING TO
INSERT
SAMPLES
5. OPERATION
PANEL
6. DISPLAY
SCREEN
7. WATER SUPPLY
TO COOL THE
INSTRUMENT
Transmission electron microscope
Specimen Preparation

For transmission electron microscopy, specimens


must be cut very thin.

1) NEGATIVE STAINING

Specimen is spread out as thin


film with phosphotungstic acid.

Heavy metal does not penetrate


specimen.

Background appears bright in


photographs.
2)Shadow Casting

Metal placed on Coated surface


On heating it Coats on side of
tungsten scatters e- Uncoated- Dark
evaporates. specimen.
filament. Bright
3)Surface replicas

Low MW wt First floated in Replica is


material(carbon) water & cleaned &
Dissolves
deposited on transferred to placed on
specimen.
specimen in stong acid or specimen grid
vaccum. alkali. for viewing.
4) Freeze-etching

5) Microtomy

Advantages

•Very high resolution—0.2nm

•Magnification is 1,000,000 times greater than the


size of the object.

•Visualization of all microorganisms,viruses & even


molecules & atoms.
Disadvantages

1) No 3-D image.

2) Absorption of electrons heats up the


sample and changes its characteristics.

3) Specimen must be thin

H.pylori Rotavirus
Scanning electron microscope

Uses electrons reflected from the surface of a specimen


to create image.

It captures the images of the specimen surface by


scanning it with a high-energy beam of electrons , in a scan
pattern.

Produces a 3-dimensional image of specimen’s surface


features.
Primary e- knocks e-
out of specimen.

Secondary e-

Collector

Detector—emits light
when struck by e-.
Light converted to
current.
Cathode ray tube
screen
Specimen preparation

Easy.

Air dried material can also be used.

Samples must be electrically conductive.

If a non conductive material has to be viewed , then


it has to be coated by a thin layer of electrically
conductive material(gold/palladium).

This coating is done using a sputter coater.


Staphylococcus Enterococcus

Avian flu
RBC
NEURON
ADVANTAGES OF SEM

 Primarly used for viewing surface details

 Used to examine specimens of large


thickness

 Image can be directly viewed

 3-Dimensional image can be obtained

 Has large depth of focus


DISADVATAGES OF SEM

• The Resolution of image is poor.

• Some samples can loose their structural


property due to their interaction with the
electrons.
Scanning transmission electron microscope(STEM)

 Combination of TEM & SEM.

 Serves as analytical device - chemical composition of


observed structure can be determined.

 Done by energy dispersive X-ray spectroscopy.

 Computers analyse the data.


Environmental Scanning Electron Microscopy
• Produces images of sufficient quality and resolution
with the samples being wet or contained in low vacuum
or gas.

• For biological samples that are unstable in the


high vacuum of conventional electron microscopes.

Reflection Electron Microscopy

• Reflected beam of elastically scattered electrons is


detected.
Low-voltage Electron Microscopy

• Low cost alternative with very similar


architecture to a conventional TEM.

• Enables to take advantage of a 5 kV electron


source.

Advantages :
5kV Low-voltage EM

1. High contrast

2. Staining is not required.

80kV Conventional TEM


Limitations of electron microscopy

• Cells cannot be examined in living state.

• Drying may alter morphology.

•Thin sections of specimen.

Problems

• Formation of artefacts
Comparision of various types of Microscopes

Type of Maximum useful Resolution (nm) Common uses


Microscope magnification
Bright field 1,500 X 100-200 Used for visualization of
microorganisms, usually necessary
to stain specimens for viewing

Dark field 1,5oo X 100-200 Used for viewing live


microorganisms, particularly those
with characterstic morphology,
staining not required, specimen
apperas bright & background is
dark

Ultraviolet 2,500 X 100 Improved resolution over normal


light microscopes, largely replaced
by elecrton microscopes.

Fluorescence 1,500 X 100-200 Uses fluorescent stains, useful in


diagnostic procedures for
identifying. Detecting
immunological reactions.
Comparision of various types of Microscopes

Type of Maximum useful Resolution (nm) Common uses


Microscope Magnification
Phase contrast 1,500 X 100-200 Used to examine structure of living
microorganisms, staining not
required.

Interference 1,500 X 100-200 Used to examine structure of


microorganisms, produce sharp
multicolored image with three
dimensional apperance.

TEM 5,00,000 to 1nm Used to view ultrastructure of


1,000,000 X microorganisms, including viruses,
greater resolving power and useful
magnification then can be achevied
by light microscope

SEM 10,000 to 1 to 10nm Used for showing detailed surface


1,000,000 X structures of microorganisms,
produces three dimensional image.
Advantages and Disadvantages of major types of Microscopy
Type of Advantages Disadvantages
Microscopy
1. Bright field 1. Easy to use. 1. Lack of contrast
2. Relatively expensive 2. Inability to resolve viruses and some
3. Allows staining reactions thin bacteria
to be interpreted 3. Most intracellular structures must be
4. Readily available stained to be observable.
4. Introduction of artifacts during
staining processes.
2. Dark field 1. Allows living specimens 1. Inability to evaluate staining
to be viewed. reactions.
2. Reduces artifacts 2. Subcellular detail not readily
observable.
3. Phase 1. Allows observation of 1. Inability to evaluate staining reactions
contrast intracellular biological
activity.
2. Enhances subcellular
details.
4. Fluorescence 1. Allows very rapid 1. Limited to viewing specimens naturally
identification of infectious fluoresce or that are stained with a
agents fluorescent dye.
5. Electron 1. Permits viewing of objects 1. Specimen must be killed.
too small to be detected by 2. Harsh preparation procedures
light microscopy increase the probability of artifacts
in the specimen.
3. Very expensive.
OTHER MICROSCOPES
POLARISING MICROSCOPE

Transverse wave light whose vibration possess direction is


called polarized light.
 Light from an ordinary light source (natural light) that
vibrates in random directions & is called nonpolarized light.
propagation
direction
plane of
vibration

light vibrates in
all planes that contain
vibration
the light ray
direction
(i.e., all planes
perpendicular to
the propagation
direction)

Polarized Light
Unpolarized Light
• Detects specimens that are birefringent(have the
characteristic of double refraction, i.e. the velocity of
light refracted by a substance is not the same in all
directions).
Compared to a typical microscope,a polarizing microscope
has the following added units:

1) Polarizing condenser that includes a polarizer

2) A rotating stage that allows the position of the specimen


to be set

3) A strain-free objective for polarized light

4) An analyzer

6) A Bertrand lens for observing the pupil of the


objective

7) A test plate, a compensator, and an eyepiece with


crosshair.
-The polarizer should be placed below the
condenser and the analyzer should be above
the objective.

-A polarizing objective differs from ordinary


objectives in a respect that it possesses a
high light polarizing capability.
•A Bertrand lens projects an interference
image of specimen, formed in the objective
pupil, onto the objective image position.

•It is located between the analyzer and


eyepiece for easy in and out of the light path

•The specimen is placed between two


polarizers crossed at 90o to each other (one
in condenser and one in objective).
USES

• Used for identification substances with highly


organized molecular structure

-crystals
-minerals
-certain foreign bodies and
-some lipids.

• Polarising microscopes are employed in gout using


synovial fluid.
• Amyloid determinations are possible by polarised
light observations of a sample using Congo Red dye
• If amyloid is present , a bright green colour will
be visible.

Lipid crystals Vitamin B1 crystals


Inverted Microscope

• Inverted microscopes are


constructed with the tip of the
objective pointing upward so as to
view the specimen from below.

•The objective is underneath the


stage and light is directed on the
specimen from above.
Inverted Vs Upright Microscope

1.This Microscope model is very good choice if sample to


be viewed in suspension or is very large & heavy.

2. The inverted is offered with 2 models


Routine.
Research.

3. An upright Microscope can be purchased as a smaller


model for student use which influences price.
Advantages

1. Observation of living specimens or tissues.

2. The process of cell division can be examined which is


not the case using a conventional compound
microscope.

3. What makes an inverted model useful is the ability to


maintain a more natural environment for the
specimen, thus extending its life.

4. The viewing of valuable life processes can be


researched longer. This is the major advantage over a
compound light microscope.
Also the inverted microscopes permit use of Kohler
Illumination which is a great option as well as DIC and
phase contrast optics(creating different shades of
brightness) greatly enhancing the image for better
viewing instead of needing to stain the specimen and thus
kill it.

 Its design is perfect for analysis.

 Additionally, models typically have connection ports,


which allow them to be attached to a variety of digital
recording equipment.
The option of recording the interaction of a specimen in its
environment allows for validation and review of the real
time observations.

Disadvantages

The largest disadvantage is the cost.

This means there are fewer used microscopes of this type on


the market, and less competitive pricing.

Additionally, all microscopes have a limited working distance


for focusing on the specimen.
 When using the inverted type, this difficulty is compounded
by the reality of looking up through containers of various
optical quality and thickness.

 You may look through plastic, which has a much different


optical correction than the thin glass slip on a standard slide
used with a conventional microscope.
USES:

1. Examination of cultures in flat bottom dishes.

2. Microdissection

3. Examination of parasites

4. Observation of agglutination in serology

5. Used to study close check of the general cellular


morphology & great patterns should be examined using
an inverted microscope.
6. View cultures using an inverted microscope to assess the
degree of cell density & confirm the absence of bacteria
and fungal contaminants.

7. View cultures using an inverted phase contrast


microscope cells growing in exponential growth phase should
be bright round & refractile. Hybridomas may be very
sticky & require a gentle knock to the flask to detach the
cells

8. EBV transformed cells can grow in very large clumps that


are very difficult to count & the center of the large clumps
may be non viable.
Cell cultures examined under inverted microscope
Latest versions

1.Confocal laser scanning microscope.

2.Total internal reflection fluorescence microscope.

3. Flow cytometry
CONFOCAL MICROSCOPE

Invented in 1955 by Marvin Minsky, who was trying


to see the interconnections between neuronal cells .
• Unlike the conventional microscope, the confocal
instrument images only the one spot—rather than
the entire field of view of the objective lens—onto
the detector.

• To generate a complete image, the spot is moved


over the specimen and the image built point by point.
Confocal laser microscope

 A laser light beam is focused onto a fluorescent specimen


through the objective lens.

 The mixture of reflected and emitted light is captured


by the same objective and is sent to the dichroic mirror.

 The reflected light is deviated by the mirror while the


emitted fluorescent light passes through a confocal
aperature (pinhole) to reduce the “out of focus” light.

 The focused light then passes through the emission filter


and proceeds to the photomultiplier.
Advantages

• Sharp images of a thin slice of a thick specimen

• Minimum background and interference from out-of-focus


parts of the specimen.

• One can build up a very clean three-dimensional image.

USES

• Observing cellular morphology in multilayered specimen

Eg. used in diagnosing Ca cervix

• Evaluation and diagnosis of basal cell carcinoma of skin


• It is used for localizing and identifying the presence of
filamentary fungal elements in the corneal stroma in
cases of keratomycosis, enabling rapid diagnosis.

• Biofilms can be visualized.


Scanning Probe Microscopy

It maps the bumps and valleys of a surface on an


atomic scale.

Resolution is more than that of electron microscopy


with no special preparation needed.

Types :

 Scanning Tunneling Microscopy : provides detailed


view of molecules such as DNA.

 Atomic Force Microscopy : produce 3D images of


surface of molecule.
Disposable microscope

•‘ Foldscope’, this invention is cheap costing less than $1 a


piece. Very easy to use and versatile enough to screen for a
variety of diseases on the field including malaria, Giardia
and leishmaniasis.

• The sample to be studied is still mounted as a stain on a


glass slide.

• The total cost varies according to the magnification to be


achieved. An apparatus that enables a 2,000x
magnification, sufficient to spot organisms like Leishmania
donovani, malarial parasites and E. coli. Weighs about 8
grams.
•The Foldscope comes as a single piece of card, with all the
necessary parts including optics, an LED and mirror built-in.
A user tears each part from the template and then matches
the pieces based on colour. The whole thing costs between 30
and 40 pence to manufacture.

•The Foldscope partially assembled to use the device, a


sample is mounted on a standard microscope slide and wedged
between the paper layers of the microscope. The user then
holds the sample up to their eye and uses their thumb and
forefinger to adjust focus by flexing and sliding the paper
platform accordingly.
DISPOSABLE MICROSCOPES
Magnetic Resonance Microscopy

Principle:

It is based on the principle of MRI in which magnetic fields


force electrons in living tissue to shift position.

When they return to their original location, computers help to


create images based on pattern of energy they release. It is
based on the principle of MRI in which magnetic fields force
electrons in living tissue to shift position.

When they return to their original location, computers help to


create images based on pattern of energy they release.
Magnetic Resonance Microscopy
Magnetic Resonance Force Microscopy in Microbiology:

Imaging of transgene expression.

To study phosphorus metabolism of micro-organisms from


waste waters.

To visualize and track stem and progenitor cells

To study the cytoplasmic membrane and total phospholipids


in micro-organisms (Escherichia coli)
Super Resolution Microscopes

Allows images to be taken with a light microscopey a higher


resolution than the diffraction limit.

These are of 2 types

1.Deterministic super resolution – MC used


2.Stochastic super resolution

True super resolution techniques


1. Near field scanning optical microscope (NSOM)
2. Local enhancement / ANSOM/Optical nano-antenas
3. Near field optical random mapping (NORM) microscopy
4. 4Pi
5. Structured illumination microscopy
6. Spatially modulated illumination
1.Deterministic functional techniques

1. Stimulated emission depletion microscopy(STED)


2. RESOLT microscopy – very high resolution that can not
imaged with conventional or confocal microscopy
3. Ground state depletion
4. Saturated structured illumination microscopy

2. Stochastic functional techniques

1. Localization microscopy
2. Localization microscopy SPDM
3. SPDM Phymod
4. STORM – Stochastic optical reconstruction
microscopy
PALM - Photoactivated localization microscopy
FPALM - Fluorescence photo activation localization
microscopy
5. Label- free localization microscopy

6. Direct stochostical optical reconstruction microscopy


(dSTORM)

Software for localization microscopy

Super resolution optical fluctuation imaging (SOFI)

Combination of techniques:

3D light microscopical nanosizing (LIMON) microscopy


1. Video enhanced contrast microscope

2. Transmission positron microscope

3. Low light dose microscope

4. Magnetic resonance microscope

5. Inferometric photo activated localization


microscope (iPalm)

6. Serial/Stretched team encoded amplification


microscopy (STEAM)
REFERENCES
Text book of Fundamentals of Microbiology Volume 1
Dr. H. A. Modi

Robert Cruickshank, Medical Microbiology, 12th edition,


volume 2.

 Mackie & McCartney Practical Medical Microbiology,


14th edition.

Ananthanarayan and Paniker’s Textbook of


Microbiology, 9th edition.

Dr. Subhash Chandra Parija’s Textbook of Practical


Microbiology, 1st edition.

Surinder Kumar Textbook of Microbiology, 1st edition


 Dr. Subhash Chandra Parija’s Textbook of Practical
Microbiology and Immunology, 1st edition.

 Video enhanced contrast microscopy – Biocyclopedia.

 David M. Shotton. Video enhanced light microscopy and


its applications in cell biology, Journal of cell science
89,129-150.

 Video microscopy – Shinya Inoue, Marine Biological


Laboratory. By Robert J. Walter et al.,

 Bio- Optics world- By Lee Dubay (14/10/2016)

 Immunoelectron Microscopy: A reliable tool for the


analysis of cellular processes- By Ana L. De Paul et al.,
 M. Firtel et al., Scanning probe microscopy in
Microbiology. Micron vol 26, issue 4, pages 347-362

 Mike Hasenberg et al., 8 visualizing immune responses in


fungal infections: Established & Novel methods. Human
fungal pathogens vol 12 of the series ‘The Mycota,’ 141-
160.

 Laser systems for optical Microscopy- By Kenneth R.


Spring

 Microscopy enhanced by the wave characteristics of


light.

Nanoscale MRI- The quest for a molecular structure


microscope- By Dr. John Mamin.
Magnetic Resonance Microscopy- Wikipedia.

 Adaptive optics for Biomedical Microscopy- By Martin J.


Booth et al.,
ACKNOWLEDGEMENT

Dr. V.Satya Chandrika SR

Dr. N.V Hemalatha SR

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