Lectures Notes Types of microscopes.pptx

DIFFERENT VARIANTS OF MICROSCOPE

BASICS
 Visible Light:
Electromagnetic spectrum detected by human eye-
400nm(deep violet) to 800nm(Far red).
Human eye doesn’t see much beyond 700nm(deep
red).
 White light:
Mixture of light containing some % of wavelength
from all visible portion.

BASICS
 Color Temperature:
Measure of mixture of light given off by light
source.
Higher the color temp closer is to natural daylight.
Natural daylight color temperature~5200kelvin
Incandescent light from Tungsten Bulb~3200k
Higher color temp- more blue/white to eye.
Lower color temp- more red to yellow to eye &
regarded as being ‘warmer’ in color.

BASICS
 The Amplitude (i.e. brightness):
Decreases as light gets further from source
because of absorption in media it passes.
 Energy Level:
Energy content of light, expressed as electron volt
per photon (the particle representation of light).
Visible light-1electron volt/photon.
Soft x-ray portion-50-100ev/photon
Energy increases moving towards violet &
ultraviolet range of spectrum.

BASICS
 Spherical aberration:
Light rays hitting periphery will be more refracted
than the rays hitting centre of lens.
 Chromatic Aberrations:
White light of passing through simple lens, each
wavelength will be refracted to different extent.
Blue brought to a shorter focus than red, results in a
un-sharp image with color fringes.
Corrected with ‘Achromat’ & ‘Apochromat’ lens.

BASICS
 Magnification:
For variable tube length=From 160-250mm
Optical tube length X Magnification of eye piece
Focal length of the objective
 Resolving Power:
Capacity of the optical system to produce separate
images of objects very close to each other.
Resolving power of std LM=200nm

BASICS
 Illumination:
Critical illumination-when the light source is
focused by condensor in the same plane as the
object, when the object is in focus.
The illuminating light consists of rays or waves that
are nearly parallel to one another.
 This quality of light is called partially coherent
light. The ultimate resolving power of the
microscope depends on the use of partially
coherent illumination.
 The condenser in Köhler illumination is used to
create and focus this light onto the specimen

BASICS
 Real Image:
The real image forms on the side of the lens
opposite the object.
A slide projector produces a real image on a
projection screen.
The objective lens produces a real image in
the intermediate image plane.

BASICS
 Virtual Image:
A virtual image cannot be seen on a screen. A
second lens is required to see a virtual image and
this would cause the image to be inverted relative
to the specimen.
To a human, the virtual image appears to be on
the same side of the lens as the object.
The image we see in a compound microscope is a
virtual image.
In upright microscopes the virtual image is
inverted. In inverted microscopes the image is
made to be upright by optics in the binocular.

BASICS
 Back Focal Plane:
The side of a lens where an image is formed is called
the image side or back side of the lens. The plane at
one focal length on this side is the back focal plane.
The back focal plane of the objective lens is on the
inside of the microscope tube.
 Coma:
Resulting in the images of structures out from the center
being smeared outwards.
 Astigmatism:
Resulting in light in the X plane being focused differently
to light in the Y plane.

•NUMERICAL APARTURE:
• Is a dimensionless number that characterizes
the range of angles over which the system can
accept or emit light.
•It is the acceptance cone of an objective (and
hence its light-gathering ability and resolution).
The numerical aperture with
respect to a
point P depends on the half-
angle θ of the maximum
cone of light that can enter
or exit the lens.
where n is the index of refraction of the medium in
which the lens is working, and θ is the half-angle
of the maximum cone of light that can enter or exit
the lens
BASICS

BASICS
 Compound Lenses:
There are four basic lens shapes: double
convex, double concave, plano convex and
plano concave.
In the 1830’s, the solution to chromatic aberrations
was found by cementing two lenses made of
different glasses (one Double convex and one
Plano-concave) together into an Achromatic
Doublet to make a compound lens.
Today, compound lens consists of many lens
elements. These elements are necessary for
magnification and correction of various lens
aberrations.

REFRACTIVE INDEX
•Refractive index is the light-
bending ability of a medium.
• The light may bend in air
so much that it misses the
small high-magnification
lens.
• Immersion oil is used to
keep light from bending.
RI of Air- 1.0
RI of Water -1.3
RI of Glycerol- 1.47
RI of Glass(avg)- 1.5
RI of Oil- 1.52

UNITS OF MEASUREMENT
 1 µm micrometer = 1/1000 mm
 1 nm nanometer = 1/1000 µm
 Parfocal distance: Distance between objective
shoulder and specimen(45 mm for most
microscopes).
 Working Distance Focal Length
Scanning- 17-20mm 40mm
Low power- 4-8mm 16mm
High power- 0.5-0.7mm 4mm
Oil immersion- 0.1mm 1.8-2.0mm

INFINITY CORRECTED MICROSCOPES
 A majority of modern research microscopes are
equipped with infinity-corrected objectives that no
longer project the intermediate image directly into
the intermediate image plane. Light emerging from
these objectives is instead focused to infinity, and a
second lens, known as a tube lens, forms the
image at its focal plane.

 Wave trains of light leaving the infinity-focused
objective are collimated, allowing beam-splitters,
polarizers, Wollaston prisms, etc., to be introduced
into the space between the objective and tube lens.

 In a microscope with infinity-corrected optics,
magnification of the intermediate image is
determined by the ratio of the focal lengths of the
tube lens and objective lens.
 Because the focal length of the tube lens varies
between 160 and 250 millimeters (depending upon
the manufacturer and model), the focal length of the
objective can no longer be assumed to be 160
millimeters divided by its magnification.
 Thus, an objective having a focal length of 8
millimeters in an infinity-correct microscope with a
tube lens focal length of 200 millimeters would have
a lateral magnification of 25x (200/8).

MODERN TECHNOLOGY IMPROVING MICROSCOPY
 The invention of the microscope allowed scientists and
scholars to study the microscopic creatures in the world
around them.
 When learning about the history of the microscope it is
important to understand that until these microscopic
creatures were discovered, the causes of illness and
disease were theorized but still a mystery.
 The microscope allowed human beings to step out of the
world controlled by things unseen and into a world where the
agents that caused disease were visible, named and, over
time, prevented.

MICROSCOPES CAN BE SEPARATED INTO SEVERAL
DIFFERENT CLASSES
 One grouping is based on
what interacts with the
sample to generate the
image:
-Light or photons(optical
microscopes)
-Electrons (electron
microscopes)
-Probe (scanning probe
microscopes).
 Whether they analyze the
sample via:
-Scanning point (confocal
optical microscopes,
scanning electron
microscopes and scanning
probe microscopes)
-Analyze the sample all at
once (wide field optical
microscope and
transmission electron
microscopes).

BRIGHT-FIELD MICROSCOPY
 The useful magnification of Light microscope is limited
by its resolving power.
 The resolving power in limited by wavelength of
illuminating beam.
 Resolution is determine by certain physical parameters
like wave length of light and light generating power of
the objective & condenser lens.
 Higher N.A Better light generation Better Resolution
Shorter the Wavelength Better Resolution.
 The ordinary microscope is called as a bright field
microscope. It forms dark image against bright
background.

Dark objects are visible
against a bright
background.
Light reflected off the
specimen does not enter
the objective lens.

ADVANTAGES
 Bright field compound microscopes are commonly
used to view live and immobile specimens such as
bacteria, cells, and tissues.
 For transparent or colorless specimens, however, it
is important that they be stained first so that they
can be properly viewed under this type of a
microscope.
 Staining is achieved with the use of a chemical dye.
By applying it, the specimen would be able to adapt
the color of the dye. Therefore, the light won’t
simply pass through the body of the specimen
showing nothing on the microscope’s view field

PRINCIPLE
 When observing unstained/living cells, such specimens & their
components have refractive indices close to that of the medium in
which they are suspended & are thus difficult to see by bright field
techniques, due to their lack of contrast.
 Dark field microscopy overcomes these problems by
preventing direct light from entering the front of the objective
& the only light gathered is the reflected or diffracted by
structures within the specimen.
This causes the specimen to appear as a bright image on a
dark background, the contrast being reversed & increased.
 Dark field permits the detection of particles smaller than the optical
resolution that would be obtained in bright field, due to high
contrast of scattered light.
 In the microscope, oblique light is created by using a modified
or special condensor that form a hollow cone of direct light
which will pass through the specimen but outside the
objective.

DARK FIELD MICROSCOPY
 Light enters the microscope for illumination of the
sample.
 A specially sized disc, the patch stop blocks some
light from the light source, leaving an outer ring of
illumination.
A wide phase annulus can also be reasonably
substituted at low magnification.
 The condenser lens focuses the light towards the
sample.

 The light enters the sample. Most is directly
transmitted, while some is scattered from the
sample.
 The scattered light enters the objective lens, while
the directly transmitted light simply misses the
lens and is not collected due to a direct
illumination block.
 Only the scattered light goes on to produce the
image, while the directly transmitted light is omitted.

 Dark field microscopy is a very simple yet effective
technique and well suited for uses involving live
and unstained biological samples:
-Spirochetes
-Flagellates
-Cell Suspension
-Flow Cell Techniques
-Parasites
-Autoradiographic grain counting
-Once used in Fluorescence Microscopy

 In Dark Field Illumination the objective must have
a lower Numerical Aperture than the condensor.
 In Bright Field Illumination optimum efficiency is
obtained when the Numerical Aperture of both the
Objective and the Condensor are matched.
 Dark Field Condensor may be:
-Dry, low power objective
-Oil immersion, high power objective

ADVANTAGES
 The advantage of darkfield microscopy also becomes its
disadvantage: not only the specimen, but dust and other
particles scatter the light and are easily observed For
example, not only the cheek cells but the bacteria in
saliva are evident. The dark field microscopes divert
illumination and light rays thus, making the details of the
specimen appear luminous.
 Dark field light microscopes provide good results,
especially through the examination of live blood
samples. It can yield high magnifications of living
bacteria and low magnifications of the tissues and cells
of certain organisms. Certain bacteria and fungi can be
studied with the use of dark field microscopes.

DEFINITION
 Phase contrast microscopy is an optical illumination
technique in which small phase shifts in the light
passing through a transparent specimen are
converted into amplitude or contrast changes in the
image. The technique was invented by Frits
Zernite in the 1930s for which he received the
Nobel prize in physics in 1953 .

Accentuates
diffraction of the
light that passes
through a
specimen. Direct
and reflected
light rays are
combined at the
eye. Increasing
contrast
PHASE CONTRAST MICROSCOPY

APPLICATION
 Applications for phase contrast microscopy
equipment range from the study of living biological
specimens, medical applications, study of live blood
cells, and other biological and science applications.
 Most commonly used to provide contrast of
transparent specimens such as living cells or small
organisms.
 Useful in observing cells cultured in vitro during
mitosis.

 Unstained/living biological specimens have little contrast
with their surrounding medium.
To see them clearly involves:
A-Closing down the iris diaphragm of condensor which
decreases the Numerical Aperture producing diffraction
effects & destroy the Resolving Power of the Objective.
B-Or when using dark field illumination, which enhances
contrast by reversal, but often fails to reveal internal
details.
 The Phase Contrast microscopy overcomes these
problems by controlled illumination using the full
aperture & thus improving the Resolution.
The high the refractive index of the structure, the darker
it will appear against the light background, i.e. with more
contrast.

 If a diffraction grating is examined under the
microscope, diffraction spectra are formed in the
Back Focal Plane of the objective due to
interference between the direct & diffracted rays of
light.
 The grating consists of alternate strips of material
with slightly different refractive indices, through
which light acquire small phase difference & these
form the images.

 By converting the phase differences, between light
passing through a specimen and that passing
through the surrounding medium, into amplitude
(brightness) differences, phase contrast microscopy
provides a difference in brightness between the
object and the background, which the eye can then
see.

 Phase contrast microscopy uses an annular stop in the
condenser and a phase plate within the objective lens,
which is aligned with the annular stop.
In this configuration, the light path can be split and
each of the separated beams will pass through the
same transparent medium at the specimen stage.
The light passes through the annular stop and forms a
cone of light, which comes to its vertex at the focal point
of the specimen.
Any background light, which is un-deviated by the
specimen, will go through the phase ring in the phase
plate and is advanced by a quarter of a wavelength.
Deviated light passing through the specimen is retarded
by a quarter of a wavelength and passes through the
phase plate without going through the ring.

 When the beams are recombined further along the
light path, the differences in the phase of the
deviated and un-deviated light beams become
additive and subtractive.
 The resultant wave is the sum of the two waves
which have their crests and troughs opposite each
other. The difference in amplitude can be seen as a
change in brightness, since brightness is
proportional to the square of the amplitude.
The net result is that features of the object are
either lighter or darker than the surrounding field.

Plane Light
Polarized Light
High Relief

PRINCIPLE
 Light is a series of pulse of energy radiating away from a
source & shown diagrammatically as sine curve, with
wavelength & amplitude.
 Light can also be described as electromagnetic
vibration, which travels outwards from source of its
propagation, much in the same way as vibration along a
rope.
 Natural light vibrates in many directions but polarized
light in only one direction.

POLARIZED LIGHT MICROSCOPY
 Polarization can be achieved with the use of
substance/crystals which allow vibration only in one plane &
called birefringent.
 Light entering a birefringent crystal such as calcite is split into
two light paths, each determined by a different refractive
index & each vibrating in one direction only, but at right angle
to each other.
 The higher the RI, the greater the retardation of the ray, so
that each ray leaves the crystal at a different velocity.
 The high RI ray is called slow and low RI ray is called fast.
 There is also a phase difference between the rays, so that if
they are recombined interference occurs & various spectral
colors are seen.

 Two discs made up of prism are placed in a path of
light, one below the objective known as Polarizer &
another placed in the body tube which is known as
Analyzer.
 Polarizer sieves out ordinary light rays vibrating in
all directions allowing light waves of one orientation
to pass through.
 The lower disc (polarizer) is rotated to make the
light plane polarized. During rotation, when
analyzer comes perpendicular to polarizer, all light
rays are cancelled or extinguished.
 Birefringent objects rotate the light rays & therefore
appear bright in a dark background.

 Analyzer (upper polarizer) -- a
polarizing prism located above the
microscope stage, between the
objective lens and the eyepiece. This
restricts the transmission of light
vibrating perpendicular to the polarizer.
The analyzer can be slipped in or out
of the light path or rotated for partially
crossed polarized light. Light passing
through the polarizer will not pass
through the analyzer unless the
vibration direction of the light is
changed between the two prisms.
Anisotropic minerals can perform this
deed.
 Polarizer (lower polarizer) -- a
polarizing prism located beneath the
microscope stage (between the light
source and the object of study). This
restricts transmission of light to that
vibrating in only one (N-S) direction.
Some microscopes have a different
orientation direction. In effect, it plane
polarizes the incident light beam.

 Numerous crystals, fibrous structures (both natural &
artificial), pigment, lipid, protein, bone & amyloid deposits
exhibit birefringence.
 Originally, polarizer's, made from calcite & known as Nicol
Prism, were cemented together with Canada balsam in such
away that the slow rays was deflected away from the optical
path & into the mount of the prism, leaving only the polarized
fast rays to pass through (optic axis).
 Some substances & crystals can produce plane polarized light
by differential absorption & give rise to phenomenon of
Dichorism. These absorb the slow rays & are pleochoric
(absorbing all colors equally) and are the most useful in
microscopy.
Such crystals suspended in thin plastic films & oriented in one
direction have replaced bulky Nicol Prism.

 Two phenomenon detected in polarized light-
Birefringence & Dichroism.
 When a birefringent substance rotated between two
crossed polarizers, the image appears & disappears
four times each in 360 deg rotation (at 45̊ each).
 Dichroism- Only the polarizer is used &, if no rotatory
stage is available the polarizer itself can be rotated.
Changes in intensity & color is seen during rotation at 90
deg. This is due to differential absorption of light,
depending upon the vibration direction of the two rays in
a birefringent substance.
 Weak birefringence in biological specimen is increased
by addition of dyes or impregnating metals, in a orderly
linear alignment e.g. amyloid fibrils.

DIFFERENTIAL INTERFERENCE CONTRAST

DIFFERENTIAL INTERFERENCE CONTRAST
 In this complex form of polarised light microscopy two
slightly separate, plane polarised beams of light are
used to create a 3D-like image with shades of grey.
 Wollaston prisms situated in the condenser and above
the objective produce the effect, and additional elements
add color to the image.
 Care must be taken to interpreting DIC images as the
apparent hills and valleys in the specimen can be
misleading. The height of a "hill" (e.g. the nucleus) is a
product of both the actual thickness of the feature (i.e.
ray path length) and its refractive index.
 Variations of the DIC system are named after their
originators, Nomarski and de Senarmont. Options can
be selected to maximize either resolution or contrast.

Accentuates
diffraction of the
light that passes
through a
specimen; uses
two beams of
light. Adding
color
Differential Interference Contrast Microscopy

INTRODUCTION
 Electron microscope is a type of microscope that
uses a particle beam of electrons to illuminate a
specimen & create a highly-magnified image. Co-
invented by Germans, Max Knoll and Ernst
Ruska in 1931

ELECTRON GUN
 2 types of guns are used in electron microscopy- Thermionic
Emission Gun & Field Emission Gun.
 Thermionic: Electrons emitted from heated filament (
tungsten, Lanthanum Hexaboride). Most common, cheap &
ultra high vacuum not required.
 Field Emission: Strong electron field used to extract
electrons from filament. High vacuum needed.

ELECTROMAGNETIC LENS
 An electromagnet designed to produce a suitably
shaped magnetic field for focusing &
deflection of electrons in electron optical
instruments.
 A strong magnetic field is generated by passing a
current through a set of windings.
 This field acts as a convex lens in case of electron
microscope.

CONDENSER LENS
 The first lens(controlled by "spot size knob")
largely determines the "spot size"; the general size
range of the final spot that strikes the sample.
 Second condenser lens: The second
lens(controlled by the "intensity/ brightness knob"
changes the size of the spot on the sample;
changing it from a wide dispersed spot to a pinpoint
beam.

OTHER PARTS
 The other parts include:
-Condenser aperture,
-Objective lens,
-Objective aperture,
-Selected area aperture(to examine diffraction
patterns),
-Intermediate lens(magnifies initial image formed by
objective lens) &
-Projector lens.

TYPES OF ELECTRON MICROSCOPES
 There are 2 types of electron microscopes:
 Transmission Electron Microscope: Process is
carried out under vacuum to avoid friction. The
"Virtual Source" at the top represents the electron
gun, producing a stream of monochromatic
electrons. The usual potential is around 10000 –
15000V.
 SCANNING ELECTRON MICROSCOPE

TRASMISSION ELECTRON MICROSCOPY

•Ultrathin sections of specimens.
•Light passes through specimen, then an
electromagnetic lens, to a screen or film.
•Specimens may be stained with heavy metal salts.
Transmission Electron Microscopy (TEM)

TRANSMISSON ELECTRON MICROSCOPE
 This transmitted portion is focused by the objective lens
into an image.
 The Objective & Selected Area metal apertures restrict
the beam.
 The image is passed down the column through the
intermediate and projector lenses, being enlarged all the
way.
 The image strikes the phosphor image screen & light is
generated, allowing the user to see the image.
 The darker areas represent areas that fewer electrons
were transmitted (thicker or denser). The lighter areas
represent areas that more electrons were transmitted
(thinner or less dense)

TRANSMISSION ELECTRON MICROSCOPE
 Transmission electron microscopy (TEM) involves a high voltage
electron beam emitted by a cathode and formed by magnetic
lenses. The electron beam that has been partially transmitted
through the very thin (and so semitransparent for electrons)
specimen carries information about the structure of the specimen.
 The spatial variation in this information (the "image") is then
magnified by a series of magnetic lenses until it is recorded by
hitting a fluorescent screen, photographic plate, or light sensitive
sensor such as a CCD (charge-coupled device) camera. The image
detected by the CCD may be displayed in real time on a monitor or
computer.
 Transmission electron microscopes produce two-dimensional, black
and white images.
 Resolution of the TEM is also limited by spherical and chromatic
aberration, but a new generation of aberration correctors has been
able to overcome or limit these aberrations.

•An electron gun produces a beam of electrons that scans the surface of
a whole specimen.
•Secondary electrons emitted from the specimen produce the image.
Scanning Electron Microscopy (SEM)

SCANNING ELECTRON MICROSCOPE
 Unlike the TEM, where the electrons in the primary beam are
transmitted through the sample, the Scanning Electron
Microscope (SEM) produces images by detecting secondary
electrons which are emitted from the surface due to excitation
by the primary electron beam.
 In the SEM, the electron beam is scanned across the surface
of the sample in a raster pattern, with detectors building up an
image by mapping the detected signals with beam position.

SCANNING ELECTRON MICROSCOPE
 The 1st scanning electron microscope (SEM)
debuted in 1938 by Von Ardenne with the first
commercial instruments out around 1965.
 In this case electrons are not used to directly image
the specimen, but to excite it in such a way that it
gives out secondary electrons which are collected
by detectors & used to form the image.
 The first scanning electron microscope (SEM)
debuted in 1938 ( Von Ardenne) with the first
commercial instruments around 1965.

SPECIMEN
 For electron microscopy, tissue is fixed in 4%
glutaraldehyde at 4°C for 4 hours.
 Fixation - In chemical fixation for electron microscopy,
glutaraldehyde is often used to crosslink protein
molecules and osmium tetroxide to preserve lipids.
 Dehydration - Organic solvents such as ethanol or
acetone for SEM specimens or infiltration with resin and
subsequent embedding for TEM specimens.
 Embedding - Resin (for electron microscopy) such as
araldite or LR White, which can then be polymerised into
a hardened block for subsequent sectioning.

SPECIMEN
 Sectioning - Typically around 90nm cut on an
ultramicrotome with a glass or diamond knife. Glass
knives can easily be made in the laboratory and are
much cheaper than diamond, but they blunt very quickly
and therefore need replacing frequently.
 Staining - uses heavy metals such as lead and uranium
to scatter imaging electrons and thus give contrast
between different structures, since many (especially
biological) materials are nearly "transparent" to the
electron beam.
 By staining the samples with heavy metals, electron
density is added to it which results in there being more
interactions between the electrons in the primary beam
and those of the sample, which in turn provides us with
contrast in the resultant image.

ARTIFACTS
 It must be emphasized from the outset that every
electron micrograph is, in a sense, an artifact.
 Artifacts present themselves in many ways:
-There could be loss of continuity in the
membranes,
-Distortion or disorganization of organelles,
-Empty spaces in the cytoplasm of cells or
sharp bends or curves in filamentous structures that
are usually straight, such as microtubules and so
on.
 With experience, microscopists learn to recognize
the difference between an artifact of preparation
and true structure.

DISADVANTAGES OF ELECTRON MICROSCOPY
 Electron microscopes are very expensive to buy and maintain.
 They are dynamic rather than static in their operation: requiring
extremely stable high voltage supplies, extremely stable currents to each
electromagnetic coil/lens, continuously-pumped high/ultra-high vacuum
systems and a cooling water supply circulation through the lenses and
pumps.
 As they are very sensitive to vibration and external magnetic fields,
microscopes aimed at achieving high resolutions must be housed in
buildings with special services.
 A significant amount of training is required in order to operate an
electron microscope successfully and electron microscopy is considered a
specialised skill.
 The samples have to be viewed in a vacuum, as the molecules that
make up air would scatter the electrons. This means that the samples
need to be specially prepared by sometimes lengthy and difficult
techniques to withstand the environment inside an electron microscope.
 Recent advances have allowed some hydrated samples to be imaged
using an environmental scanning electron microscope, but the
applications for this type of imaging are still limited.

PRINCIPLE
 Confocal microscopy is an optical imaging
technique used to increase optical
resolution and contrast of a micrograph by using
point illumination and a spatial pinhole to
eliminate out-of-focus light in specimens that are
thicker than the focal plane.
 It enables the reconstruction of three-dimensional
structures from the obtained images.

•Uses fluorochromes and
a laser light.
•The laser illuminates
each plane in a specimen
to produce a 3-D image.
Confocal Microscopy

INTRODUCTION
 Although conventional light and fluorescence microscopy
allow the examination of both living and fixed specimens,
certain problems exist with these techniques.
-One of the main problems is out-of-focus blur degrading the
image by obscuring important structures of interest,
particularly in thick specimens.
 In conventional microscopy, not only is the plane of focus
illuminated, but much of the specimen above and below this
point is also illuminated resulting in out-of-focus blur from
these areas.
 This out-of-focus light leads to a reduction in image contrast
and a decrease in resolution.

APPLICATIONS IN CONFOCAL MICROSCOPY
 The broad range of applications available to laser
scanning confocal microscopy includes a wide
variety of studies in neuroanatomy and
neurophysiology, as well as morphological studies
of a wide spectrum of cells and tissues.
 In addition, the growing use of new fluorescent
proteins is rapidly expanding the number of original
research reports coupling these useful tools to
modern microscopic investigations.

OTHER APPLICATIONS
 Include-
-Resonance energy transfer,
-Stem cell research,
-Photobleaching studies,
-Lifetime imaging,
-Multiphoton microscopy,
-Total internal reflection,
-DNA hybridization,
-Membrane and ion probes,
-Bioluminescent proteins, and
-Epitope tagging.

 Confocal reflection microscopy can be utilized to gather
additional information from a specimen with relatively little
extra effort, since the technique requires minimum specimen
preparation and instrument re-configuration.
 In addition, information from unstained tissues is readily
available with confocal reflection microscopy, as is data from
tissues labeled with probes that reflect light.
 The method can also be utilized in combination with more
common classical fluorescence techniques.
 Examples of the latter application are detection of unlabeled
cells in a population of fluorescently labeled cells and for
imaging the interactions between fluorescently labeled cells
growing on opaque, patterned substrata

FLUORESCENT MICROSCOPY
 This method is used for demonstration of naturally
occurring fluorescent material and other non-
fluorescent substances or micro-organisms after
staining with some fluorescent dyes e.g.
Mycobacterium tuberculosis, amyloid, lipids, elastic
fibres etc.
 UV light is used for illumination.

•Uses UV light.
• Fluorescent
Substances absorb
UV light and emit
visible light.
• Cells may be stained
with fluorescent dyes
(fluorochromes).
Fluorescence Microscopy

WHY FLUORESCENCE MICROSCOPY?
 In all types of microscopes, cell constituents are not
distinguishable, although staining dose, but not
totally.
 In fluorescent microscopy, various fluorescent dyes
are used which gives property of fluorescence to
only specific part of the cell and hence it can be
focused.
 Fluorescent microscopy depends upon illumination
of a substance with a specific wavelength (UV
region i.e. invisible region) which then emits light at
a lower wavelength (visible region).

FLUORESCENCE PRINCIPLE
 When certain compounds are illuminated with high
energy light, they then emit light of a different, lower
frequency. This effect is known as fluorescence.
 Often specimens show their own characteristic
autofluorescence image, based on their chemical
makeup.
 The key feature of fluorescence microscopy is that
it employs reflected rather than transmitted light,
which means transmitted light techniques such as
phase contrast and DIC can be combined with
fluorescence microscopy.

COMPONENTS
 Typical components of a fluorescence microscope
are:
 the light source (xenon arc lamp or mercury-vapor
lamp),
 the excitation filter,
 the dichroic mirror and
 the emission filter.

STAINING
 Many different fluorescent dyes can be used to
stain different structures or chemical compounds.
 One particularly powerful method is the
combination of antibodies coupled to a
fluorochrome as in immunostaining.
 Examples of commonly used fluorochromes are
fluorescein or rhodamine.

FUNCTIONING
 A component of interest in the specimen is
specifically labeled with a fluorescent molecule
called a fluorophore.
 The specimen is illuminated with light of a specific
wavelength (or wavelengths) which is absorbed by
the fluorophores, causing them to emit longer
wavelengths of light (of a different color than the
absorbed light).

APPLICATIONS
 Fluorescence microscopy is a critical tool for
academic and pharmaceutical research, pathology,
and clinical medicine.

The distance between the focal plane of the objective (f1) and the focal plane
of the eyepiece (f2) is called the tube length (l).
The object to be viewed is placed just outside the focal point at the left side
of the objective lens. The enlarged image is formed at a distance l + f1 from
the objective.

X-RAY MICROSCOPY
 less common,
 developed since the late 1940s,
 resolution of X-ray microscopy lies between that of
light microscopy and the electron microscopy.
 X-rays are a form of electromagnetic radiation with
a wavelength in the range of 10 to 0.01
nanometers, corresponding to frequencies in the
range 30 PHz to 30 EHz.

RECENT ADVANCES IN MICROSCOPY
 In the recent times, computers and chip technology
have helped in developing following advances in
microscopy.

IMAGE ANALYSERS AND MORPHOMETRY
 In these techniques, microscopes are attached to
video monitors and computers with dedicated
software systems.
 Microscopic images are converted into digital
images and various cellular parameters (e.g.
nuclear area, cell size etc) can be measured. This
quantitative measurement introduces objectivity to
microscopic analysis.

TELEPATHOLOGY (VIRTUAL MICROSCOPY)
 It is the examination of slides under microscope set
up at a distance. This can be done by using a
remote control device to move the stage of the
microscope or change the microscope field or
magnification called as robobic telepathology.
 Alternatively and more commonly, it can be used by
scanning the images and using the highspeed
internet server to transmit the images to another
station termed as static telepathology.
Telepathology is employed for consultation for
another expert opinion or for primary examination

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