MICROSCOPY FOR MEDICAL AND LIFE SCIENCE STUDENTS

1. MICROSCOPY
Neethu soman
MSc Medical Biochemistry

2. INDEX
❑INTRODUCTION
❑HISTORY OF MICROSCOPE
❑PRINCIPLES OF MICROSCOPY
❑TYPES OF MICROSCOPE
❑APPLICATIONS OF MICROSCOPY

3. INTRODUCTION
➢Microscope (Greek: mikron = small and Scopes = to look)
➢It is an optical instrument used to magnify (enlarge) minute objects or
microorganisms which cannot be seen by naked eye.
➢Microscopy is the scientific field that involves the use of microscopes to
investigate objects and details that are too small to be seen with the naked eye.
➢Microscopic means invisible to the eye unless aided by a microscope

4. HISTORY OF MICROSCOPE
• Zacharias Jansen (1580—1638) of Holland invented a compound light
microscope, one that used two lenses, with the second lens further magnifying
the image produced by the first.
• Englishman Robert Hooke (1635—1703) further refined the compound
microscope, adding such features as a stage to hold the specimen, an illuminator,
and coarse and fine focus controls. until 1800, compound microscopes designed
by Hooke and others were limited to magnifications of 30x to 50x, and their
images exhibited aberrations.
• Carl Zeiss (1816—1888) and Ernst Abbe (1840—1905) added the substage
condenser and developed superior lenses that greatly reduced chromatic and
spherical aberration, while permitting vastly improved resolution and higher
magnification.

5. ➢Physicist Ernst Ruska and the electrical engineer Max Knoll (1931) developed
the first prototype electron microscope which was capable of four-hundred-
power magnification
➢Ernst Lubke of Siemens & Halske (1932) built and obtained images from a
prototype electron microscope, applying the concepts described in Rutenberg's
patent.
➢Ruska (1933) built the first electron microscope that exceeded the resolution
attainable with an optical (light) microscope.
➢Manfred von Ardennes (1937) pioneered the scanning electron microscope.
➢Siemens (1939) produced a transmission electron microscope (TEM) in 1939.

6. Father of Microscopy
• ANTONY VAN LEEUWENHOOK was
a Dutch scientist . In 1674 , viewing a
drop of rainwater, he observed things
moving which he called “Animalcules”
• First to experimented with microbes ,
using single lensed microscopes of his
own design invented in 1670.
• Magnified up to 200x and achieved
twice the resolution of the best
compound microscopes , mainly
because he crafted better lenses.

8. PRINCIPLES OF MICROSCOPY
MAGNIFICATION
RESOLUTION
NUMERICAL APERTURE
ILLUMINATION

9. MAGNIFICATION
❖ Magnification is the factor by which an image appears to be enlarged. It is
dependent upon the curvature and size of the lens. The image formed is
enlarged to a particular degree called Magnifying Glass the “Power of
Magnification”.
❖ When light passes through the objective lens and reaches your eye through
the eyepiece, it appears larger than its actual size.
❖ The total magnification of an object is the result of the magnification of the
objective lens multiplied by the magnification of the eyepiece.
❖ For example, if you have a microscope with a 10x eyepiece and a 40x
objective lens.
❖ Total Magnification = 10x (eyepiece) × 40x (objective) = 400x

10. RESOLUTION
➢Resolution or resolving power is the ability of a lens to show two adjacent objects
as discreate entities.
➢it is the microscope's ability to show fine detail and clarity in the images it
produces.
➢The resolving power of a microscope can be calculated using the formula:
R=0.61λ​/NA
➢Where: R is the resolving power,
λ (lambda) is the wavelength of light used, and
NA is the numerical aperture of the lens.
➢For example, if a microscope uses green light with a wavelength of 500
nanometres (0.5 micrometres) and has a numerical aperture of 1.4, the resolving
power would be: R=0.61×0.5μm/ 1.4 ​= 0.22μm

11. NUMERICALAPERTURE
• The Numerical Aperture (NA) of a microscope is a critical parameter that
determines its ability to gather light and resolve fine details in the specimen
being observed.
• It is a dimensionless number that describes the light-gathering ability of the
objective lens of the microscope
• Numerical Aperture (NA) is defined as the product of the refractive index of the
medium between the lens and the specimen (n) and the sine of the half-angle of
the maximum cone of light that the lens can gather (θ).
• NA=n⋅ sin(θ)

12. IILUMINATION
➢Effective illumination is required for efficient magnification and resolving
power. Artificial light from a tungsten lamp is the most commonly used light
source in microscopy. Illumination in microscopy is a crucial aspect of obtaining
clear, high-quality images of specimens.
➢It involves providing the right amount and type of light to illuminate the
specimen for observation. Proper illumination enhances contrast, resolution, and
overall image quality.
1. Transmitted Illumination: Light passes through the specimen from below, transmitted
through the sample. Commonly used in compound light microscopes for observing thin,
transparent specimens like biological samples on glass slides
2. Reflected Illumination: Light is directed onto the specimen from above, reflecting off the
surface. Suitable for opaque or thick specimens, such as metals, ceramics, or thick biological
samples.

13. ABERRATION
• Aberration is a property of optical system such as lenses that causes light to be
spread out over some region of space rather than focused to a point.
• An aberration in the context of microscopes refers to any departure from ideal
imaging conditions, resulting in distortion or blurring of the observed image.
• These aberrations can be caused by imperfections in the lenses or other optical
components of the microscope.
• Aberration causes the image formed by a lens to be blurred or distorted.
• Aberration associated with microscope are spherical aberration and chromatic
aberration.

14. SPHERICALABERRATION
• Spherical aberration is a common
optical aberration that occurs when
light rays passing through the
periphery of a lens or mirror are
focused at a different point than those
passing through the centre.
• In microscopes, spherical aberration
can degrade image quality by causing
blurring and distortion, particularly at
high magnifications
• Creates a curved image rather than
flat.

15. CHROMATIC ABERRATION
• Creates a blurry image like a rainbow, caused by the lens acting as a prism
• which occurs due to differences in the curvature of a lens or mirror, chromatic
aberration arises from the dispersion of light, where different wavelengths of
light are refracted differently as they pass through a lens
• Achromatic objective and Apochromatic objectives.
1. Apochromatic lens systems: These are designed to minimize both spherical
and chromatic aberrations by combining multiple lens elements made from
different types of glass with varying dispersion properties.
2. Achromatic lenses: These are designed to reduce chromatic aberration by
combining two or more lens elements made from different types of glass to
bring two or more wavelengths of light into focus at the same point

16. PARTS OF MICROSCOPE

17. MICROSCOPE

18. 1. Eyepiece (Ocular):The eyepiece is the lens at the top of the microscope that
you look through. Typically provides 10x magnification. Some microscopes
have adjustable eyepieces to accommodate users with different vision.
2. Objective Lenses: The objective lenses are located on the revolving nosepiece
beneath the eyepiece. These lenses are responsible for magnifying the specimen.
• Microscopes usually have multiple objective lenses with different
magnification powers (e.g., 4x, 10x, 40x, 100x).
• High-quality microscopes may feature specialized objectives such as oil
immersion lenses for high-resolution imaging.

19. 3. Stage:
• The stage is the platform where the specimen is placed for observation.
• It often includes mechanical controls (e.g., knobs or controls) for precise
movement of the specimen in both the X and Y directions.
4. Condenser:
• The condenser is located beneath the stage and helps focus light onto the
specimen.
• It may have adjustable diaphragms to control the amount of light reaching
the specimen, improving contrast and resolution.

20. 5. Illumination System: Microscopes feature various illumination methods, including:
a) Brightfield illumination: The most common method where light passes through the
specimen and is collected by the objective lens.
b) Darkfield illumination: Illumination from the sides, allowing objects to appear bright
against a dark background.
c) Phase contrast and differential interference contrast (DIC): Techniques used for observing
transparent or unstained specimens.
d) Fluorescence illumination: Excites fluorescent molecules in the specimen to produce
fluorescent signals.
e) Light sources can include halogen bulbs, LED lights, or specialized lamps depending on
the illumination technique.

21. 6. Fine and Coarse Focus Adjustments:
• These knobs or controls are used to bring the specimen into sharp focus.
• The coarse adjustment moves the stage up and down rapidly for rough
focusing, while the fine adjustment allows for precise focusing.
7. Base:
• The base provides stability and support for the entire microscope.
8. Body Tube:
• The body tube connects the eyepiece to the objective lenses, maintaining
the correct distance and alignment between them.

22. 9. Diaphragm: The diaphragm is located below the condenser lens and controls
the amount of light that reaches the specimen. It can be adjusted to change the
brightness and contrast of the image.
10. Stage Clips: These are used to hold the specimen slide in place on the stage

23. TYPES OF MICROSCOPE

24. COMPOUND LIGHT MICROSCOPE
Principle: Compound light microscopes use visible light and a series of lenses
to magnify small specimens. They work by passing light through the specimen
and magnifying it with objective and eyepiece lenses.
Applications: Widely used in biology, medicine, education, and research for
viewing cells, tissues, microorganisms, and other small objects. They are
versatile tools for studying biological samples, including live specimens.
Advantages: Relatively simple to use, cost-effective, and suitable for observing
live specimens. They provide high-quality images with sufficient magnification
for most biological studies.
Limitations: Limited resolution compared to electron microscopes, typically up to
around 0.2 micrometres. They are not suitable for observing structures smaller than
the wavelength of visible light

25. BRIGHT FIELD MICROSCOPE
• Bright field Microscope is also known as the Compound Light Microscope.
• It is the simplest of all the optical microscopy illumination techniques.
• Sample illumination is transmitted (i.e., illuminated from below and observed
from above) white light, and contrast in the sample is caused by attenuation Of
the transmitted light in dense areas Of the sample.
• It is an optical microscope that uses light rays to produce a dark image against a
bright background.
• It is used in Biology, Cellular Biology, and Microbiological Laboratory studies.
• This microscope is used to view fixed specimens, that have been stained with
basic stains, gives a contrast between the image and the image background.
• It is specially designed with magnifying glasses known as lenses that modify
the specimen to produce an image seen through the eyepiece.

26. PRINCIPLE
• In Bright field Microscope, the specimen must pass through a uniform beam of the
illuminating light to be the focussed and produce an image.
• The microscope will produce a contrasting image through differential absorption and
differential refraction
• The specimens used are stained to introduce colour for easy contracting
characterization.
• The coloured specimens will have a refractive index that will differentiate it from the
surrounding, presenting a combination of absorption and refractive contrast.
• The microscope function is based on its ability to produce a high-resolution image
from an adequately provided light source, focused on the image, producing a high-
quality image.
• The specimen which is placed on a microscopic slide is viewed under Oil immersion or/and
covered with a cover slip. Oil immersion improve resolution by reducing light scatter.

27. APPLICATIONS
1. Used to understand cell structures in cell Biology, Microbiology,
Bacteriology to visualizing parasitic organisms in Parasitology.
2. Most Of the specimens to viewed are stained using special staining to
enable visualization. Examples: Negative staining and Gram staining.
3. Some of its applications include:
• To visualize and study the animal cells
• To visualize and study plant cells.
• To visualize and study the morphologies of bacterial cells
• TO identify parasitic protozoans such as Paramecium.

28. DARK FIELD MICROSCOPY
• Microscopes are designated as either light microscopes or electron
microscopes.
• Light microscopes use visible light or ultraviolet rays to illuminate specimens.
• This is similar to the ordinary light microscope; however, the condenser system
is modified so that the specimen is not illuminated directly.
• The condenser directs the light obliquely so that the light is deflected or
scattered from the specimen, which then appears bright against a dark
background.
• Living specimens may be observed more readily with dark field than with
brightfield microscopy.

29. PRINCIPLE
❖ In dark field microscope, the light source is blocked Off, causing light to scatter as it
hits the specimen.
❖ This is ideal for making objects with refractive values similar to the background
appear bright against a dark background.
❖ When light hits an Object, rays are scattered in all azimuths Or directions.
❖ The design Of the dark field microscope is such that it removes the dispersed light, or
zeroth order, so that only the scattered beams hit the sample.
❖ The introduction Of a condenser and/or stop below the Stage ensures that these light
rays will hit the specimen at different angles, rather than as a direct light source
above/below the object.
❖ The result is a "cone of light" where rays are diffracted, reflected and/or refracted off
the object, ultimately, allowing the individual to view a specimen in dark field.

30. USES
• Demonstration of very thin bacteria not visible under ordinary illumination since
the reflection of the light makes them appear larger.
• Frequently used for demonstration of Treponema pallidum in clinical specimens.
• Demonstration of the motility of flagellated bacteria and protozoa.
• Dark field is used to study marine organisms such as algae, plankton, diatoms,
insects, fibbers, hairs, yeast and protozoa as well as some minerals and crystals,
thin polymers and some ceramics.
• Used to study mounted cells and tissues.
• Useful in examining external details, such as outlines, edges, grain boundaries
and surface defects than internal structure

31. COMPOUND LIGHT MICROSCOPE

32. STEREO MICROSCOPE
(DISSECTING MICROSCOPE)
Principle: Stereo microscopes provide a three-dimensional view of larger
specimens by using two separate optical paths. They use incident light to illuminate
the specimen from multiple angles, allowing for depth perception.
Applications: Ideal for tasks such as dissecting, examining surfaces, electronics
inspection, material sciences, and other applications where manipulation or
dissection of samples is required.
Advantages: Offer a wide field of view, long working distances, and a large depth
of field. They are well-suited for viewing opaque objects and specimens that require
manipulation.
Limitations: Lower magnification compared to compound microscopes, typically
up to around 100x. Limited resolution compared to electron microscopes.

33. ELECTRON MICROSCOPE:
Principle: Electron microscopes use a beam of electrons instead of light to
illuminate the specimen. They offer much higher resolution than light microscopes
due to the shorter wavelength of electrons.
Types:
1. Transmission Electron Microscope (TEM): allows one the study of the inner
surface
2.Scanning Electron Microscope (SEM):. used to visualize the surface of
objects.
• Three-dimensional imaging

34. ELECTRON MICROSCOPE
The main components of an electron microscope are:
• An electron gun
• Electromagnetic lens system
• Vacuum system
• Camera/detector
• Computer

35. Basic Principles
• The gun consists of an electron source, electrode, Wenhelt assembly and anode.
• A current is run through the filament/crystal to heat it, resulting in the emission
of electrons from the tip. The high voltage difference between the cap and the
anode causes the electrons to accelerate and form a beam
• TEM lenses are electromagnetic, creating precise, circular magnetic fields that
manipulate the electron beam, much the same way that optical lenses focus and
direct light.
• similarly to optical lenses, electromagnetic lenses are also susceptible to
aberrations

36. ELECTRON MICROSCOPE:
Applications: Used in materials science, nanotechnology, biology, and other
fields where high-resolution imaging is necessary. TEM is suitable for studying
internal structures, while SEM is ideal for surface imaging.
Advantages: Exceptional resolution, capable of imaging structures at the atomic
level. Can achieve magnifications exceeding 1,000,000x.
Limitations: Requires extensive sample preparation, including dehydration and
coating with conductive materials. Expensive equipment and specialized training
are needed for operation and maintenance.

37. TRANSMISSION ELECTRON
MICROSCOPE (TEM)

38. TRANSMISSION ELECTRON
MICROSCOPE (TEM)
• TEM is a microscopy technique where a beam of electrons is transmitted through
an ultra thin specimen.
• An image is formed from the interaction Of the electrons transmitted through the
specimen;
• The image is magnified and focused onto an imaging device, such as a
fluorescent screen, on a layer of photographic film, or to be detected by a sensor
such as a CCD camera.
.

39. HOW IT WORKS
• The condenser lens system focuses the emitted electrons into a coherent beam.
• The first condenser controls the spot size of the beam. This is controlled by the spot size
setting in the TEM software.
• The second condenser focuses the beam onto the sample (this is controlled by the
‘brightness’ knob on the microscope). The condenser aperture restricts the beam by
excluding high angle electrons. Usually a middle sized condenser aperture is suitable.
• The objective lens focuses the electrons transmitted through the sample into a magnified
image.

40. • The objective aperture can be used to increase contrast by excluding high angle
transmitted electrons.
• The intermediate and projection lenses enlarge the image. When the electrons hit
the phosphorescent screen, it generates light which allows the human eye to view
it.
• Images can be acquired using a high resolution

41. SAMPLE PREPARATION FOR TEM

42. SAMPLE PREPARATION FOR
ELECTRON MICROSCOPY
• TEM specimens must be:
• Very thin ,Well preserved ,Electron dense and Stable in the vacuum
• The degree of specimen preparation for biological TEM depends on the
specimen
• Particulate samples (eg: protein and viruses) can be stained and viewed quickly
• Cells and tissue samples require extensive preparation for TEM

43. SAMPLE PREPARATION FOR ELECTRON
MICROSCOPY
1. DEHYDRATION : The wet sample is dehydrated by keeping in increasing
concentration of ethanol or acetone
2. FIXATION
❖Fixation is done by immersing the specimen in chemical preservatives
called fixative.
❖Osmium tetroxide, glutaraldehyde, potassium permanganate, formalin, etc.
are common fixatives.
❖These fixatives form covalent bond with biological molecules like proteins
and lipids.
❖They stabilize the structural organization in the specimen.

44. • Fixation stops cellular processes and
aims to preserve the specimen as close as
possible to its natural state.
Characteristics of a good fixative:
• Permeates cells readily and acts quickly
• Is irreversible
• Does not cause fixation artifacts
Methods of fixation include:
• Chemical fixation with aldehydes
• Cryo-fixation with liquid nitrogen
• Microwave fixation

45. • Tissue can be cry-fixed using LN2 in the High Pressure Freezer and then further processed for
TEM (adds 1 week)
• Specimens are mounted into specimen carriers and cryo-fixed with LN2 under high pressure
(~2000 bar) to prevent damaging ice crystal formation up to 200 μm into the tissue
• Samples are then carefully transferred to the AFS and freeze-substituted with solvent (+
osmium and/or glutaraldehyde or uranyl acetate) at sub-zero temperatures.
• Cons of cryofixation: time consuming, finicky and restrictions on sample size, possible ice
crystal issues
• Pros of cryo-fixation: best possible ultrastructural preservation, maintains fluorescence and
antigenicity

46. secondary fixation
• Osmium tetroxide (very toxic!) is a heavy metal that fixes
unstaturated lipids and is also electron dense.
• Used as both a secondary fixative and an electron stain and
significantly improves specimen preservation (especially
membranes) and contrast.
Microwave processed liver tissue, E Johnson

47. • Dehydration is the process of gradually replacing water in the sample
with a solvent (usually acetone or ethanol).
• The solvent is then gradually replaced with resin. This process can
be lengthy and depends on both the sample and type of resin used.
Resin blocks Poor resin infiltration

48. EMBEDDING
• The specimen is embedded in a hard embedding medium like
araldite vestopaI-W or Epson-812 or plastic medium
• Thickness using a glass or diamond knife fixed in an
ultramicrotome, The thin section is mounted on a copper grid Of
3mm diameter and covered With parlodoan-

49. Leica Ultracut 7 ultramicrotome , Dunn School Introduction to ultramicrotomy video, University of Sydney

50. NEGATIVE STAINING
• Contrast can be increased by post-staining sections with salts of heavy metals,
specifically uranyl acetate and lead citrate solutions.
• Uranyl acetate stains protein and DNA and also acts as a mordant for lead citrate,
which is a more general stain.
• Coat grids with plastic film and carbon
• Apply the particulate specimen
• Stain with heavy metal solution, eg: uranyl acetate, phosphotungstic acid,
sodium silica tungstate
• Blot dry and view in the TEM

51. SCANNING ELECTRON
MICROSCOPE (SEM)

52. SAMPLE PREPARATION FOR SEM
• well preserved with no surface contamination or damage
• Stable in the vacuum ,Conductive , Composed of high atomic number elements
• The conventional preparation for SEM samples is similar to that for TEM, although the
resin and sectioning steps are omitted.
• There are less size restrictions on SEM samples compared to TEM.
• It is used for studying surfaces and revealing surface morphology at nanoscale resolutions
• This microscopy technique involves using a focused beam of electrons to scan across the
surface of a specimen

54. • Once the dehydration series is complete, the solvent itself must be removed from the tissue
without introducing surface tension/drying artifacts into your sample. This is achieved through
the use of a transitional fluid, most commonly hexamethyl disilazane (HMDS) or liquid CO2.
Air drying is not recommended, as ethanol evaporation generally causes severe surface tension
artifacts.
• Liquid CO2 can be used to flush the solvent from tissue using a technique called Critical
Point Drying (CPD).
• if a biological specimen is not mounted and coated correctly, it will react to the electron beam
(an effect called charging), resulting in sample damage and/or image distortion.
• Mounting immobilizes the sample on a conductive backing, grounding it. Ensure that your
sample is in full contact with the conductive backing; if not, use conductive glue (eg: carbon
and silver) to ensure conductive continuity.

55. • Sputter coating with metal ions deposits a thin continuous conductive layer over the sample,
such that charge from the electron beam flows to the ground and does not build up on the
sample.
• Sputter coating also increases the SE signal (and therefore contrast), high Z elements have a
higher yield of SEs than low Z elements (biological material!).
• Variable pressure and environmental SEM (ESEM) allows untreated, hydrated specimens to
be imaged at high resolution.
• Utilises a specialised detector and vacuum system that enables imaging under low pressure
conditions (ie; not a vacuum!).

56. CONFOCAL MICROSCOPE:
➢An optical imaging technique for increasing optical resolution and contrast of a
micrograph.
➢Radiations emitted from laser cause sample to fluoresce
➢uses pinhole screen to produce high resolution images.
➢Eliminates out of focus.
➢So images have better contrast and are less hazy.
➢A series of thin slices of the specimen are assembled to generate a 3 dimensional
image.
➢Is an updated version of fluorescence microscopy.

57. ❖ In confocal microscopy two pinholes are typically used:
❖ A pinhole is placed in front of the illumination source to allow transmission
only through a small area
❖ This illumination pinhole is imaged onto the focal plane of the specimen, i.e.
only a point of the specimen is illuminated at one time.
❖ Fluorescence excited in this manner at the focal plane is imaged onto a
confocal pinhole placed right in front of the detector
❖ Only fluorescence excited within the focal plane of the specimen will go
through the detector pinhole.
❖ Scanning of small sections is done and joined them together for better view.

58. WORKING MECHANISM
• Confocal microscope incorporates 2 ideas:
1. Point-by-point illumination of the specimen.
2. Rejection of out of focus of light.

59. • Light source of very high intensity is used—zirconium arc lamp in Minsky's
design & laser light source in modern design.
a)Laser provides intense blue excitation light.
b)The light reflects off a dichroic mirror, which directs it to an assembly of
vertically and horizontally scanning mirrors.
c)These motor driven mirrors scan the laser beam across the specimen.
d) The specimen is scanned by moving the stage back & forth in the vertical &
horizontal directions and optics are kept stationary.

60. Dye in the specimen is excited by the laser light & fluoresces.
• The fluorescent (green) light is descanted by the same mirrors that are used
to scan the excitation (blue) light from the laser beam then it passes through the
dichroic mirror then it is focused on to pinhole.
• the light passing through the pinhole is measured by the detector such as
photomultiplier tube.
• For visualization, detector is attached to the computer, which builds up the
image at the rate of 0.1-1 second for single image

61. ADVANTAGES
The specimen is everywhere illuminated axially, rather than at different angles,
thereby avoiding optical aberrations.
Entire field of view is illuminated uniformly.
The field of view can be made larger than that of the static objective by
controlling the amplitude of the stage movements.
Image formed are of better resolution.
Cells can be live or fixed.
Serial optical sections can be collected.
Taking a series of optical slices from different focus levels in the specimen
generates a 3D data set.

62. DRAWBACKS
• Resolution : It has inherent resolution limitation due to diffraction. Maximum
best resolution of confocal microscopy is typically about 200nm.
• Pin hole size : Strength of optical sectioning depends on the size of the pinhole.
• Intensity of the incident light Fluorophores :
a)The fluorophore should tag the correct part of the specimen.
b)Fluorophore should be sensitive enough for the given excitation wave length.
c) lt should not significantly alter the dynamics of the organism in the living
specimen.
• Photobleaching: photochemical alteration of a dye or a fluorophore molecule
such that it permanently is unable to fluoresce

63. CONFOCAL MICROSCOPE:

64. PHASE-CONTRAST MICROSCOPE
• The phase contrast microscope is a light microscope.
• It is modification of compound microscope.
• It contains all the component of a compound microscope in addition to an
annular ring and a phase plate.
• It magnifies not only object but also changes in brightness.
• Phase -contrast microscope enhance contrast in transparent specimens By
exploiting differences in refractive index
.
.

65. Principle
❑ The phase contrast microscope separates the illuminating background light
and the specimen scattering light.
❑ Phase contrast microscope is used to visualize transparent, colourless,
unstained, living biological specimens. These objects are called phase objects.
❑ Light is bend (diffracted) and retarded based on the refractive index of the
object.
❑ Highly refractive structures bend and retard light much.
❑ This principle is used in phase contrast microscope.

66. PHASE-CONTRAST MICROSCOPE
Principle: Phase-contrast microscopes enhance the contrast of transparent and
colourless specimens by exploiting differences in refractive index. They convert
phase differences in light passing through the specimen into brightness
variations.
Advantages: Allows for the visualization of live, unstained specimens without
the need for special preparation techniques such as fixing and staining. Suitable
for observing cellular structures and dynamics.
Applications: Commonly used in cell biology, microbiology, and other fields
where live-cell imaging is required, such as observing cell division and motility.
Limitations: Limited to transparent specimens with subtle density variations.
Lower resolution compared to electron microscopes

68. FLUORESCENCE MICROSCOPE
Principle: Fluorescence microscopes excite fluorescent dyes in the specimen with
specific wavelengths of light, producing fluorescent emissions that are captured to
create an image.
Advantages: Enables the visualization of specific molecules or structures labelled
with fluorescent markers, allowing for precise localization and tracking within cells
and tissues. It is commonly used for observing living cells in real-time.
Applications: Widely used in cell biology, immunology, genetics, and other fields
for visualizing specific molecules and structures within cells and tissues, such as
studying protein localization and dynamics.
Limitations: Requires fluorescent labelling of the specimen, which can affect
sample integrity and may introduce artifacts. Limited to specimens with suitable
fluorescent markers.

69. ATOMIC FORCE MICROSCOPY
• AFM is a type of scanning probe microscopy(SPM), with demonstrated
resolution on the order of fractions of a nanometre, more than 1000 times better
than the optical diffraction limit.
• The information is gathered by "feeling" or "touching" the surface with a
mechanical probe
• AFM provides a 3D profile of the surface on a nanoscale, by measuring forces
between a sharp probe and the surface.
• The AFM has three major abilities: force measurement, imaging, and
manipulation.
• It is powerful because an AFM can generate images at atomic resolution with
angstrom scale resolution height information, with minimum sample
preparation.

70. • The main principle behind atomic force microscopy is measuring the forces between a
sharp probe tip and a sample surface.
• Mode of operation in atomic force microscopy are Contact Mode , Tapping Mode and
Non-contact Mode
• The typical range of resolution achievable with atomic force microscopy is Sub-
nanometre.
• The material is commonly used for the probe tip in atomic force microscopy is Silicon.
• The primary advantage of dynamic or tapping mode AFM over contact mode AFM is
to reduce sample damage.
• "force curve" refer to the curve representing the force between the tip and the sample as
a function of distance

72. APPLICATION
❖ The AFM has been applied to problems in a wide range of disciplines of the natural sciences,
including solid-state physics, semiconductor science and technology, molecular engineering,
polymer chemistry and physics, surface molecular-biology, cell-biology, and medicine.
❖ It gives information about the toughness, roughness and smoothness value of surface.
❖ Applications in the field of solid state physics include (a) the identification of atoms at a
surface, (b) the evaluation of interactions between a specific atom and its neighbouring atoms.
❖ In molecular biology, AFM can be used to study the structure and mechanical properties of
protein complexes and assemblies. For example, AFM has been used to image microtubules
and measure their stiffness.
❖ In cellular biology, AFM can be used to attempt to distinguish cancer cells and normal cells
based on a hardness of cells, and to evaluate interactions between a specific cell and its neigh
boring cells in a competitive culture system.
❖ Soft surfaces are analysed by this technique without damaging it like Lipids.
❖ Covalent bond strength is measured by this Technique

73. LIMITATIONS OF AFM
• Slow Imaging Speed: AFM typically operates at slower imaging speeds
compared to other microscopy techniques like scanning electron microscopy
(SEM) or optical microscopy.
• Complexity of Operation: Operating an AFM requires a certain level of
expertise and training due to its complex setup and delicate probe-sample
interactions. This can result in longer learning curves for users new to the
technique.
• Surface Sensitivity: AFM is highly sensitive to surface conditions, including
sample preparation, cleanliness, and even atmospheric conditions. Variations in
these factors can affect imaging results and introduce artifacts.
• Probe Wear and Damage: The sharp probe tip used in AFM can experience
wear or damage during imaging, especially in contact mode AFM where the tip
physically interacts with the sample surface. This can lead to decreased imaging
quality and increased experimental variability over time.

74. LIMITATIONS OF AFM
• Limited Imaging Range: The imaging range of AFM is typically limited to the
micrometer scale, which may not be suitable for studying larger samples or
structures. Additionally, AFM may struggle with imaging samples that have
significant height variations or roughness beyond its range.
• Sample Compatibility: AFM is not always compatible with all types of samples
or environments. For example, samples that are soft, sticky, or highly mobile may
pose challenges for AFM imaging, and certain environmental conditions (such as
high humidity) can affect imaging results.
• Cost: While the cost of basic AFM systems has decreased over the years, high-
resolution and specialized AFM setups can still be relatively expensive, making
them less accessible for some researchers or laboratories.

75. REFERENCES
• Text Book Of Microbiology By Surinder Kumar
• Prescott’s Microbiology , 10th Edition , Willey Sherwood Woolverton
• Microbiology 5th Edition Lansing M. Prescott.
• Essential Microbiology By Stuart Hogg
• Laboratory Exercises In Microbiology 5th Edition, Harley
• Principle and Techniques of Biochemistry and Molecular Biology. Keith Wilson and John Walker (Eds). 6th Edition.
Cambridge University Press.
• Physical Biochemistry (Application to Biochemistry and Molecular Biology).David Frei Felder (Ed.) WH Freeman and
Company, San Francisco.
• Parija S.C. (2012). Textbook Of Microbiology ed.), India: Elsevier India.
• Cappuccino, J. and Welsh. C. (2014). Microbiology: A Manual, Global Edition. 1st Pearson Education.
• Sastry A.S. & Bhat SK. (2016). Essentials of Medical Microbiology. New Delhi : Jaypee Brothers Medical pub I IS hers.
• Trivedi P.C., Pandey S, and Bhaduria S. (2010). Textbook Of Microbiology. Pointer Publishers; First edition
• Patskovsky; et al. (2014). "Wide-field hyperspectral 3D imaging. of functionalized gold nanoparticles targeting cancer cells by
reflected light microscopy". Bioptomes. 8