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MICROSCOPY

CONTENT

• INTRODUCTION• DEFINITION• HISTORICAL BACKGROUND• VARIBLES USED IN MICROSCOPY• COMPOUND MICROSCOPE - Structure

and Function• USE OF MICROSCOPE• VARIOUS TYPES OF MICROSCOPES• CARE OF MICROSCOPE 2

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INTRODUCTION TO MICROSCOPY

• Understanding the optical principles and construction of microscopes

• Role of microscopy• Microscopic

techniques and application

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DEFINITION

• A microscope (Greek: micron = small and scopos = aim)

• MICROSCOPE - An instrument for viewing objects that are too small to be seen by the naked or unaided eye

• MICROSCOPY - The science of investigating small objects using such an instrument is called microscopy

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HISTORICAL BACKGROUND

• 1590 - Hans Janssen and his son Zacharias Janssen, developed first microscope.

• 1609 - Galileo Galilei - occhiolino or compound microscope.

• 1620 - Christian Huygens, another Dutchman, developed a simple 2-lens ocular system that was chromatically corrected.

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Anton van Leeuwenhoek(1632-1723)

• Anton van Leeuwenhoek is generally credited with bringing the microscope to the attention of biologists.

• A tradesman of Delft, Holland.

1661 - He discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells, microscopic nematodes etc.

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Microscope used by Anton von Leeuwenhoek

An old pocket Microscope

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VARIABLES USED IN

MICROSCOPY

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MAGNIFICATION

• Degree of enlargement• No of times the length, breadth or

diameter, of an object is multiplied.

MAGNIFICATION VS SHARPNESS

• USEFUL MAGNIFICATION AND EMPTY MAGNIFICATION

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• RESOLUTION – Ability to reveal closely adjacent structural details as separate and distinct

• LIMIT OF RESOLUTION (LR) – The min distance between two visible bodies at which they can be seen as separate and not in contact with each other

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ABERRATION

1. Chromatic aberration • Correction of aberration – Achromatic

objective and Apochromatic objectives.

Blue focusRed focus

Incident light

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2. Spherical aberration

Focus of marginal rays

Focus of axial rays

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TYPES OF MICROSCOPE• Simple microscope• Compound microscope• Phase Contrast

Microscope• Dark Ground Microscope• Fluorescent Microscope• Electron Microscope• Others

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COMPOUND MICROSCOPE

Compound microscope made by John Cuff 1750

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PARTS OF COMPOUND MICROSCOPE

• Ocular (Eye piece)   • Body or Tube   • Coarse focusing knob• Fine focusing knob   • Objective  Lens • Movable stage   • Condenser Lenses   • Field (Iris)

Diaphragm   • Mirror and light

source 

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OBJECTIVE LENS

• It forms magnified real image.

• Mounted on Nose piece

• Magnification of objective

= Optical Tube length Focal Length

• Scan - 4X

• Low Power - 10X

• High Power - 40X

• Oil immersion - 100X

TYPES

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EYE PIECE

• Forms magnified virtual & erect image

• TYPES (a) Monocular(b) Binocular(c) Trinocular

or(a) Huygenian(b) Ramsden(c) Compensating

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PARTS OF MICROSCOPE (Cont)

Iris Diaphragm

Condenser

Field Diaphragm

Diaphragm control ring

Focusing knob

illumination source

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OPTICAL PATH IN COMPOUND MICROSCOPE

Compound Microscope

• The Compound Microscope is the type of microscope we use in the lab. It uses visible light as it’s light source for illumination.

• The average compound microscope has resolving power of .2m (1m = .000001m)

• The resolving power is the ability of the microscope to distinguish between 2 pts, or to show detail.

• Staining is used to make the specimen contrast sharply with the medium so that it is easier to see. 20

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FLUORESCENCE MICROSCOPY• PRINCIPLE

UV light

Fluorochrome

Visibleradiation

FITC EX - 495 nm EM - 520nm

TRITC EX – 540 nm EM – 590 nm

Texas Red Ex – 600 nm EM – 615 nm

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Fluorescence microscopy(cont.)

• This particular microscope uses fluorochromes to attach to organisms and allow them to fluoresce.

• Fluorochromes are small molecules that only fluoresce once they have attached to their target.

• Immunofluorescence is a particular type of marker used in fluorescence microscopy.

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Fluorescence microscopy(cont.)

• UV rays passes through exciter filter

• Dark ground condenser

• Micro organisms stained with fluorescent dye, when examined under microscope with ultraviolet light are seen as bright object against dark background

Fluorescence microscopy(cont.)

• The fluorochromes are first attached to a specific antibody (Ab). The antibody is usually specific for a particular type of cell, virus, or other type of specimen.

• This type of microscopy is useful for detecting viruses and other org. within cells, tissue, or other clinic specimen that normally we wouldn’t be able to see or detect otherwise

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Uses Of Fluorescence microscopy

•Establish the intracellular distribution of a protein

•Characterize the dynamics of the protein distribution

•Suggest potential functions of the protein

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The advantages of fluorescence microscopy

1.High contrast

2.High specificity

3.Subcellular resolution

4.Multiple parameters

5.Vital microscopy –physiological studies

6.Quantitative analyses

7.3-dimensional analyses–Bio-Rad Microsciences, Inc.

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PHASE CONTRAST MICROSCOPY

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PRINCIPLE OF PHASE CONTRAST MICROSCOPY

• Phase contrast microscope employs an optical mechanism to translate minute variations in phase into corresponding changes in intensity of image.

• Unstained micro-organisms (bacteria) have constituents of different refractive index .

• Diffraction of light

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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

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IMAGES OF PHASE CONTRAST MICROSCPY

Clostridium botulinumSpirilium volutans

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COMPARISION OF IMAGES OF BRIGHT FIELD AND PHASE CONTRAST

MICROSCOPY

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USES OF PHASE CONTRAST MICROSCOPY

• Phase contrast enables visualization of internal cellular components.

• Diagnosis of tumor cells .• Examination of growth, dynamics, and

behavior of a wide variety of living cells in cell culture

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DARK GROUND MICROSCOPY

• The microscopes that require a dark background in order to bring out the finer details of the specimen is called dark field microscope.

• Optical system to enhance the contrast of unstained bodies .

• Specimen appears gleaming bright against dark background

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USES OF DARK GROUND MICROSCOPY

Treponema pallidum

• Useful in demonstrating

-Treponema pallidum

- Leptospira

- Campylobacter jejuni

- Endospore

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ELECTRON MICROSCOPE

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ELECTRON MICROSCOPE

• Electron Microscopes uses a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the info about– Topography– Morphology– Composition– Crystallographic structure

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TYPES OF EM

• Transmission Electron Microscope (TEM)

• Scanning Electron Microscope (SEM)

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TRANSMISSION ELECTRON MICROSCOPE (TEM)

• Stream of electrons is formed

• Accelerated using a positive electrical potential

• Focused by metallic aperture and Electro magnets

• Interactions occur inside the irradiated sample which are detected and transformed into an image .

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TEM (Cont)

• Projector Lens forms image on Fluorescent viewing screen

• 2D Image

• Magnification

10,000 X to 100,000 X

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• Scan a gold-plated specimen to give a 3-D view of the surface of an object which is black and white.

• Used to study surface features of cells and viruses.

• Scanning Electron microscope has resolution 1000 times better than Light microscope .

SCANNING ELECTRON MICROSCOPE

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SEM IMAGES

Vibrio cholerae with polar flagellaTreponema pallidum

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INVERTED MICROSCOPE

• Used in metallurgy• Examination of cultures in flat bottom

dishes• Micro dissection • Examination of parasites• Observation of agglutination in serology

OTHER MICROSCOPES

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STEREO MICROSCOPE

• Double Microscope• Produces 3D images

POLARIZING MICROSCOPE

• Uses two Polariser

• Gives info about Birefringence of a body

• Used in Crystallography, Urine examination

• Apple Green Birefringerence in AMYLODOSIS

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• Uses a laser beam to illuminate a specimen whose image is then digitally enhanced for viewing on a computer monitor.

• Laser beam scans single plane of 1µm thickness.

CONFOCAL SCANNING LASER MICROSCOPE

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Comparison of Depth of Light Collection and Image clarity

Light Microscope Confocal Scanning Laser Microscope

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PRINCIPLE OF CONFOCAL MICROSCOPY

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USES OF CONFOCAL MICROSCOPE

• Observing cellular morphology in multilayered specimen

• Eg. used in diagnosing Ca cervix• Evaluation and diagnosis of basal cell

carcinoma of skin

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NEWER MICROSCOPE

• SCANNING PROBE MICROSCOPE -Class of Microscope that measures surface features by moving a sharp probe over object surface. Used to visualize atoms and molecules

– Scanning Tunneling Microscope (STM)

– Atomic Force Microscope (AFM)

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