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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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