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MICROSCOPYA Brief Overview

Dr Saleh M.Y.

Practical Lab Experiment -1-

MBBS-Phase II

19/10/2010

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- Introduction

- Definition

- Historical Background

- Varibles Used In Microscopy

- Compound Microscope - Structure And Function

- Use Of Microscope

- Various Types Of Microscopes

- Care Of Microscope

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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 = to look)

- MICROSCOPE: Is 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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Antony 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 enlargementDegree of enlargement

No of times the length, breadth or diameter, No of times the length, breadth or diameter, of an object is multiplied.of an object is multiplied.

MAGNIFICATION VS SHARPNESS

USEFUL MAGNIFICATION AND EMPTY USEFUL MAGNIFICATION AND EMPTY MAGNIFICATIONMAGNIFICATION

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RESOLUTION: Ability to reveal closely : Ability to reveal closely adjacent structural details as separate and adjacent structural details as separate and distinctdistinct

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

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Types of microscope Resolving power

Compound Microscope 200 nanometers

Scanning Electron Microscope 10 nanometers

Transmission Electron Microscope 0.2 nanometers

LR = 0.61 x W W = Wavelength

NA = Num apertureNA

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NUMERICAL APERTURE(NA)

- Ratio of diameter of lens to its focal length- Ratio of diameter of lens to its focal length

- NA = n Sin - NA = n Sin θθ/2/2

n = refractive index,n = refractive index,

θθ = angle of aperture (CAD) = angle of aperture (CAD)

θ/2

A

BDC

n of air = 1

n of oil = 1.5

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DEFINITION

- Capacity of an objective to render outline - Capacity of an objective to render outline of the image of an object clear and distinctof the image of an object clear and distinct

- Depends of elimination of Spherical and - Depends of elimination of Spherical and Chromatic aberrationChromatic aberration

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ABERRATION

- Chromatic aberration - Chromatic aberration

- Correction of aberration – Achromatic - Correction of aberration – Achromatic objective and Apochromatic objectives.objective and Apochromatic objectives.

Blue focusRed focus

Incident light

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Spherical aberration

Focus of marginal rays

Focus of axial rays

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TYPES OF MICROSCOPE

- Simple microscope- Simple microscope

- Compound microscope- Compound microscope

- Phase Contrast - Phase Contrast MicroscopeMicroscope

- Dark Ground Microscope- Dark Ground Microscope

- Fluorescent Microscope- Fluorescent Microscope

- Electron Microscope- Electron Microscope

- Others- 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)   - Ocular (Eye piece)   - Body or Tube   - Body or Tube   - Coarse focusing knob- Coarse focusing knob- Fine focusing knob   - Fine focusing knob   - Objective  Lens - Objective  Lens - Movable stage   - Movable stage   - Condenser Lenses   - Condenser Lenses   - Field (Iris) Diaphragm   - Field (Iris) Diaphragm   - Mirror and light source  - Mirror and light source 

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OBJECTIVE LENS

It forms magnified real image.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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OIL IMMERSION OBJECTIVE

- Highest magnification- Highest magnification

- Oil prevents refraction of light outwards and - Oil prevents refraction of light outwards and allows it to pass straight in to objectiveallows it to pass straight in to objective

GLASS

OIL

AB

C

D

E

G

F

FBEG - OIL ABCD - AIR

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EYE PIECE

- Forms magnified virtual & erect - Forms magnified virtual & erect imageimage

- TYPES - TYPES (a)(a) MonocularMonocular(b)(b) BinocularBinocular(c)(c) TrinocularTrinocular

oror(a)(a) HuygenianHuygenian(b)(b) RamsdenRamsden(c)(c) CompensatingCompensating

What’s the power of this lens?

To calculate the power of magnification, multiply the power of the ocular lens by the power of the objective, e..g.: 10x40=400 times

What are the powers of magnification for each

of the objectives we have on our microscopes?

Fill in the table on your worksheet.

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Comparing Powers of Magnification

Which of these images would be viewed at a

higher power of magnification?

We can see better details with higher the powers of magnification, but we cannot see as much of the image.

10x 40x

Let’s give it a try ...1 – Turn on the microscope and then rotate the nosepiece to click the red-banded objective into place.

2 – Place a slide on the stage and secure it using the stage clips. Use the coarse adjustment knob (large knob) to get it the image into view and then use the fine adjustment knob (small knob) to make it clearer.

4 – When you are done, turn off the microscope and put up the slides you used.

3 – Once you have the image in view, rotate the nosepiece to view it under different powers. Draw what you see on your worksheet!

Be careful with the largest objective! Sometimes there is not enough room and you will not be able to use it!

How to make a wet-mount slide …

1 – Get a clean slide and coverslip from your teacher.

2 – Place ONE drop of water in the middle of the slide. Don’t use too much or the water will run off the edge and make a mess!

3 – Place the edge of the cover slip on one side of the water drop.

You do not need to use the stage clips when viewing wet-mount slides!

5 – Place the slide on the stage and view it first with the red-banded objective. Once you see the image, you can rotate the nosepiece to view the slide with the different objectives.

4 - Slowly lower the cover slip on top of the drop.

Cover Slip

Lower slowly

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ILLUMINATION - Lamp, sunlight, battery operated lamp, 60 W bulb, Quartz halogen light.

FILTERS - Blue, Green, Heat absorbing filters, Barrier filters.

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Multiple step operation employed to attain Multiple step operation employed to attain optimal illumination:optimal illumination:

1.1. Remove any diffusing filter. Remove any diffusing filter. 2.2. Put a slide on the stage and Put a slide on the stage and focus. focus. 3.3. Completely close the field Completely close the field diaphragm. diaphragm. 4.4. Move the condenser until the Move the condenser until the border border or the iris hexagon is neat and or the iris hexagon is neat and clear. clear. 5.5. Center if necessary. Center if necessary. 6.6. Open the field diaphragm until Open the field diaphragm until the tip the tip of the hexagon touches the field of the hexagon touches the field limitlimit

KOHLER’S ILLUMINATION

The Parts of a Microscope

Body Tube

Objective

Lenses

Stage

Clips

Diaphragm

Light Source

Ocular Lens

Arm

Stage

Coarse Adj

Fine Adjustment

Base

Nose Piece

The Parts of a Microscope

Body Tube

The body tube holds the objective lenses The body tube holds the objective lenses and the ocular lens at the proper distanceand the ocular lens at the proper distance

Diagram

Nose Piece

The Nose Piece holds the objective lenses The Nose Piece holds the objective lenses and can be turned to increase the and can be turned to increase the magnificationmagnification

Diagram

Objective Lenses

The Objective Lenses increase The Objective Lenses increase magnification (usually from 10x to 40x)magnification (usually from 10x to 40x)

Diagram

Stage Clips

These 2 clips hold the slide/specimen in These 2 clips hold the slide/specimen in place on the stage.place on the stage.

Diagram

Diaphragm

The Diaphragm controls the amount of light The Diaphragm controls the amount of light on the slide/specimenon the slide/specimen

Turn to let more light in or tomake dimmer.

Diagram

Light Source

Projects light upwards through the Projects light upwards through the diaphragm, the specimen and the lensesdiaphragm, the specimen and the lenses

Some have lights, others have mirrors Some have lights, others have mirrors where you must move the mirror to reflect where you must move the mirror to reflect lightlight

Diagram

Ocular Lens/Eyepiece

Magnifies the specimen imageMagnifies the specimen image

Diagram

Arm

Used to support the microscope when Used to support the microscope when carried. Holds the body tube, nose piece carried. Holds the body tube, nose piece and objective lensesand objective lenses

Diagram

Stage

Supports the slide/specimen Supports the slide/specimen

Diagram

Coarse Adjustment Knob

Moves the stage up and down (quickly) for Moves the stage up and down (quickly) for focusing your imagefocusing your image

Diagram

Fine Adjustment Knob

This knob moves the stage SLIGHTLY to This knob moves the stage SLIGHTLY to sharpen the imagesharpen the image

Diagram

Base

Supports the microscopeSupports the microscope

Diagram

MagnificationTo determine your magnification…you To determine your magnification…you

just multiply the ocular lens by the just multiply the ocular lens by the objective lensobjective lens

Ocular 10x Objective 40x:10 x 40 = 400Ocular 10x Objective 40x:10 x 40 = 400

Objective Lens have their magnificationwritten on them.

Ocular lenses usually magnifies by 10x

So the object is 400 times “larger”

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HOW A MICROSCOPE WORKS ?

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OPTICAL PATH IN COMPOUND MICROSCOPE

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Method of using Compound Microscope

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1.1. Grasp the microscopes Grasp the microscopes armarm with with one hand and place your other one hand and place your other hand under the hand under the basebase. .

2. Place the microscope on a bench. 2. Place the microscope on a bench. Adjust seatAdjust seat

3. Clean Lenses.3. Clean Lenses.

4. Turn the coarse adjustment knob 4. Turn the coarse adjustment knob to raise the body tubeto raise the body tube

armarm

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5. Revolve the nose piece to set low-5. Revolve the nose piece to set low-power objective lens.power objective lens.

6. Adjust the Condenser lenses and 6. Adjust the Condenser lenses and diaphragm . diaphragm .

7. Place a slide on the stage and secure 7. Place a slide on the stage and secure with stage clips.with stage clips.

8. Switch on the light at low intensity and 8. Switch on the light at low intensity and then increase intensity.then increase intensity.

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9. Turn the coarse 9. Turn the coarse adjustment knob to lower adjustment knob to lower the body tube until the low the body tube until the low power objective reaches power objective reaches its lowest point. its lowest point.

10. Looking through the eyepiece, very slowly move the coarse adjustment knob until the specimen comes into focus.

11. Adjust distance between eye piece.

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12. Switch to the high power objective lens only 12. Switch to the high power objective lens only after adjusting condenser and iris diaphragm.after adjusting condenser and iris diaphragm.

13. Place a drop of oil over specimen before using 13. Place a drop of oil over specimen before using oil immersion objective. oil immersion objective.

14. Lower the objective until oil makes contact with 14. Lower the objective until oil makes contact with objective.objective.

15. Looking through the eyepiece, very slowly 15. Looking through the eyepiece, very slowly focus the objective away from the slide i.e by focus the objective away from the slide i.e by raising the objective lens. raising the objective lens.

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How to observe a slide ?

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Causes of Error in Focusing

- Revolving Nose Piece is off centre- Revolving Nose Piece is off centre

- Preparation is upside down- Preparation is upside down

- Thick cover slip- Thick cover slip

- Dirt or Dried oil over Lens- Dirt or Dried oil over Lens

- Air bubble in immersion oil- Air bubble in immersion oil

- Poor illumination – Condenser not fully - Poor illumination – Condenser not fully racked upracked up

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PHASE CONTRAST MICROSCOPE

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Phase Contrast Microscope

- First described in 1934 by Dutch physicist - First described in 1934 by Dutch physicist Frits ZernikeFrits Zernike

- Produces high-contrast images of - Produces high-contrast images of transparent specimens transparent specimens

- Advantage - Living cells can be examined - Advantage - Living cells can be examined in their natural state in their natural state

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Principle Of Phase Contrast Microscopy

- Unstained bacteria have constituents of - Unstained bacteria have constituents of different refractive index .different refractive index .

- Diffraction of light- Diffraction of light

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

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Requisite For Phase Contrast Microscopy

- Annular Diaphragm

- Phase Plate

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Condenser Annulus

- The - The condenser annuluscondenser annulus or or annular annular diaphragmdiaphragm is opaque flat-black (light is opaque flat-black (light absorbing) plate with a transparent absorbing) plate with a transparent annular ring. annular ring.

- Produces hollow cone of light. - Produces hollow cone of light.

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Condenser annulus

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Phase Plate

- Placed in back focal plane of objective. - Placed in back focal plane of objective. - Function:

1. Enhances phase difference by retarding diffracted wave front by one quarter

of wavelength .2. Reduces intensity of direct rays and equalizes it with diffracted rays intensity.

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Phase plate

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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 - Phase contrast enables visualization of internal cellular components.internal cellular components.

- Diagnosis of tumor cells .- Diagnosis of tumor cells .

- Examination of growth, dynamics, and - Examination of growth, dynamics, and behavior of a wide variety of living cells in behavior of a wide variety of living cells in cell culture cell culture

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Dark Ground Microscope

- Optical system to enhance the contrast of - Optical system to enhance the contrast of unstained bodies .unstained bodies .

- Specimen appears gleaming bright against - Specimen appears gleaming bright against dark background dark background

PRINCIPLE OF DGI

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Optical path in Dark Ground Microscopy

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Requisites for Dark Ground Microscopy

- Dark ground - Dark ground condenser condenser

- High intensity - High intensity lamplamp

- Funnel stop- Funnel stop

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Uses of Dark Ground Microscopy

Treponema pallidum

Useful in demonstrating

-Treponema pallidum

- Leptospira

- Campylobacter jejuni

- Endospore

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Fluorescence MicroscopyPRINCIPLE

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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- UV rays passes through exciter filter- UV rays passes through exciter filter

- Dark ground condenser- Dark ground condenser

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

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Use of Fluorescence Microscopy

- Auramine Rhodamine – Yellow - Auramine Rhodamine – Yellow Fl - Tubercle bacilliFl - Tubercle bacilli

- Acridine Orange R - gives - Acridine Orange R - gives orange red Fl with RNA and orange red Fl with RNA and yellow green Fl with DNAyellow green Fl with DNA

- QBC- QBC

- IMMUNOFLUORESCENCE- IMMUNOFLUORESCENCE

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Electron Microscope

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ELECTRON MICROSCOPE

-Electron Microscopes uses a beam of highly -Electron Microscopes uses a beam of highly energetic electrons to examine objects on a energetic electrons to examine objects on a very fine scale. This examination can yield the very fine scale. This examination can yield the info aboutinfo about

- Topography- Topography

- Morphology- Morphology

- Composition- Composition

- Crystallographic structure- Crystallographic structure

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TYPES OF EM

- Transmission Electron Microscope (TEM)- Transmission Electron Microscope (TEM)

- Scanning Electron Microscope (SEM)- Scanning Electron Microscope (SEM)

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Transmission Electron Microscope (TEM)

- Stream of electrons is formed - Stream of electrons is formed

- Accelerated using a positive electrical - Accelerated using a positive electrical potentialpotential

- Focused by metallic aperture and Electro - Focused by metallic aperture and Electro magnets magnets

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

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TEM (Cont)

-Projector Lens forms -Projector Lens forms image on Fluorescent image on Fluorescent viewing screenviewing screen

- 2D Image- 2D Image

- Magnification - Magnification

10,000 X to 100,000 X10,000 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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Working of SEM

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SEM IMAGES

Vibrio cholerae with polar flagellaTreponema pallidum

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INVERTED MICROSCOPE

- Used in metallurgy- Used in metallurgy

- Examination of cultures in flat bottom - Examination of cultures in flat bottom dishesdishes

- Micro dissection - Micro dissection

- Examination of parasites- Examination of parasites

- Observation of agglutination in serology- Observation of agglutination in serology

OTHER MICROSCOPES

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STEREO MICROSCOPE- Double Microscope- Double Microscope

- Produces 3D images- 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 - Uses a laser beam to illuminate a specimen whose image is then specimen whose image is then digitally enhanced for viewing on digitally enhanced for viewing on a computer monitor. a computer monitor.

- Laser beam scans single plane of - Laser beam scans single plane of 1µm thickness.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 - Observing cellular morphology in multilayered specimenmultilayered specimen

- Eg. used in diagnosing Ca cervix- Eg. used in diagnosing Ca cervix

- Evaluation and diagnosis of basal cell - Evaluation and diagnosis of basal cell carcinoma of skincarcinoma of skin

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What is the advantage of using a confocal microscope?

- By having a confocal pinhole, the - By having a confocal pinhole, the microscope is really efficient at rejecting microscope is really efficient at rejecting out of focus fluorescent light so that very out of focus fluorescent light so that very thin section of a sample can be analyzed.thin section of a sample can be analyzed.

- By scanning many thin sections through a - By scanning many thin sections through a sample, one can build up a very clean sample, one can build up a very clean three-dimensional image .three-dimensional image .

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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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CARE OF THE MICROSCOPE

- Handling

- Proper storage

- Care of Lenses

- Care of oil emersion objective

- Care of lamp

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