types of Microscopy and its parts and uses
- 1. M I C R O S C O P Y A N D I T S C L I N I C A L U S E S D R S C R A V I N D R A K U M A R , 1 S T Y E A R M . D . M I C R O B I O L O G Y , K I L P A U K M E D I C A L C O L L E G E .
- 2. P R I N C I P L E S O F O P T I C S The refractive index is a measure of how greatly a substance slows the velocity of light; the direction and magnitude of bending are determined by the refractive indices of the two media forming the interface. When a ray of light passes from one medium to another, refraction occurs that is, the ray is bent at the interface of the two mediums through which light passes.
- 3. T E R M I N O L O G I E S T O R E M E M B E R The focal length of a lens system is defined as the distance from the lens center to a point where parallel rays are focused on the optical axis termed as the principal focal point. An imaginary plane perpendicular to the principal focal point is called the focal plane of the lens system.
- 4. H I S T O R Y O F M I C R O S C O P E The first microscope was developed by Anton Von Leeuwenhook in late 16th centurywith magnification and resolution nearly 200 times. He was a Dutch buisnessman who used to grind lenses. He made lenses with short focal length and high resolution which was preferred over the earlier compound microscopes which had the chromatic abberations .
- 5. T Y P E S O F M I C R O S C O P E S Bright field or Light Microscopy Dark field Microscopy Phase Contrast Microscopy Fluorescence Microscopy Electron microscopy
- 6. P A R T S O F A L I G H T M I C R O S C O P E Click to add text
- 8. PA R T S O F A M I C R O S C O P E • The following are the important parts of a commonly used compound microscope The stage of a microscope where the lens is held has a hole in the centre to allow the illuminating light from the light source. The stage has adjustment knobs to adjust the slide anteroposteriorly and sidewards for changing the observing field. The illuminator is the light source is from the base of the microscope passing through an ABBE condenser and a diaphragm which adjusts amount of light passing through it.
- 9. A B B E C O N D E N S E R
- 10. N U M E R I C A L A P E R T U R E O F A C O N D E N S E R • The numerical apperture of the condenser system is also important for calculating the resolution of a microscope. • A condenser is a series of lenses placed above the light source to converge a cone of light from the light source to the objective lens. • The numerical apperture of a condenser commonly used in light microscope is between 1.2 amd 1.4
- 11. A D J U S T M E N T O F L I G H T S O U R C E • The light source from the illuminator is adjusted by the diaphragm with the help of a knob . • The height of the condenser can also be adjusted up and down . When the condenser is kept at a lower level the amount of light reaching the stage will be less. • So the condenser is kept at a higher level to ensure maximum light from the light source can be utilised. • A rheostat at the base of the microscope is used to control the light intensity of the light source. • The light source of microscope is an incandescent tungsten halogen bulb
- 12. R E S O L U T I O N O F A M I C R O S C O P E • Resolution of a microscope was explained by Mr. Ernest Abbe in 1870s. • He explains that the resolution is determined by the distance between two objects and the wavelenght of the light passing through the specimen and the numeric aperture of the lens used . This can be explained by the formula d= 0.5delta /n sineθ Here d is the distance between two particles and delta is the wavelength of the illuminating light and nsineθ is the numeric aperture of a lens. While explaining the numerical aperture of the lens n is the refractive index of the medium in which the objective lens works and sineθ is ½ the angle of the cone of light entering the objective . If the angle of the cone of light entering is narrow the resolution is less and if the angle of the cone of light entering the objective is wide then the resolution is higher.
- 13. R E S O L U T I O N • Resolution of a lens is defined as the ability of a lens to clearly distinguish between two different ponits in the field of study clearly as two different objects . • By increasing the numerical apperture of a lens the resolution of the microscope can be increased . As the refractive of air is 1 and no lens can have a numerical aperturture more than 1 a colourless liquid having same refractive index as glass can be used so that maximum light rays from light source enters the objective lens and the resolution of the magnification system is increased .
- 14. C A L C U L AT I O N O F R E S O L U T I O N • Calculation of resolution of a microscope depends on the numerical aperture of both condensing lens and the objective lens . But for practical purposes the numerical aperture of the objective lens is taken into consideration as per ABBE equation . • The numerical apperture of the objective lens is taken 1.25 and the wavelength of the light used for veiwing is in a blue green spectrum is 530 nm d= 0.5(530) /1.25 =0.2 micro meters So the maximum resolution of a light microscope is to clearly distinguish objects 0.2 μm.
- 15. W O R K I N G D I S TA N C E O F A L E N S Working distance of a lens is defined as the distance between the objective lens and the coverslip on the stage . The working distance of a lens with good resolution will be lesser . The numerical apperture of the lens depends upon the working distance involved .
- 16. M A G N I F I C A T I O N O F A L I G H T M I C R O S C O P E • The standard microscopes in day today practice have eye piece with magnification of 10x • Hence when used with the combination of objective the magnification obtained is 1000 times the numerical aperture of the objective lens Hence a 10x can produce a maginification of 1000x with oil immersion A maximum of 10000x can be obtained but due to overmagnification the image obtained will be a blur .
- 17. O T H E R F O R M S O F L I G H T M I C R O S C O P E For observation of organisms under a microscope the common technique of staining is used which kills the organism while staining. So in order to study living organisms under a microscope various principles are used to differentiate between host cells and the organism under study. The following are the light microcopes working under different principles for studying living microscopic organisms 1. Darkfield microscope 2. Phase contrast microcope 3. Differential interface contrast microscope.
- 18. D A R K F I E L D M I C R O S C O P Y Optics of dark field microscopy • In dark field microscopy a cone of refracted and reflected are only allowed to enter the objective lens making the ouline of the organism for example T.pallidum in a dark background visible for examination. • The internal structures of few larger eukaryotes can be studied by dark field microscopy.
- 19. D A R K F I E L D M I C R O S C O P Y
- 20. P H A S E C O N T R A S T M I C R O S C O P E • The principle of phase contrast microscopy was explained by dutch scientist FRITS ZERNIKE in 1934 • A phase-contrast microscope converts slight differences in refractive index and cell density into easily detected variations in light intensity. • The condenser of a phase-contrast microscope has an annular stop, an opaque disc with a thin transparent ring, that produces a hollow cone of light which passes through the specimen to be examined . • The undiflected light passes through a phase plate and falls on the image plate to form the baground of the image while the refracted waves with a reduction in wavelength forms the image on the image plane. • Phase contrast microscopy can be used to visualize living cells with good resolution without fixing them which kills the cells.
- 21. P H A S E C O N T R A S T M I C R O S C O P E
- 22. O P T I C S O F P H A S E C O N T R A S T M I C R O S C O P Y • The light from the light source of a phase contrast microscope divides into two the zeroth order or the surround wave S forming the surrounding image and the diffracted D wave forming the image of the specimen under study after leaving the specimen plane the S and D waves combine to form the particle P wave to form the image on the image plane . Hence the particle P wave is an addition of S and D waves to form the image of the specimen under examination . • Detection of the specimen depends on the difference in the intensity of the S and D waves. • If the difference in the intensity of both waves are larger the image of the specimen under under study will be with good resolution. • Optical path length is the term defined as the product of the refractive index and thickness of the specimen under study
- 24. P H A S E C O N T R A S T M I C R O S C O P E O P T I C A L T R A I N
- 25. D I F F E R E N T I A L I N T E R F E R E N C E C O N T R A S T M I C R O S C O P Y • In a differential interference microscope two different polarised beams of light at right angles are produced by prisms which pass through the specimen and both the beams join together to form an image in which the image is found to be in a plane higher than the surrounding plane producing a three dimensional effect.
- 26. F L U O R E S C E N T M I C R O S C O P Y • Fluorescent microscope uses the principle that an illuminated object absorbs light and in turn emits light which is used to interpret the image . • In a fluorescent microscope the light source emitted excites the molecules of the specimen which is stained with fluorochromes which in turn emits light with a longer wavelength to return to a stable state. This emited wave is interpreted in the image plane to form the image • The most commonly used one is the epifluorescence microscope .
- 27. O P T I C S O F F L U O R E S C E N T M I C R O S C O P E • Light from an mercury arc lamp passes through an exciter filter to remove the longer wavelength , which then passes through a dichromatic mirror and the light waves with short wavelengths with higher energy hits the specimen and the light from the specimen passes through the dichromatic mirror to form the image • The barrier filter filters the harmful rays to allow only the light in the visible spectrum.
- 28. I M A G E S
- 29. C O N F O C A L M I C R O S C O P Y I N V E N T E D B Y M A R V I N M E L I N S K Y I N 1 9 5 5 • Confocal microscopy Is one where in the light from the light source is used to scan different layers of the specimen under study . • In the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labeled with one or more fluorescent probes. • Advantages of confocal microscopy is 1. Adjustable depth of field of study 2. Eliminating out of focus information degrading the view of field of intereset 3. Ability to collect serial images from various planes of the specimen under study
- 30. O P T I C S O F C O N F O C A L M I C R O S C O P Y • When a fluorescent stained microscope is used to visualize a fluorescent stained specimen light waves from various structures which are not in the field of interest also interfere with the image formed and hence the clarity of the image produced is lesser . • Hence the confocal microscopy uses the principle where the light rays from a laser source passes through a pinhole apertute to filter out the scattered light beams , which is then focused on to the specimen by a dichromatic mirror . • The light passing through the specimen scans different layers of the specimen under study and the light rays from the specimen passes through a pin hole aperture again in the detector end where the divergent rays are filtered out and the the image is interpreted
- 31. L I G H T S O U R C E • The light source in confocal microscope is laser . • This laser beam is reflected on to the specimen to form different optical section. • An optical section is one where defferent levels of the specimen is studied by the light source without making sections of the specimen physically. • In a confocal microscope the light used to focus on the specimen is kept in continuous movement scanning across the object to be studied. • The image is interpreted by interpreting the light hitting the photoelectric plate in a serial manner to form the final image . • The final resolution achieved by the instrument is governed by the wavelength of light, the objective lens, and the properties of the specimen itself.
- 32. E N H A N C E M E N T O F R E S O L U T I O N • The apparent increase in resolution was enabled by digital enhancement of images that were captured using a low light level silicon intensified target (SIT) video camera connected to a digital image processor. • The cellular structures were imaged using differential interference contrast (DIC) optics, and the images were further enhanced using digital processing methods.
- 34. E L E C T R O N M I C R O S C O P E • The shorter the wavelength the greater the resolution is the principle involved in a light microscope . • The principle is used in electron microscope where instead of light electron beam can be used to strike the specimen under study. • Electron microscopy prototype with 400x magnification was invented by Ernest Ruska in 1931. • The wavelength of an electron beqm is 100000 times lesser than an ordinary light wave hence increasing the resolution to 1000 times more than a light microscope.
- 35. O P T I C S O F E L E C T R O N M I C R O S C O P E • In an electron microscope the beam of electons is produced by a heated tungsten filament. • The electron beam is focused on the specimen by a condenser . • As the electron beam cannot pass through glass slide a donut shapped magnet is used to focus the electron beam on the specimen. • The electron beam focusedon the specimen is scattered by the specimen causing variations or reduction in the electron beam reaching the interpretating fluorescent plate. • The received electron beam is analysed to produce an image on the fluorescent plate . • The more thicker the region of the cell under bstudy the more the electrons getting scattered making it an electron dense area in the interpretation plane. • As air and dust can difflect electron bean the lenses and objective planes are kept under strict vacuum condition.
- 36. L I G H T M I C R O S C O P E V S T R A N S M I S S I O N E L E C T R O N M I C R O S C O P E
- 37. S P E C I M E N P R E P A R A T I O N F O R T E M • For a specimen to be kept under study it should be made as thin slice . • Fixation with chemicals such as glutaraldehyde and osmium tetroxide to stabilize cell structure is the first step in preparation of slide • Then the specimen is dehydrated by using acetone or ethanol • Then the specimen is soaked in poly epoxy plastic and allowed to form the block • Thin slices are made by using a microkeratome • The specimen has to be stained as in a light microscopy. • staining is done with lead citrate and uranyl acetate. • The stains get attached to the cellular structure and makes it more opaque for the electrons and thus increase the contrast of the organelles • After fixation the specimen is mountedon a copper grid for examination.
- 38. O T H E R M E T H O D S O F S T A I N I N G U S E D • Negative staining in which the stain used is not absorbed by the specimen and for the surrounding dark region making the the specimen to be viewed distinctly . • Shadowing in shadowing the specimen is stained on the oppssite side of the slide with a heavy metal like platinum at 45 degrees making the study plane visible with a dark background formed by themetal and the object under under study to be visible with good resolution.
- 39. F R E E Z E E T C H I N G P R E PA R AT I O N • In order to visualise internal structure freeze etching is adopted for fixing structure 1. First the specimen is freeze dried with aliquid nitrogen making the specimen brittle which creates a break near the outer cell membrane. 2. The exposed surface is coated with carbon and platinum to form a replica of the surface 3. After obtaining the replica the organism is removed chemically . 4. The replica is studied under TEM . 5. This procedure avoids the risk of artifacts in the field of study
- 40. I M A G E S S E E N I N A T E M Freeze etching
- 41. S C A N N I N G E L E C T O N M I C R O S C O P E • In a scanning electon microscope a beam of electron scans the creates under study and creates specimen back and forth and forms the secondary electron which are trapped by the detector and forms an image to be finally interpreted on the image detector . • The nature of the secondary electron depends on the nature of the surface of the specimen under study. • If the surface of the specimen is elevated more secondary electrons are produced making it brighter in the image and in contrary if the surface is deep then the secondary electons produced are lesser making the surface appear darker. • This creates a three dimensional image of the specimen under study
- 42. O P T I C S O F S E M
- 43. S C A N N I N G P R O B E M I C R O S C O P Y • In a scanning probe microscopy a sharp tip of the size of holding a single atom at its tip is used to move up and down and back and forth at a constant height and the signals received are used to construct the surface on which the prob is moving. • In a scanning probe microscopy the objects that can conduct energy can only be imaged • For imaging non conducting specimen another technique called as ATOMIC FORCE MICROSCOPY where the probe movement over the surface causes a deflection in the laser beam which is used to form the image of the surface of the specimen under study.
- 44. Scanning probe microscopy Atomic force microscopy
- 45. S T A I N I N G O F S P E C I M E N In brief staining of a specimen is done to visualize the organisms under study. For fixing of the organism under study two techniques r used Namely 1.heat fixation 2.chemical fixation Dyes used are namely acid and basic dyes depending on the chemical composition and affinity properties .
- 46. • Binding of the dyes can happen ionically as n acidic and basic dyes • Or can happen covalently as in schiffs reagent. The staining of the specimen can b 1. simple staining 2. Differential staining 3. Negative stainning
- 47. D I F F E R E N T I A L S T A I N N I N G
- 48. A C I D FA S T S TA I N I N G • Here once the organism which is initially stained with carbol fuschin resists decolourisation by acid hence named so. • This property is exhibited by mycobacterium group of species • This property is rendered to the presence of mycplic acid acid in the walls of the organism
- 49. N E G A T I V E S T A I N I N G • The dye is used to stain the surrounding srtructures rather than the organism to make it distinctly visible on the image plane . Dye used commonly are india ink and nigrosine
- 50. R E F R E N C E S • Prescotts_Microbiology_by_Joanne_Willey_Linda_Sherwood_Christopher • https://www.microscopyu.com/techniques/confocal/introductory-confocal-concepts • Lab_Manual_and_Workbook_in_Microbiology_Applications_to_Patient • Apurba Sankar Sastry Essentials of Medical Microbiology • Ananthanarayan_and_Paniker's_Textbook of microbiology
Editor's Notes
- #6: Stereo Microscope are used to look at a variety of samples that you would be able to hold in your hand Inverted Microscope are used for in-vitro fertilization, live cell imaging, developmental biology, cell biology, neuroscience, and microbiology. Biological inverted microscopes are used to view living samples that are in a petri dish Metallurgical Microscope examine metals Polarizing Microscope to examine rocks,chemicals and minerals