Compound microscopy


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  • © Ryan Barrow 2008
  • Compound microscopy

    1. 1. UNITS OF MEASUREMENT <ul><li>1m = 10 3 mm (millimetres) </li></ul><ul><li>1m = 10 6 µm (micrometres) </li></ul><ul><li>1m = 10 9 nm (nanometres) </li></ul><ul><li>Sometimes in old texts Angstroms (Å) are used (the diameter of a hydrogen atom) </li></ul><ul><li>1m = 10 10 Å </li></ul>
    2. 2. History <ul><li>In 14 th century spectacles & lenses were used to magnify objects. </li></ul><ul><li>During the later part of the 15 th century, da Vinci stressed the important of lenses for studying small object </li></ul><ul><li>The earliest recorded use of a magnifying lens goes back to Conrad Gesber (1558), a swiss biologist---- observed protozoan </li></ul><ul><li>It was Zacharias Janssen, a Dutch astronomer in 1650 added another lens to the telescope & thus provided 1 st time prototype of present day telescope & compound microscope. </li></ul>
    3. 3. <ul><li>The microscope constructed by early scientist had a magnifying power between 10X & 80X </li></ul><ul><li>Galileo had also constructed a microscope at the same time (1610) similar to that of Janssen & it was employed for the study of arrangement of the compound eye of insects </li></ul><ul><li>He improved earlier type of telescope by adding focusing. </li></ul><ul><li>Between 1590 & middle of 17 th century, several improvement were made in terms of magnification & refinement of lenses </li></ul><ul><li>Italian scientist- showed that glasses could be ground to any curvature for increasing the magnification. </li></ul>
    4. 4. <ul><li>1673 - Antioni van Leeuwenhoek – father of biology – 1 st to use microscope for biological studies </li></ul><ul><li>A simple microscope, high power magnification with some microscope having magnification 0f 300 X </li></ul><ul><li>He studied everything he could think of e.g stagnant water, saliva, blood, sperm, insect etc </li></ul><ul><li>He made 247 microscopes & published 450 letters describing protozoa, bacteria, sperms, RBC & nuclei of fish RBCs </li></ul><ul><li>He made many observation of biological importance. </li></ul><ul><li>Leeuwenhoek’s contributions have been recorded in a series of reports of the royal society of London </li></ul>
    5. 6. <ul><li>Compound microscope was constructed by Robert Hooke (1665) & is forerunner of present day compound microscope. </li></ul><ul><li>Many of Leeuwenhoek,s reports were confirmed by Robert Hooke </li></ul><ul><li>book Micrographia , published in 1665, devised the compound microscope, most famous microscopical observation was his study of thin slices of cork. </li></ul>
    6. 7. <ul><li>Improve & refinement added—more detail of cell available </li></ul><ul><li>1830 Achromatic lens were made </li></ul><ul><li>1870 good quality of lens with oil immersion lens </li></ul><ul><li>Abbe is credited with design of 1 st modern microscope </li></ul><ul><li>Addition of condenser was last imp contribution to development of compound microscope </li></ul>
    7. 8. Early Compound Microscopes <ul><li>Like a telescope in reverse </li></ul><ul><li>Could magnify about 30 times </li></ul><ul><ul><li>A magnifying glass can magnify about 10 times </li></ul></ul>
    8. 9. Microscope Microbiology Cytology Histology Embryology
    9. 10. Simple to Complex – Life’s Levels of Organization Our journey begins here.
    10. 12. Properties of light <ul><li>Amplitude : minimum displacement of wave from an equilibrium position </li></ul><ul><li>When a light wave decrease or increases in amplitude, it is observed by our eye in the form of a difference in the intensity of light i.e darkness or brightness. </li></ul><ul><li>Light ray passing through medium– there is decrease in amplitude </li></ul><ul><li>The decrease – greater/lesser depending on refracting index of media </li></ul><ul><li>Light ray can pass through a cell but various parts of cell have different refractive indices (RI). </li></ul><ul><li>Cytoplasm – causes little or no decrease in amplitude- transparent </li></ul><ul><li>Nucleus has higher RI- decrease in amplitude – darker in microscope </li></ul>
    11. 14. <ul><li>2) Frequency: It refers to the number of times a wave crest passes a particular point in one second. </li></ul><ul><li>Frequency remains constant for a light wave. </li></ul><ul><li>Light waves or identical frequency of coherent rays can combine or interfere with each other. </li></ul><ul><li>3) Wavelength: it is the distance between two wave crests </li></ul><ul><li>Our eyes are sensitive to different wavelengths of light in the visible range (380-740 nm) & register them in the form of colour </li></ul>
    12. 15. Fig. 3-4
    13. 16. The light spectrum <ul><li>Wavelength ---- Frequency </li></ul>Blue light 488 nm short wavelength high frequency high energy (2 times the red) Red light 650 nm long wavelength low frequency low energy Photon as a wave packet of energy
    14. 17. Properties of Light
    15. 18. <ul><li>Reflection:- light bounces off an object </li></ul><ul><li>We see colors of objects based on the wavelengths of light reflected by the surfaces of the object </li></ul><ul><li>Absorption:- black object absorbs light than reflect light will gain heat more rapidly than white objects which reflect than absorb light </li></ul>
    16. 19. <ul><li>Refraction </li></ul><ul><ul><li>Bending of light as it passes through one medium to another of different density </li></ul></ul><ul><ul><li>Such as from air in to glass microscope lens </li></ul></ul><ul><li>Degree of bending depends on relative refractive indices of the media </li></ul>
    17. 21. Principles of Microscopy <ul><li>Refractive Index :- bending of light from one medium to another of different densities </li></ul><ul><li>Given by RI= speed of light in a given medium </li></ul><ul><li>speed of light in vacuum </li></ul><ul><li>Light is also bent as it passes through a glass microscope lens </li></ul><ul><li>So shape of lens determines how exactly light is bent </li></ul><ul><li>A convex-convex lens (one that is curved outward on both on both sides) will bent parallel light rays so that light theoretically is focused at a single (focal) point. </li></ul>
    18. 22. <ul><li>Magnification </li></ul><ul><li>In light microscopy, visible light is bent by a series of ground glass microscope lens to achieve magnification </li></ul><ul><li>Magnification- enlargement of object </li></ul><ul><li>Two lenses magnify images – ocular lens & objective lens </li></ul><ul><li>Image of specimen much larger than object itself </li></ul><ul><li>Total magnification is the product of magnification of individual lens </li></ul>
    19. 23. <ul><li>Resolution: degree to which detail in specimen is retained in magnified image </li></ul><ul><li>Resolving power: </li></ul><ul><li>Capacity of microscope or any other instrument to distinguish between images of two pointed objects lying very close together </li></ul><ul><li>Unaided eye – 0.1 mm apart </li></ul><ul><li>Microscope - 0.2 µm apart </li></ul><ul><li>Limit of resolution </li></ul><ul><li>Limit of resolution: </li></ul><ul><li>Minimum distance at which two objects appears as two distinct object </li></ul>
    20. 24. Resolution Actual What We Might See Even if we magnify an image of two objects, we can not distinguish them unless we have adequate resolution .
    21. 25. <ul><li>Limit of resolution = 0.61 λ </li></ul><ul><li>NA </li></ul><ul><li>NA is light gathering capacity of objective </li></ul><ul><li>NA (Numerical aperture) = n sin α </li></ul>constant Wavelength of illumination Numerical aperture Refractive index of air or liquid between specimen & lens Sine of semi-angle of the aperture
    22. 26. <ul><li>Limit of resolution inversely proportional to resolving power (N.A) </li></ul><ul><li>So higher N.A lower limit of resolution </li></ul><ul><li>Sin α value cannot exceed 1 & R.A of instrument is constant </li></ul><ul><li>40X objective RA= 1.6, 100X objective RA= 1.6 </li></ul><ul><li>So limit of resolution = 0.61 X wavelength ( λ ) </li></ul><ul><li>1 X 1.4 </li></ul>
    23. 27. <ul><li>So limit of resolution directly dependent on wavelength </li></ul><ul><li>In case of red light with a wavelength of 6000 ---- limit of resolution = 0.25 µm </li></ul><ul><li>But if violet light which has wavelength of 4000 is used, then limit of resolution = 0.17 µm </li></ul><ul><li>Thus NA is constant & only way to decrease the wavelength of the light </li></ul><ul><li>For this purpose UV light can be used </li></ul><ul><li>But UV cannot pass through glass lenses thus necessitating the use of quartz </li></ul>
    24. 30. Resolution vs. Magnification
    25. 31. <ul><li>Light Microscope </li></ul><ul><ul><li>light as source of </li></ul></ul><ul><ul><li>illumination </li></ul></ul><ul><ul><li>glass lenses </li></ul></ul><ul><ul><li>limited resolution (loses resolving power at magnifications above 2000X) </li></ul></ul>
    26. 32. THE MICROSCOPE <ul><li>EYEPIECE </li></ul>
    27. 33. THE MICROSCOPE <ul><li>ARM </li></ul>
    28. 34. THE MICROSCOPE <ul><li>BASE </li></ul>
    29. 35. THE MICROSCOPE <ul><li>BINOCULAR TUBE </li></ul>
    30. 36. THE MICROSCOPE <ul><li>REVOLVING NOSEPIECE </li></ul>
    31. 37. THE MICROSCOPE <ul><li>OBJECTIVE LENS </li></ul>
    32. 38. THE MICROSCOPE <ul><li>MECHANICAL STAGE </li></ul>
    33. 39. THE MICROSCOPE <ul><li>STAGE CLIPS </li></ul>
    34. 40. THE MICROSCOPE <ul><li>IRIS DIAPHRAGM </li></ul>
    35. 41. THE MICROSCOPE <ul><li>COARSE ADJUSTMENT KNOB </li></ul>
    36. 42. THE MICROSCOPE <ul><li>FINE </li></ul><ul><li>ADJUSTMENT </li></ul><ul><li>KNOB </li></ul>
    37. 43. THE MICROSCOPE <ul><li>LAMP </li></ul>
    38. 44. THE MICROSCOPE <ul><li>BULB </li></ul>
    39. 45. THE MICROSCOPE <ul><li>ON/OFF SWITCH </li></ul>
    40. 46. Reading an objective
    41. 47. How Does a Microscope Work? the eye the image the specimen Each lens magnifies the image, increasing its overall size A lens is a bi-convex disk that bends light The farther the light rays are bent, the larger the image appears The bent rays produces an image
    42. 48. How Does a Microscope Work? the eye the image the specimen The image is always seen upside down and backwards from its actual position
    43. 49. Know and be able to use the vocabulary of the microscope <ul><li>Magnification </li></ul><ul><li>Resolution </li></ul><ul><li>Numerical aperture </li></ul><ul><li>image larger </li></ul><ul><li>image clearer </li></ul><ul><li>ability of lens to gather light </li></ul>
    44. 50. Know and be able to use the vocabulary of the microscope contd… <ul><li>Working distance </li></ul><ul><li>Depth of field </li></ul><ul><li>Parfocality </li></ul><ul><li>between lens & stage </li></ul><ul><li>bottom to top of slide </li></ul><ul><li>all objectives in reasonable focus at the same time </li></ul>
    45. 51. Care of microscope <ul><li>Carrying a microscope: one hand grabs the arm and the other hand supports the bottom of the base. </li></ul><ul><li>Lens care </li></ul><ul><ul><li>DO NOT TOUCH THE LENS! The oil from your hands can etch the glass. </li></ul></ul><ul><ul><li>CLEAN THE LENS WITH LENS PAPER ONLY! Other paper has fiber that can scratch the lens. </li></ul></ul><ul><li>Putting away the microscope: rotate to the 4X objective and roll the nosepiece away from the stage so that the space between the stage and nosepiece is at a maximum. </li></ul>
    46. 52. ThAnk YoU