Microscope ug

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Microscope ug

  1. 1. 9/29/2010 MICROSCOPE Dr.T.V.Rao MD BASICS 1 Dr.T.V.Rao MD
  2. 2. THE HISTORY 9/29/2010  Many people experimented with making microscopes Dr.T.V.Rao MD  Was the microscope originally made by accident? (Most people were creating telescopes)  The first microscope was 6 feet long!!!  The Greeks & Romans used “lenses” to magnify objects over 1000 years ago. 2
  3. 3. THE HISTORY  Hans and Zacharias Janssen of Holland 9/29/2010 in the 1590’s created the “first” compound microscope Dr.T.V.Rao MD  Anthony van Leeuwenhoek and Robert Hooke made improvements by working on the lenses Robert Hooke 3 Anthony van Leeuwenhoek Hooke Microscope 1632-1723 1635-1703
  4. 4. ANTIONI VAN LEEUWENHOEK 9/29/2010 • Leeuwenhoek is called "the inventor of the microscope" Dr.T.V.Rao MD • Created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683 • Could reach magnifications of over 200x with simple ground lenses 4
  5. 5. HOW A MICROSCOPE WORKS 9/29/2010 Convex Lenses are curved glass used to Dr.T.V.Rao MD make microscopes (and glasses etc.) Convex Lenses bend light and focus it in one spot. 5
  6. 6. HOW A MICROSCOPE WORKS WITH.. 9/29/2010 Ocular Lens Objective Lens (Magnifies Image) (Gathers Light, Dr.T.V.Rao MD Magnifies And Focuses Image Body Tube Inside Body Tube) (Image Focuses) •Bending Light: The objective (bottom) convex lens magnifies and focuses (bends) the image inside the body tube and the ocular convex (top) lens of a 6 microscope magnifies it (again).
  7. 7. INTRODUCTION 9/29/2010 A microscope is an optical instrument that uses a lens or a combination of lenses to Dr.T.V.Rao MD magnify and resolve the fine details of an object.  The magnified image seen by looking through a lens is known as a virtual image, whereas an image viewed directly is known as a real image.  The object to be magnified is placed under the lower lens, called the objective and viewed through the upper lens, called the eyepiece. 7
  8. 8. DEFINITIONS  Absorption  When light passes through an object the intensity is reduced 9/29/2010 depending upon the color absorbed. Thus the selective absorption of white light produces colored light.  Refraction Dr.T.V.Rao MD  Direction change of a ray of light passing from one transparent medium to another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface, with greater deviation for shorter wavelengths  Diffraction  Light rays bend around edges - new wavefronts are generated at sharp edges - the smaller the aperture the lower the definition  Dispersion  Separation of light into its constituent wavelengths when entering a transparent medium - the change of refractive index with wavelength, such as the spectrum produced by a prism or a rainbow 8
  9. 9. REFRACTION 9/29/2010 Short wavelengths are “bent” more than long Dr.T.V.Rao MD wavelengths dispersion Light is “bent” and the resultant colors separate (dispersio Red is least refracted, violet most refracted. 9
  10. 10. REFRACTION & DISPERSION 9/29/2010 Short wavelengths are “bent” more than long wavelengths Dr.T.V.Rao MD dispersion Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted. 10
  11. 11. BECAUSE OF REFRACTION WE CANNOT SHOOT SOMETHING IN WATER 010 9/2 9/2 Dr.T.V.Rao MD He sees the fish here…. . 11 But it is really here!!
  12. 12. LENSES AND THE BENDING OF LIGHT 9/29/2010  lightis refracted (bent) when passing Dr.T.V.Rao MD from one medium to another  refractive index  a measure of how greatly a substance slows the velocity of light  direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface 12
  13. 13. LENSES 9/29/2010  focus light rays at a specific place called the focal point Dr.T.V.Rao MD  distance between center of lens and focal point is the focal length  strength of lens related to focal length  short focal length more magnification 13
  14. 14. PRINCIPLES IN MAGNIFICATION 9/29/2010 Dr.T.V.Rao MD 14
  15. 15. THE LIGHT MICROSCOPE 9/29/2010 many types Dr.T.V.Rao MD  bright-fieldmicroscope  dark-field microscope  phase-contrast microscope  fluorescence microscopes  compound microscopes  image formed by action of 2 15 lenses
  16. 16. COMPOUND MICROSCOPES 9/29/2010 In compound  microscopes Dr.T.V.Rao MD  image formed by action of 2 lenses 16
  17. 17. THE COMPOUND MICROSCOPE 9/29/2010 The Optical System Dr.T.V.Rao MD  Objective Lens: the lens closest to the specimen; usually several objectives are mounted on a revolving nosepiece.  Parafocal: when the microscope is focused with one objective in place, another objective can be rotated into place and the specimen remains very nearly in correct focus.  Eyepiece or Ocular Lens: the lens closest to the eye.  Monocular: a microscope having only one eyepiece 17  Binocular: a microscope having two eyepieces.
  18. 18. THE COMPOUND MICROSCOPE 9/29/2010 The Optical System Dr.T.V.Rao MD  Objective Lens: the lens closest to the specimen; usually several objectives are mounted on a revolving nosepiece.  Parafocal: when the microscope is focused with one objective in place, another objective can be rotated into place and the specimen remains very nearly in correct focus.  Eyepiece or Ocular Lens: the lens closest to the eye.  Monocular: a microscope having only one eyepiece 18  Binocular: a microscope having two eyepieces.
  19. 19. 9/29/2010 Dr.T.V.Rao MD 19 COMPOUND MICROSCOPE
  20. 20. DARK FIELD MICROSCOPE 9/29/2010 Dr.T.V.Rao MD 20
  21. 21. PRINCIPLES OF PHASE CONTRAST MICROSCOPE 9/29/2010 Dr.T.V.Rao MD 21
  22. 22. FLUORESCENT MICROSCOPE Arc Lamp 9/29/2010 EPI-Illumination Excitation Diaphragm Excitation Filter Dr.T.V.Rao MD Ocular Dichroic Filter Objective 22 Emission Filter
  23. 23. 9/29/2010 Dr.T.V.Rao MD 23 ELECTRON MICROSCOPE
  24. 24. THE BRIGHT-FIELD MICROSCOPE 9/29/2010 produces a dark image against a brighter background Dr.T.V.Rao MD has several objective lenses  parfocal microscopes remain in focus when objectives are changed total magnification  product of the magnifications of the ocular lens and the objective lens 24
  25. 25. THE CONVENTIONAL MICROSCOPE 9/29/2010 Mechanical Dr.T.V.Rao MD tube length = 160 mm Object to Image Distance = 195 mm Focal length of objective 25 = 45 mm Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press
  26. 26. 9/29/2010 Eyepiece Body Tube Dr.T.V.Rao MD Revolving Nosepiece Arm Objective Lens Stage Stage Clips Coarse Focus Diaphragm Fine Focus Light Base 26
  27. 27. Ocular Lens Body Tube 9/29/2010 Nose Piece Arm Dr.T.V.Rao MD Objective Lenses Stage Stage Clips Coarse Adj. Diaphragm Fine Adjustment Light Source Base 27 Skip to Magnification Section
  28. 28. SOME PRINCIPLES 9/29/2010  Rule of thumb is is not to exceed 1,000 times Dr.T.V.Rao MD the NA of the objective  Modern microscopes magnify both in the objective and the ocular and thus are called “compound microscopes” - Simple microscopes have only a single lens 28
  29. 29. BINOCULAR MICROSCOPE 9/29/2010  Schematic diagram of a stereoscopic microscope. This Dr.T.V.Rao MD microscope is actually two separate monocular microscopes, each with its own set of lenses except for the lowest objective lens, which is common to both microscopes. 29
  30. 30. DIAGRAMMATIC REPRESENTATION OF MICROSCOPE 9/29/2010 Dr.T.V.Rao MD 30
  31. 31. 9/29/2010 Dr.T.V.Rao MD 31 The principle of the compound microscope. The passage of light through two lenses forms the virtual image of the object seen by the eye.
  32. 32. MAGNIFICATION  An object can be focused generally no closer 9/29/2010 than 250 mm from the eye (depending upon how old you are!) Dr.T.V.Rao MD  this is considered to be the normal viewing distance for 1x magnification  Young people may be able to focus as close as 125 mm so they can magnify as much as 2x because the image covers a larger part of the retina - that is it is “magnified” at the place where the image is formed 32
  33. 33. MAGNIFICATION 9/29/2010  To determine your magnification…you just multiply the ocular lens by the objective lens Dr.T.V.Rao MD  Ocular 10x Objective 40x:10 x 40 = 400 So the object is 400 times “larger” Objective Lens have their magnification written on them. Ocular lenses usually magnifies by 10x 33
  34. 34. MAGNIFICATION 1000mm 9/29/2010 There used to be things called “slide Projectors” 35 mm slide 24x35 mm Dr.T.V.Rao MD 1000 mm M = 35 mm = 28 p The projected image is 28 times larger than we would see it at 250 mm from our eyes. If we used a 10x magnifier we would have a magnification of 280x, but we would reduce the field of view by a factor of 10x. 34
  35. 35. MICROSCOPE RESOLUTION 9/29/2010  Abilityof a lens to separate or Dr.T.V.Rao MD distinguish small objects that are close together  Wavelength of light used is major factor in resolution shorter wavelength greater resolution 35
  36. 36. INFINITY OPTICS 9/29/2010 Ocular Primary Image Plane Dr.T.V.Rao MD The main advantage of infinity corrected lens systems Tube Lens is the relative insensitivity to additional optics within the Infinite tube length. Secondly one can Other optics focus by moving the objective Image Distance Other optics and not the specimen (stage) Objective Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press Sample being imaged 36
  37. 37. OBJECTIVES 9/29/2010 Limit for smallest d = 1.22 Dr.T.V.Rao MD resolvable distance d between 2 points is (Rayleigh criterion): This defines a “resel” or “resolution element” Thus high NUMERICAL APERTURE is critical for high magnification In a medium of refractive index n the wavelength gets shorter: n 37
  38. 38. NUMERICAL APERTURE 9/29/2010  Resolving power is directly related to numerical aperture. Dr.T.V.Rao MD  The higher the NA the greater the resolution  Resolving power: The ability of an objective to resolve two distinct lines very close together NA = n sin u  (n=the lowest refractive index between the object and first objective element) (hopefully 1)  u is 1/2 the angular aperture of the objective 38
  39. 39. NUMERICAL APERTURE 9/29/2010 A Dr.T.V.Rao MD Light cone NA=n(sin ) (n=refractive index) 39
  40. 40. MICROSCOPE OBJECTIVES 9/29/2010 Standard Coverglass Thickness #00 = 0.060 - 0.08 #0 = 0.080 - 0.120 Dr.T.V.Rao MD #1 = 0.130 - 0.170 Microscope 60x 1.4 NA Objective #1.5 = 0.160 - 0.190 PlanApo #2 = 0.170 - 0.250 #3 = 0.280 - 0.320 #4 = 0.380 - 0.420 #5 = 0.500 - 0.60 mm Oil Stage Coverslip Specimen 40
  41. 41. Refractive Index 9/29/2010 Dr.T.V.Rao MD n = 1.52 Objective n = 1.5 n = 1.52 Oil Air n = 1.0 n=1.52 n = 1.52 Coverslip n=1.52 Specimen Water n=1.33 41
  42. 42. OIL IMMERSION INCREASES MAGNIFICATION 9/29/2010 Dr.T.V.Rao MD 42
  43. 43. CARING FOR A MICROSCOPE 9/29/2010  Clean only with a soft cloth/tissue Dr.T.V.Rao MD  Make sure it’s on a flat surface  Don’t bang it  Carry it with 2 HANDS…one on the arm and the other on the base 43
  44. 44. CARRY A MICROSCOPE CORRECTLY 9/29/2010 Dr.T.V.Rao MD 44
  45. 45. USING A MICROSCOPE 9/29/2010  Start on the lowest magnification  Don’t use the coarse adjustment knob Dr.T.V.Rao MD on high magnification…you’ll break the slide!!!  Place slide on stage and lock clips  Adjust light source (if it’s a mirror…don’t stand in front of it!)  Use fine adjustment to focus 45
  46. 46. TEACHING MICROSCOPY IS A ART 9/29/2010 Dr.T.V.Rao MD 46
  47. 47. Created by Dr.T.V.Rao MD for 9/29/2010 “ e “ Learning for Basic Medical Dr.T.V.Rao MD Graduates in Developing countries email doctortvrao@gmail.com 47

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