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Polarized microscopy seminar/prosthodontic courses

The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.for more details please visit 
www.indiandentalacademy.com

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Polarized microscopy seminar/prosthodontic courses

  1. 1. POLARIZED MICROSCOPY INDIAN DENTAL ACADEMY Leader in continuing Dental Education www.indiandentalacademy.com
  2. 2. REFERENCES • Theory and Practice of Histological Techniques-Bancroft • Cellular Pathology Technique-Culling • Microscope-Basics & Beyond • J.K. Soneja, Microscopy www.indiandentalacademy.com
  3. 3. CONTENTS • History & Introduction of polarized microscopy • Polarized light • Birefringence • Constituents of polarized microscope • Dichroism • Applications of polarized microscope • Advantages & disadvantages of polarized microscope • Conclusion www.indiandentalacademy.com
  4. 4. ADVANCES IN MICROSCOPY Over the last 150 years , there has been a painstaking development of new techniques for light and electron microscopy, which facilitates the precise analysis of cell & tissue structure and demonstrate more clearly the changes , which pathological processes bring about. www.indiandentalacademy.com
  5. 5. ADVANCES IN MICROSCOPY • Dark field microscopy • Fluorescence microscopy • Phase contrast microscopy • Polarized microscopy • Electron microscopy • Stereomicroscopy www.indiandentalacademy.com
  6. 6. POLARIZED MICROSCOPY www.indiandentalacademy.com
  7. 7. How to compare & contrast polarized microscope and light microscope ?? www.indiandentalacademy.com
  8. 8. LIGHT SOURCE STAGE ANALYZER How to compare & contrast polarized microscope and light microscope ?? www.indiandentalacademy.com
  9. 9. HISTORY • Polarizing microscope was developed in the late nineteenth century. • It was initially made for studying rocks and minerals. • It may also be used in the biology and medical fields. • Polarizing microscopes are also known as petrographic microscopes. www.indiandentalacademy.com
  10. 10. INTRODUCTION • A polarizing microscope is a microscope that uses polarized light for looking at specimens. • It is used for detailed examination of specimens. • Numerous crystals , fibrous structures, pigments , lipids , proteins, bone and amyloid deposits exhibit birefringence. www.indiandentalacademy.com
  11. 11. POLARIZED LIGHT • Transverse wave light whose vibration possess direction is called polarized light. • Light from an ordinary light source (natural light) that vibrates in random directions is called non-polarized light. • Natural light vibrates in many planes or vibration directions whereas polarized light vibrates in only one plane . www.indiandentalacademy.com
  12. 12. plane of vibration vibration direction propagation direction light vibrates in all planes that contain the light ray (i.e., all planes perpendicular to the propagation direction POLARIZED LIGHT UNPOLARIZED LIGHT www.indiandentalacademy.com
  13. 13. POLARIZER • Polarized light can be produced for microscopy purpose by passing natural light through a polarizer. • Polarizer is an optical component made from a substance that will allow vibrations of only one vibration direction to pass. www.indiandentalacademy.com
  14. 14. Light passes through the polarizer West (left) east (right) Plane polarized lightUnpolarized light www.indiandentalacademy.com
  15. 15. BIREFRINGENCE • Birefringence or double refraction is the phenomenon, which occurs when light that is launched through a crystal material is divided into two linearly polarized light rays having mutually crossing vibration directions, and then refracted. www.indiandentalacademy.com
  16. 16. • Their velocities and refractive indices(RI) differ from one another. • Higher the RI greater the retardation of the ray. • Substances or crystals capable of producing plane polarized light are called birefringent . www.indiandentalacademy.com
  17. 17. mineral grain plane polarized light fast ray (low RI ) slow ray (high RI) lower polarizer D=retardation d Observation: frequency of light remains unchanged during splitting, regardless of material F= V/ l if light speed changes, l must also change l is related to color; if l changes, color also changes Violet (400 nm)  Red (700 nm) BIREFRINGENCE www.indiandentalacademy.com
  18. 18. www.indiandentalacademy.com
  19. 19. BIREFRINGENCE WAVELENGTH IN NANOMETRES Thicknessinmicrons T www.indiandentalacademy.com
  20. 20. NICOL PRISMS • Named after the inventor . • Polarizers made from calcite, were cemented together with Canada balsam in such a way that slow ray was reflected away from optical pathway , and into the mount of prism , leaving only polarized fast ray to pass through. www.indiandentalacademy.com
  21. 21. www.indiandentalacademy.com
  22. 22. POLAROID DISCS • In 1935, polaroids- glass or celluloid covered discs with the ability to polarize light were made available for use in place of Nicol prisms. • Polaroids are made by suspending ultramicroscopic crystals of herapathite in nitrocellulose. • Some substances and crystals which can produce plane polarized light by differential absorption give rise to the phenomenon called dichroism. www.indiandentalacademy.com
  23. 23. • Such crystals suspended in thin plastic films in one direction have now replaced the bulky and expensive Nicol prisms. • These thin films totally absorb the slow rays and are pleochroic (absorbing all colors equally) . www.indiandentalacademy.com
  24. 24. POLARIZED LIGHT MICROSCOPE CONFIGURATION www.indiandentalacademy.com
  25. 25. CONSTITUENTS OF POLARIZED MICROSCOPE Polarizing microscope uses two polarizers- 1. Polarizer & Analyzer Polarizer • Placed beneath the substage condenser • Held in rotatable graduated mount • Can be removed from light path when not required www.indiandentalacademy.com
  26. 26. ANALYZER • It is the upper polarizer • Placed between the objective and the eyepiece • Also graduated for measurement to be taken . www.indiandentalacademy.com
  27. 27. A microscope equipped for polarized light www.indiandentalacademy.com
  28. 28. www.indiandentalacademy.com
  29. 29. 2. Polarizing objective ( strain free objective) • An objective lens for polarization is designed and manufactured to have low turbulence by refraction in the polarizing state on the lens surface . • Also, to have low lens strain . • A polarizing objective can be distinguished from the ordinary ones by the label P, PO, Pol . www.indiandentalacademy.com
  30. 30. 3. Polarizing condenser A polarizing condenser has following 3 characteristics – • Built-in rotatable polarizer • Top lens-out construction when parallel light illumination at low magnification is required • Strain-free optical system, like the objectives www.indiandentalacademy.com
  31. 31. 4. Polarizing rotating stage • Rotating an anisotropy between crossed nicols changes the brightness. • For this reason, in polarized light observation, the specimen is often rotated to the diagonal position (the position where the anisotropy is brightest). www.indiandentalacademy.com
  32. 32. • In other words, rotatability of the polarizing stage and centerability are fundamental . • 360° angle gradations are indicated in the area surrounding the rotating stage . www.indiandentalacademy.com
  33. 33. 5. Bertrand lens • A Bertrand lens projects an interference image of the specimen, formed in the objective pupil, onto the objective image position . • It is located between the analyzer and eyepiece for easy in and out of the light path. www.indiandentalacademy.com
  34. 34. 6. Centerable revolving nosepiece • Since the stage needs to be rotated for polarizing observation, the objective and the optical axis of the tube must coincide exactly with one another. • In order for the optical axis to completely match even when the lens‘ magnification is changed, a revolver with optical centering mechanism is installed in each hole. www.indiandentalacademy.com
  35. 35. 7. Eyepiece with crosshair • The eyepiece has a built-in focusing plate containing a crosshair. • By inserting the point pin into the observation tube sleeve, the vibration direction of the polarizer and analyzer can be made to agree with the crosshair in the visual field. www.indiandentalacademy.com
  36. 36. 8. Test plate with compensator • These are phase plates used for verifying the double refractivity of specimens, • determining their vibration directions , and for retardation measurement . www.indiandentalacademy.com
  37. 37. Polarizer and Analyzer are the essential components of the polarizing microscope When we look through 2 polarizers, there can be 3 conditions:- 1. When polarizer and analyzer are parallel Rays vibrating in the parallel plane are able to pass. - www.indiandentalacademy.com
  38. 38. 2. When polarizer and analyzer are crossed Rays able to pass the polarizer are blocked by analyzer. The condition when no light reaches the observer is known as Extinction . www.indiandentalacademy.com
  39. 39. west (left) east (right) south (front) north (back) Black!! POLARIZER ANALYZER The condition when no light reaches the observer is known as EXTINCTION. www.indiandentalacademy.com
  40. 40. Now insert a thin section of a rock west (left) east (right) Unpolarized light Light vibrating in many planes and with many wavelengths Light and colors reach eye! Light vibrating E-W www.indiandentalacademy.com
  41. 41. 3. When a birefringent substance is rotated between crossed polarizer It is visible when it is in the diagonal position (i.e., when it is halfway (45º) between the vibration planes of polarizer). www.indiandentalacademy.com
  42. 42. • At 45 degree, however phase differences between 2 rays which can develop are able to combine in the analyzer and form a visible image. • Image appears and disappears alternately at each 45 degree of rotation. www.indiandentalacademy.com
  43. 43. Two Phenomenon in polarizing microscope that are important for Histopathologists are :- a) Birefringence - Property of resolving a beam of plane polarized light into 2 rays at different velocities. b) Dichroism www.indiandentalacademy.com
  44. 44. Dichroism • Some birefringent substances are dichroic. • When a dichroic substance is rotated in polarized light (i.e. using polarizer only). • Changes of color and intensity can be seen after rotating 90 degrees. • The original color returns after a further 90 degree rotation. • This is due to the differential absorption of light.www.indiandentalacademy.com
  45. 45. DICHROISM www.indiandentalacademy.com
  46. 46. APPLICATIONS OF POLARIZING MICROSCOPY IN HISTOPATHOLOGY • Artifacts : Formalin pigment, sutures , starch • Crystals : Talc , urate , pyrophosphate , silica , etc. URIC ACID CRYSTALSwww.indiandentalacademy.com
  47. 47. • Protein : Collagen , amyloid , keratin Glomerulus from renal biopsy stained with congo red and examined by polarization microscopy. The characteristic "apple-green" birefringence of amyloid is apparent . www.indiandentalacademy.com
  48. 48. APPLICATIONS OF POLARIZING MICROSCOPY IN HISTOPATHOLOGY • Lipids : Myelin • Bone structure : Osteoid seams , woven bone • Miscellaneous : Muscle striations , charcot- layden crystals • On routine light microscopy, the distinction between dentin and cementum is often difficult, but the use of polarized light clearly separates the two different layers. www.indiandentalacademy.com
  49. 49. • Graphite implantation within oral mucosa appears similar microscopically to amalgam but can be differentiated by its pattern of birefringence after treatment with ammonium sulfide and by the lack of staining of the reticulin fibres. • In OSMF, to analyze collagen distribution in different stages using the picrosirius red stain under polarized microscope. www.indiandentalacademy.com
  50. 50. In Dentigerous cysts, thin fibres showed greenish yellow color and thick fibres showed orange- red color. • To compare the pattern of collagen fibres in odontogenic cysts . Section of OKC showing orangish red birefringence pattern of collagen fibres www.indiandentalacademy.com
  51. 51. ADVANTAGES OF POLARIZING MICROSCOPE • They have a compact size, making them convenient for transporting. • A polarized microscope uses an optical device known as a "polarizer “ which configures the movement of light and forces its vibration in a single direction. www.indiandentalacademy.com
  52. 52. • Polarizing microscopes are often customized, designed and constructed to suit the application which will be viewed. • Polarized light generates clearer images and higher magnification of the specimen than a general compound light microscope. • Moreover, polarizing microscopes are equipped with a rotating stage which helps in generating an intensified image of the entire specimen. www.indiandentalacademy.com
  53. 53. Disadvantages of polarized microscope • Even using phase-polar illumination, not all the fibers present may be seen. • The method requires a great degree of sophistication on the part of the microscopist. • The method uses only a tiny amount of material for analysis. This may lead to sampling bias and false results. www.indiandentalacademy.com
  54. 54. CONCLUSION • Microscopes dedicated for use with polarized light are very sophisticated instruments having components specifically designed to minimize strain and provide sharp, crisp and clear images of birefringent specimens. • For simple qualitative work , a standard miroscope can be converted for polarized light studies . www.indiandentalacademy.com
  55. 55. THANK YOU !! I E R N E C www.indiandentalacademy.com
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The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and offering a wide range of dental certified courses in different formats.for more details please visit  www.indiandentalacademy.com

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