Download
phase contrast optics n.
Skip this Video
Loading SlideShow in 5 Seconds..
Phase Contrast Optics PowerPoint Presentation
Download Presentation
Phase Contrast Optics

Phase Contrast Optics

729 Views Download Presentation
Download Presentation

Phase Contrast Optics

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Phase Contrast Optics Theory & Appl. Light Microscopy

  2. Abbé Theory • Designed optics for amplitude objects • Absorb light without change in phase of light waves • Based on assumption of no difference in index of refraction between specimen and background Theory & Appl. Light Microscopy

  3. Theory & Appl. Light Microscopy

  4. Theory & Appl. Light Microscopy

  5. Theory & Appl. Light Microscopy

  6. Theory & Appl. Light Microscopy

  7. Criterion for Resolution • Lens must capture undiffracted light plus at least first order of diffracted rays • Combine these in image plane by interference • But — most biological specimens (esp. living) are not amplitude objects • Phase Objects Theory & Appl. Light Microscopy

  8. Phase Objects • Do not absorb light • Difference in index of refraction between specimen and background Theory & Appl. Light Microscopy

  9. Theory & Appl. Light Microscopy

  10. Example: Cell • Object 1.25 m thick, i.r. = 1.35; i.r. water = 1.30 (0.05 difference) • Difference in path length for light = 1.25 (0.05) = 0.0625 m • 62.5/500 nm = 1/8 wavelength • /8 = /4 radians = 45° • This is difference in phase of wave passing through cell against wave passing next to cell Theory & Appl. Light Microscopy

  11. Theory & Appl. Light Microscopy

  12. Phase Differences • Our eyes cannot see this • Eyes set for amplitude differences, so cell is essentially transparent • But — information is present in light beams from specimen and in image • How do we see this? Theory & Appl. Light Microscopy

  13. Frits Zernike (1888–1966) • Dutch physicist • Developed vector notation for theory of light propagation through phase objects • Invented phase contrast optics in 1930; not manufactured until 1941 by Zeiss Theory & Appl. Light Microscopy

  14. Zernike Phase Vector Diagram For propagation of light through phase object S S = incindent wave P = particle wave P = phase shift of ray through specimen (S = U, undiffracted (0-order) ray P  Length of P = amplitude specimen/amplitude medium = transmission ratio Theory & Appl. Light Microscopy

  15. Calculate P by vector addition D U + D = P By the law of sines  U P D =  of all diffracted orders of light from specimen U = undiffracted light P = resulting specimen light, produced by interference between U and D in image formation Theory & Appl. Light Microscopy

  16. Theory & Appl. Light Microscopy

  17. Brightfield Optics • Shifts all vectors in phase equally, and may change all amplitudes equally: U + D = P U = P • No amplitude image • Information in P is present in , not in amplitude — eye cannot see this Theory & Appl. Light Microscopy

  18. Theory & Appl. Light Microscopy

  19. Phase Contrast Imaging • Basic principle: • Shift phases (s) and/or amplitudes of U and D differentially • This can produce a change in amplitude of P (length of vector) Theory & Appl. Light Microscopy

  20. In microscope At image plane In specimen D' D' D D P'  U U' U' P U' P' Amplitude! U = P

  21. Phase Contrast Optics • Physically separates U and D light and subjects one or the other to phase shift and/or amplitude shift • In theory, any shift of U and D are possible • In practice, a shift of  90° (/4) is appropriate for most biological specimens Theory & Appl. Light Microscopy

  22. Theory & Appl. Light Microscopy

  23. Optical Arrangements • Several possible, but major design challenge to keep U and D rays separate and handled differently • In practice, use a hollow cone of light to illuminate specimen • Phase Annulus below condenser • Phase plate at back focal plane of objective • Only 0 order rays from annulus pass through plate Theory & Appl. Light Microscopy

  24. Theory & Appl. Light Microscopy

  25. Theory & Appl. Light Microscopy

  26. Theory & Appl. Light Microscopy

  27. Phase Plate • Rings in phase plate can include • Attenuating layer (absorption but no phase shift), or • Phase-shifting layer (no absorption, phase shift only), or • Any combination of the two Theory & Appl. Light Microscopy

  28. Positive/Negative Phase • Positive Phase Specimen dark against light background (usual now) • Negative Phase Specimen bright against dark background (looks like darkfield optics) Theory & Appl. Light Microscopy

  29. Positive Phase D D' U'  U P P' U = P U'> P' Retard D relative to U (move D vector clockwise)

  30. Negative Phase D' D P' U'  U P U = P U'< P' Advance D relative to U (move D vector counterclockwise)

  31. Theory & Appl. Light Microscopy

  32. Theory & Appl. Light Microscopy

  33. Theory & Appl. Light Microscopy

  34. Theory & Appl. Light Microscopy

  35. Example Systems • Anoptral Phase Contrast Change amplitude of U (soot on ring), no phase shifts for either U or D rays. Bright image — negative phase Popular among algae workers in Great Britain in 50s–60s Theory & Appl. Light Microscopy

  36. Anoptral Phase D No phase shifts on ring D'  U U' P P' U = P U'< P' Produces delicate image against brown background

  37. Theory & Appl. Light Microscopy

  38. Example Systems • Zernike Phase Contrast Differential changes in amplitude and phase of U and D rays. • All combinations possible: • Amplitude absorption with no phase shift (metal coating) • Phase shift wavefront with no absorption (silica coating) Theory & Appl. Light Microscopy

  39. From: Rose & Pomerat (1960) J. Biophys. Biochem. Cytol. 8:423.

  40. Use/Limitation of Phaseco • Use for qualitative, not quantitative evaluation of specimens • Reasons: • Intensity differences in image not uniquely related to index of refraction differences of specimen • Phase halo— optical artifact Cannot completely separate U and D rays in optics Theory & Appl. Light Microscopy

  41. Intensity Differences • Two points may have same image intensity, but have different  values (different i.r.s) • I.e., if IP/IU of  at 240° identical to ratio at 320°, then how distinguish different i.r.? Theory & Appl. Light Microscopy

  42. Phase Halo • Serious artifact, most prominent at boundaries of sharp differences in i.r. • Exceeds ability of optics to produce an accurate image • So identification of exact boundary of specimen from image is very difficult Theory & Appl. Light Microscopy

  43. Theory & Appl. Light Microscopy

  44. Theory & Appl. Light Microscopy

  45. Reducing Phase Halo • Modification of design of phase plate • Apodized Phase Contrast Addition of neutral density filters to phase plate to suppress halo • Optical Process Theory & Appl. Light Microscopy

  46. Theory & Appl. Light Microscopy

  47. Theory & Appl. Light Microscopy

  48. Reducing Phase Halo • Modification of specimen and medium • Worst halo comes from abrupt i.r. difference between specimen (cell) and medium it is in • Match i.r. of medium to i.r. of specimen to reduce halo • Barer & Joseph (1957) Symp. Soc. Exp. Biol. 10:160–184. • Use of non-osmotic solutes to increase medium index of refraction Theory & Appl. Light Microscopy

  49. Interference Microscopy • Like phaseco in that imaging produces amplitude differences from phase differences in specimen • Quantitative Techniques • Qualitative Techniques Theory & Appl. Light Microscopy

  50. Optical Path Difference • Specimen vs. medium • ' = (s - m)t ' = optical path length t = physical thickness Can measure ', then calculate s = ('/t) + m Theory & Appl. Light Microscopy