OPTICAL PRINCIPLES OF MICROSCOPY. Interuniversity Course 28 December 2003 Aryeh M. Weiss Bar Ilan University

Transcription

1 OPTICAL PRINCIPLES OF MICROSCOPY Interuniversity Course 28 December 2003 Aryeh M. Weiss Bar Ilan University

2 FOREWORD This slide set was originally presented at the ISM Workshop on Theoretical and Experimental Microscopy Techniques, Eilat, 2 May Most of the graphics in this presentation were taken from the references given at the end of this presentation.

3 TYPES OF MICROSCOPY Light (UV, visible, IR) Electron (SEM, TEM) Near-field scanning microscopy - scanning tunneling - atomic force - near field optical

4 OVERVIEW Properties of light Optical image formation Microscope basics Contrast generation

5 ELECTROMAGNETIC WAVE W avelength (period T) Axis of Magnetic Field Axis of Propaga tion

6 SOME PROPERTIES OF LIGHT Index of refraction (n) Speed of light (c/n) Polarization Frequency (ν = Hz) Wavelength (λ = nm)

7 SOME OPTICAL PHENOMENA Reflection Refraction Interference Diffraction Polarization

8 REFLECTION AND REFRACTION θ i θ r θt Snell s Law: The angle of reflection (Ø r ) is equal to the angle of incidence (Ø i ) regardless of the surface material The angle of the transmitted beam (Ø t ) is dependent upon the composition of the material n 1 sin Ø i = n 2 sin Ø t The velocity of light in a material of refractive index n is c/n

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10 TOTAL INTERNAL REFLECTION

11 INTERFERENCE 0 o 90 o 180 o 270 o 360 o Wavelength A A+B Amplitude The frequency does not change, but the amplitude is doubled B C C+D Constructive Interference Here we have a phase difference of 180 o (2π radians) so the waves cancel each other out D Destructive Interference Figure modified from Shapiro Practical Flow Cytometry Wiley-Liss, p79

12 INTERFERENCE FILTERS

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14 DIFFRACTION

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16 POLARIZATION

17 POLARIZERS Glan-Thompson polarizer

18 POLARIZATION VECTORS

19 BIREFRINGENCE

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21 PARTICLE CHARACTER OF LIGHT Light is made up of photons Energy/photon (e) is proportional to frequency e=hν or e=hc/λ Important in: -noise analysis -interaction with matter (eg absorption, fluorescence) -many commonly used detectors (eg, PMT, CCD, film, etc)

22 LIGHT SOURCES Black body sources (halogen lamps) -spectrum is continuous -spectrum peak depends on temp. ( color temperature ) Spectral sources (Hg, Xe, other arc lamps, lasers) -spectrum has structure (peaks) -spectrum is a function of the electronic properties of the gas

23 Halogen Xenon Hg

24 OPTICAL IMAGE FORMATION Imaging with one lens lens equation Magnification Compound microscope Diffraction limit Abbe theory of image formation Aberrations (chromatic, spherical)

25 PROPERTIES OF THIN LENSES f f p q = p q f Magnification = q p

26 VIRTUAL IMAGES

27 THE COMPOUND MICROSCOPE

28 DIFFRACTION LIMITED IMAGING

29 NUMERICAL APERTURE High N.A. θ Low N.A. θ N.A.= n sin(θ)

30 RAYLEIGH CRITERION r = 0.61λ/NA NA = n sin(θ) For high NA objectives ( ) 1.5λ > r > 0.44λ

31 EFFECT OF NA ON RESOLUTION

32 ABBE THEORY OF IMAGE FORMATION Image is formed by diffraction of light by the object. Minimal requirement for resolution is ability to collect at least one diffracted order in addition to zero order.

33 ORDERS OF DIFFRACTION

34 DIFFRACTION LIMIT IN ABBE THEORY

35 ABERRATIONS CHROMATIC ABERRATION - due to n(λ) SPHERICAL ABERRATION - paraxial approx.

36 ANATOMY OF A MICROSCOPE Objectives Oculars Upright or Inverted Illumination

37 UPRIGHT MICROSCOPE

38 INVERTED MICROSCOPE

39 INFINITY OPTICS WHAT AND WHY 1 b 1 f 1 a 1 1 f 1 f

40 OBJECTIVES

41 WHY SO EXPENSIVE?

42 WAVELENGTH RANGE OF OBJECTIVES

43 OCULARS

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45 IMPORTANT PART OF SYSTEM

46 ILLUMINATION Critical or Kohler

47 CONDENSER

48 RAYLEIGH CRITERION FOR TRAN-ILLUMINATED SYSTEMS r = 1.22 λ/(na obj + NA cond ) Methods which reduce the condenser NA reduce resolution. Worst case is a factor of two, for coherent illumination.

49 KOHLER ILLUMINATION Field diaphragm conjugate to object and determines area of illumination Condensor diaphragm conjugate to source and controls NA of condenser.

50 Wide Field Iris Narrow Field Iris

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52 OTHER ILLUMINATION ISSUES Halogen lamp color spectrum changes with temp. Use ND filters to adjust illumination, NOT diaphragms or lamp voltage. There are usually color filters to adjust illuminations for: - monochromatic light (green filters) - daylight correction (blue filter).

53 PUTTING IT ALL TOGETHER NA of objective and condenser determine resolution. Ocular must create an image suitable for viewing by eye. Requirements will differ for film, CCD, or other detectors.

54 EMPTY MAGNIFICATION Magnification greater than the resolution of the system is useless.. Human eye resolves 1-2 minutes of arc.. Maximum useful magnification is about x NA.

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56 DEPTH OF FIELD

57 GENERATION OF CONTRAST Darkfield Rheinberg illumination Phase contrast microscopy DIC (Nomarski)

58 DARKFIELD IMAGING

59 DARKFIELD IMAGES

60 RHEINBERG ILLUMINATION

61 RHEINBERG IMAGE

62 PHASE CONTRAST MICROSCOPY

63 PHASE CONTRAST ALLIGNMENT

64 PHASE CONTRAST IMAGE

65 APODIZED PHASE CONTRAST

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67 Holzwarth, Webb, Kubinski, and Allen, J. Microscopy, p (1997)

68 DIC IMAGES X-pol Y-pol

69 WHAT IS CONTRAST

70 MTF

71 MTF WITH CONTRAST GENERATION

72 OTHER CONTRAST GENERATION METHODS Polarization Hoffman modulation Interference Fluorescence

73 TAKEHOME MESSAGES Numerical aperture determines resolution Empty magnification is bad Contrast generation often lowers resolution, but it is usually worth it Keep dirt off of the image planes Use ND filters to adjust illumination intensity

74 REFERENCES OPTICAL MICROSCOPY Michael W.Davidson and Mortimer Abramowitz The Florida State University FROM LENSES TO OPTICAL INSTRUMENTS Giorgio Carboni, Fun Science Gallery funsci.com/fun3_en/lens/lens.htm VIDEO MICROSCOPY 2 nd Ed. S. Inoue and K.R. Spring Plenum Press, NY 1997

75 REFERENCES Introduction to Confocal Microscopy and Image Analysis (Powerpoint slide sets) Dr. J. Paul Robinson Purdue University Cytometry Laboratories Handbook of Optical Filters Chroma Technology Corp.

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