1. Slide 1 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Lecture 2 The Principles of Microscopy BMS 524 - “Introduction to Confocal Microscopy and Image Analysis” Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine J.Paul Robinson, Ph.D. Professor of Immunopharmacology Director, Purdue University Cytometry Laboratories These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not be used for any commercial purpose. The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and figures in these lecture notes are taken from this text. UPDATED January, 2000

2. Slide 2 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Review Microscope Basics, Magnification, Optical systems Properties of Light Refraction A Lens Refractive Index Numerical Aperture Resolution Aberrations Fluorescence

3. Slide 3 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Refraction & Dispersion Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted. dispersion Short wavelengths are “bent” more than long wavelengths ref rac tion

4. Slide 4 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Reflection and Refraction 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 tt ii rr Incident Beam Reflected Beam Transmitted (refracted)Beam n 1 sin Ø i = n 2 sin Ø t The velocity of light in a material of refractive index n is c/n

5. Slide 5 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Properties of thin Lenses f 1 p + 1 q = 1 f f p q Resolution (R) = 0.61 x NA Magnification = q p (lateral) (Rayleigh criterion)

6. Slide 6 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Microscope Components Ocular Objectives Condenser Numerical Aperture Refractive Index Aberrations Optical Filters

7. Slide 7 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Ocular - Eyepiece Essentially a projection lens (5x to 15x magnification) Note: there is usually an adjustment call the inter-pupillary distance on eyepieces for personal focusing Huygenian –Projects the image onto the retina of the eye –your eye should not be right on the lens, but back from it (eyecups create this space) Compensating –designed to work with specific apochromatic or flat field objectives - it is color compensated and cannot be mixed with other objectives (or microscopes) Photo-adapter –designed to project the image on the film in the camera - usually a longer distance and lower magnification from 0.5x to 5x

8. Slide 8 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Condenser Has several purposes –must focus the light onto the specimen –fill the entire numerical aperture of the objective (i.e. it must match the NA of the objective) Most microscopes will have what is termed an “Abbe” condenser (not corrected for aberrations) Note if you exceed 1.0 NA objective, you probably will need to use oil on the condenser as well (except in inverted scopes)

9. Slide 9 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Microscope Objectives

10. Slide 10 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Objectives PLAN-APO-40X 1.30 N.A. 160/0.22 Flat field Apochromat Magnification Numerical Tube Coverglass Factor Aperture Length Thickness ∞ - Infinity corrected

11. Slide 11 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Objectives Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion): d = λ/2 N.A. Thus high NUMERICAL APERTURE is critical for high magnification This defines a “resel” or “resolution element” d = 0,61λ/N.A.

12. Slide 12 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Numerical Aperture The wider the angle the lens is capable of receiving light at, the greater its resolving power The higher the NA, the shorter the working distance

13. Slide 13 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Numerical Aperture Resolving power is directly related to numerical aperture. 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  –(n=the lowest refractive index between the object and first objective element) (hopefully 1) –  is 1/2 the angular aperture of the objective

14. Slide 14 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Numerical Aperture For a narrow light beam (i.e. closed illumination aperture diaphragm) the finest resolution is (at the brightest point of the visible spectrum i.e. 530 nm)…(closed condenser). NA 2 x NA.00053 2 x 1.00 = 0.265  m.00053 1.00 = 0.53  m With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser).. = = http://www.microscopy.fsu.edu/primer/anatomy/numaperture.html

15. Slide 15 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Object Resolution Example: 40 x 1.3 N.A. objective at 530 nm light 2 x NA .00053 2 x 1.3 = 0.20  m = 40 x 0.65 N.A. objective at 530 nm light 2 x NA .00053 2 x.65 = 0.405  m =

16. Slide 16 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Microscope Objectives Specimen Coverslip Oil Microscope Objective Stage 60x 1.4 NA PlanApo

17. Slide 17 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Refractive Index Objective n=1.52 Specimen Coverslip Oil n=1.33 n = 1.52 n = 1.0 n = 1.5 Water n=1.52 Air

18. Slide 18 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Monochromatic Aberrations –Spherical aberration –Coma –Astigmatism –Flatness of field –Distortion Chromatic Aberrations –Longitudinal aberration –Lateral aberration Sources of Aberrations

19. Slide 19 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Monochromatic Aberration –Spherical aberration Generated by nonspherical wavefronts produced by the objective, and increased tube length, or inserted objects such as coverslips, immersion oil, etc. Essentially, it is desirable only to use the center part of a lens to avoid this problem. F1F2 F1 Corrected lens

20. Slide 20 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Fig 12 p117 From:”Handbook of Biological Confocal Microscopy” J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed The figure is not reproduced in this presentation because we do not have permission to place this figure onto a public site. Monochromatic Aberrations –Coma Coma is when a streaking radial distortion occurs for object points away from the optical axis. It should be noted that most coma is experienced “off axis” and therefore, should be less of a problem in confocal systems. Fig From:Handbook of Biological Confocal Microscopy J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed Note: For class use Figure is under box

21. Slide 21 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Fig 13 p118 From:”Handbook of Biological Confocal Microscopy” J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed The figure is not reproduced in this presentation because we do not have permission to place this figure onto a public site. Monochromatic Aberrations –Astigmatism Fig 13 p118 If a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated. From:Handbook of Biological Confocal Microscopy J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed. Note: For class use Figure is under box

22. Slide 22 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Monochromatic Aberrations –Flatness of Field –Distortion Lenses are spherical and since points of a flat image are focused onto a spherical dish, the central and peripheral zones will not be in focus. Complex Achromat and PLANAPOCHROMAT lenses partially solve this problem but at reduced transmission. DISTORTION occurs for objects components out of axis. Most objectives correct to reduce distortion to less than 2% of the radial distance from the axis.

23. Slide 23 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Chromatic aberrations Chromatic aberration of a single lens causes different wavelengths of light to have differing focal lengths

24. Slide 24 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Chromatic aberrations Diffractive optical element with complementary dispersion properties to that of glass can be used to correct for color aberration

25. Slide 25 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Chromatic aberrations For an achromatic doublet, visible wavelengths have approximately the same focal length

26. Slide 26 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Useful Factoids The intensity of light collected decreases as the square of the magnification The intensity of light increases as the square of the numerical aperture Thus when possible, use low magnification and high NA objectives and high NA objectives

27. Slide 27 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence Microscopes Cannot view fluorescence emission in a single optical plane Generally use light sources of much lower flux than confocal systems Are cheaper than confocal systems Give high quality photographic images (actual photographs) whereas confocal systems are restricted to small resolution images

28. Slide 28 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescent Microscope Dichroic Filter Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter EPI-Illumination

29. Slide 29 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Interference in Thin Films Small amounts of incident light are reflected at the interface between two material of different RI Thickness of the material will alter the constructive or destructive interference patterns - increasing or decreasing certain wavelengths Optical filters can thus be created that “interfere” with the normal transmission of light

30. Slide 30 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Interference and Diffraction: Gratings Diffraction essentially describes a departure from theoretical geometric optics Thus a sharp objet casts an alternating shadow of light and dark “patterns” because of interference Diffraction is the component that limits resolution

31. Slide 31 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Polarization and Phase: Interference Electric and magnetic fields are vectors - i.e. they have both magnitude and direction The inverse of the period (wavelength) is the frequency in Hz Wavelength (period T) Axis of Magnetic Field Axis of Propagation Axis of Electric Field Modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p78

32. Slide 32 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Interference Constructive Interference Destructive Interference A B C D A+B C+D Amplitude 0o0o 90 o 180 o 270 o 360 o Wavelength Figure modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p79 Here we have a phase difference of 180 o (2  radians) so the waves cancel each other out The frequency does not change, but the amplitude is doubled

33. Slide 33 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Construction of Filters Dielectric filter components Single Optical filter “glue”

34. Slide 34 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Anti-Reflection Coatings Optical Filter Multiple Elements Coatings are often magnesium fluoride Dielectric filter components

35. Slide 35 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Standard Band Pass Filters Transmitted Light White Light Source 630 nm BandPass Filter 620 -640 nm Light

36. Slide 36 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Standard Long Pass Filters Transmitted Light Light Source 520 nm Long Pass Filter >520 nm Light Transmitted Light Light Source 575 nm Short Pass Filter <575 nm Light Standard Short Pass Filters

37. Slide 37 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Optical Filters Dichroic Filter/Mirror at 45 deg Reflected light Transmitted LightLight Source 510 LP dichroic Mirror

38. Slide 38 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Filter Properties Light Transmission %T Wavelength 100 0 50 Notch Bandpass

39. Slide 39 t:/classes/BMS524/524lect2.ppt © 1995-2000 J.Paul Robinson - Purdue University Cytometry Laboratories Summary Lecture 2 Parts of the microscope (ocular, condenser) Objectives Numerical Aperture (NA) Refractive Index/refraction (RI) Aberrations Fluorescence microscope Properties of optical filters