1. Slide 1 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories 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. Lecture 2 The Principles of Microscopy UPDATED October 27, 1998

2. Slide 2 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Review Microscope Basics Magnification Optical systems

3. Slide 3 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Microscope Components Fluorescence Microscope Numerical Aperture Refractive Index Aberrations Objectives

4. Slide 4 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Definitions Refractive Index Aberrations Fluorescence

5. Slide 5 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Fluorescent Microscope Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter EPI-Illumination

6. Slide 6 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Construction of Filters Dielectric filter components Single Optical filter “glue”

7. Slide 7 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Anti-Reflection Coatings Optical Filter Multiple Elements Coatings are often magnesium fluoride

8. Slide 8 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Standard Band Pass Filters Transmitted Light White Light Source 630 nm BandPass Filter 620 -640 nm Light

9. Slide 9 t:/powerpnt/confoc/lect2nu.ppt 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

10. Slide 10 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Optical Filters Dichroic Filter/Mirror at 45 deg Reflected light Transmitted LightLight Source 510 LP dichroic Mirror

11. Slide 11 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Filter Properties Light Transmission %T Wavelength 100 0 50 Notch Bandpass

12. Slide 12 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Refraction 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

13. Slide 13 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Light striking a surface tt ii rr Incident Beam Reflected Beam Transmitted (refracted)Beam

14. Slide 14 t:/powerpnt/confoc/lect2nu.ppt 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)

15. Slide 15 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Microscope Objectives

16. Slide 16 t:/powerpnt/confoc/lect2nu.ppt 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

17. Slide 17 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Objectives Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion): d = 1.22  Thus high NUMERICAL APERTURE is critical for high magnification In a medium of refractive index n the wavelength gets shorter:  n This defines a “resel” or “resolution element”

18. Slide 18 t:/powerpnt/confoc/lect2nu.ppt 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

19. Slide 19 t:/powerpnt/confoc/lect2nu.ppt 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 =  sin  –(n=the lowest refractive index between the object and first objective element) (hopefully 1) –  is 1/2 the angular aperture of the objective

20. Slide 20 t:/powerpnt/confoc/lect2nu.ppt 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)…. 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….. = =

21. Slide 21 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Object Resolution Example: 40 x 1.3 N.A. objective 2 x NA.00053 2 x 1.3 = 0.20  m = 40 x 0.65 N.A. objective 2 x NA.00053 2 x.65 = 0.405  m =

22. Slide 22 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Microscope Objectives Specimen Coverslip Oil Microscope Objective Stage 60 1.4 NA PlanApo

23. Slide 23 t:/powerpnt/confoc/lect2nu.ppt 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 Wat er n=1.52 Air

24. Slide 24 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Monochromatic Aberrations –Spherical aberration –Coma –Astigmatism –Flatness of field –Distortion Chromatic Aberrations –Longitudinal aberration –Lateral aberration Sources of Aberrations

25. Slide 25 t:/powerpnt/confoc/lect2nu.ppt 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

26. Slide 26 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories 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. From:Handbook of Biological Confocal Microscopy J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed 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. Note: For class use Figure is under box

27. Slide 27 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories 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. 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. Note: For class use Figure is under box

28. Slide 28 t:/powerpnt/confoc/lect2nu.ppt 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.

29. Slide 29 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Useful Facts 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.

30. Slide 30 t:/powerpnt/confoc/lect2nu.ppt 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 much cheaper than confocal systems Give high quality photographic images (actual photographs) whereas confocal systems are restricted to small resolution images

31. Slide 31 t:/powerpnt/confoc/lect2nu.ppt Purdue University Cytometry Laboratories Summary Lecture 2 Properties of optical filters Objectives Numerical Aperture Refractive Index/Refraction Aberrations Fluorescence Microscope - introduction Purdue University Cytometry Laboratories