1. Slide 1 t:/PowerPoint/confoc/lect1nu.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 stated. Textbook for this lecture series in Jim Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively for material and ideas to support the class. Lecture 1 The Principles of Microscopy UPDATED October 27, 1998

2. Slide 2 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Evaluation End of term quiz - 100% grade

3. Slide 3 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Introduction to the Course Microscopy Fluorescence Basic Optics Confocal Microscopes Basic Image Analysis 3D image analysis Live Cell Studies Advanced Applications

4. Slide 4 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Introduction to Lecture 1 Early Microscope Modern Microscopes Magnification Nature of Light Optical Designs

5. Slide 5 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Microscopes Upright Inverted Köhler Illumination Fluorescence Illumination

6. Slide 6 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Earliest Microscopes 1590 - Hans & Zacharias Janssen of Middleburg, Holland manufactured the first compound microscope 1673 Antioni Van Leeuwenhoek created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683

7. Slide 7 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Early Microscopes (Hooke)

8. Slide 8 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Secondary Microscopes In 1827 Giovanni Battista Amici, built high quality microscopes and introduced the first matched achromatic microscope in 1827. He recognized the importance of coveralls thickness and developed the concept of “water immersion” Carl Zeiss and Ernst Abbe developed oil immersion systems by developing oils that matched the refractive index of glass. Dr Otto Schott formulated glass lenses that color-corrected objectives and produced the first “apochromatic” objectives in 1886.

9. Slide 9 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Modern Microscopes Early 20th Century Professor Köhler developed the method of illumination still called “Köhler Illumination” Köhler recognized that using shorter wavelength light (UV) could improve resolution

10. Slide 10 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Köhler Köhler illumination creates an evenly illuminated field of view while illuminating the specimen with a very wide cone of light Two conjugate image planes are formed –one contains an image of the specimen and the other the filament from the light

11. Slide 11 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Köhler Illumination Specimen Field stop Field iris Conjugate planes for illuminating rays Specimen Field stop Field iris Conjugate planes for image-forming rays condenser eyepiece retina

12. Slide 12 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Some Principles Rule of thumb is is not to exceed 1,000 times the NA of the objective Modern microscopes magnify both in the objective and the ocular and thus are called “compound microscopes”

13. Slide 13 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Magnification An object can be focussed generally no closer than 250 mm from the eye (depending upon how old you are!) this is considered to be the normal viewing distance for 1x magnification Young people may be able to focus as close as 125 mm so they can magnify as much as 2x because the image covers a larger part of the retina

14. Slide 14 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Magnification 1000mm 35 mm slide 24x36 mm M = 1000 mm 36 mm = 28 The projected image is 28 times larger than we would see it at 250 mm from our eyes. If we used a 10x magnifier we would have a magnification of 280x, but we would reduce the field of view by the same factor of 10x.

15. Slide 15 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Basic Microscopy Bright field illumination does not reveal differences in brightness between structural details - i.e. no contrast Structural details emerge via phase differences and by staining of components The edge effects (diffraction, refraction, reflection) produce contrast and detail

16. Slide 16 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Some Definitions Absorption –When light passes through an object the intensity is reduced depending upon the color absorbed. Thus the selective absorption of white light produces colored light. Refraction –Direction change of a ray of light passing from one transparent medium to another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface, with greater deviation for shorter wavelengths Diffraction –Light rays bend around edges - new wavefronts are generated at sharp edges - the smaller the aperture the lower the definition Dispersion –Separation of light into its constituent wavelengths when entering a transparent medium - the change of refractive index with wavelength, such as the spectrum produced by a prism or a rainbow

17. Slide 17 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Absorption Control No blue/green light red filter

18. Slide 18 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Absorption Chart Color in white light Color of light absorbed red blue green magenta cyan yellow blue green red black gray green blue pink

19. Slide 19 t:/PowerPoint/confoc/lect1nu.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

20. Slide 20 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Refraction But it is really here!! He sees the fish here….

21. Slide 21 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Upright Scope

22. Slide 22 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Inverted Microscope

23. Slide 23 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Microscope Basics Originally conformed to the German DIN standard Standard required the following –real image formed at a tube length of 160mm –the parfocal distance set to 45 mm –object to image distance set at 195 mm Currently we use the ISO standard

24. Slide 24 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories The Conventional Microscope Focal length of objective = 45 mm Object to Image Distance = 195 mm Mechanical tube length = 160 mm Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press

25. Slide 25 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Conventional Finite Optics with Telan system Sample being imaged Intermediate Image Telan Optics Objective Other optics Ocular 45 mm 160 mm 195 mm Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press

26. Slide 26 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Infinity Optics Sample being imaged Primary Image Plane Objective Other optics Ocular Other optics Tube Lens Infinite Image Distance The main advantage of infinity corrected lens systems is the relative insensitivity to additional optics within the tube length. Secondly one can focus by moving the objective and not the specimen (stage) Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press

27. Slide 27 t:/PowerPoint/confoc/lect1nu.ppt Purdue University Cytometry Laboratories Summary Lecture 1 Upright and inverted microscopes Köhler illumination Refraction, Absorption, dispersion, diffraction Magnification Optical Designs - 160 mm and Infinity optics