1. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Lecture 4 The Principles of Confocal Microscopy: Components of the microscope. BMS 524 - “Introduction to Confocal Microscopy and Image Analysis” 1 Credit course offered by Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine UPDATED October 27, 1998 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.

2. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Overview Components of a confocal microscope system Optical pathways Optical filters Resolution 3D basics Other components

3. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Benefits of Confocal Microscopy Reduced blurring of the image from light scattering Increased effective resolution Improved signal to noise ratio Clear examination of thick specimens Z-axis scanning Depth perception in Z-sectioned images Magnification can be adjusted electronically

4. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Fluorescent Microscope Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter

5. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Confocal Principle Objective Laser Emission Pinhole Excitation Pinhole PMT Emission Filter Excitation Filter

6. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Fluorescent Microscope Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter Objective Laser Emission Pinhole Excitation Pinhole PMT Emission Filter Excitation Filter Confocal Microscope

7. BMS 524: Lecture 3 Purdue University Cytometry Laboratories MRC 1024 System UV Laser Kr-Ar Laser Optical Mixer Scanhead Microscope

8. BMS 524: Lecture 3 Purdue University Cytometry Laboratories MRC 1024 System Light Path PMT

9. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Optical Mixer - MRC 1024 UV Argon Laser Argon- Krypton Laser Fast Shutter UV Correction Optics Filter Wheels To Scanhead UV Visible Beam Expander

10. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Optical Mixer - MRC 1024 UV Argon Laser Argon- Krypton Laser Fast Shutter UV Correction Optics Filter Wheels To Scanhead UV Visible 353,361 nm 488, 514 nm 488,568,647 nm

11. BMS 524: Lecture 3 Purdue University Cytometry Laboratories MRC 1024 Scanhead From Laser To and from Scope 3 2 1PMT Galvanometers Emission Filter Wheel

12. BMS 524: Lecture 3 Purdue University Cytometry Laboratories To Scanhead From Scanhead

13. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Scanning Galvanometers x y Laser in Laser out Point Scanning To Microscope

14. BMS 524: Lecture 3 Purdue University Cytometry Laboratories The Scan Path of the Laser Beam 767, 1023, 1279 511, 1023 0 0 Start Specimen Frames/Sec# Lines 1512 2256 4128 8 64 16 32

15. BMS 524: Lecture 3 Purdue University Cytometry Laboratories How a Confocal Image is Formed Condenser Lens Pinhole 1 Pinhole 2 Objective Lens Specimen Detector Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989

16. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Fundamental Limitations of Confocal Microscopy From Source To Detector. x,y,z 22 n 2 photons 2 11 n 1 photons 1 zz yy xx VOXEL PIXEL From: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989

17. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Optical Resolution Gray Level Pixelation Aberrations

18. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Gray Level & Pixelation Analogous to intensity range For computer images each pixel is assigned a value. If the image is 8 bit, there are 2 8 or 256 levels of intensity If the image is 10 bit there are 1024 levels, 12 bit 4096 levels etc. The intensity analogue of a pixel is its grey level which shows up as brightness. The display will determine the possible resolution since on a TV screen, the image can only be displayed based upon the number of elements in the display. Of course, it is not possible to increase the resolution of an image by attributing more “pixels” to it than were collected in the original collection!

19. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Pixels Pixels & image structure Hardcopy usually compromises pixel representation. With 20/20 vision you can distinguish dots 1 arc second apart (300  m at 1 m) so 300 DPS on a page is fine. So at 100 m, you could use dots 300 mm in size and get the same effect! Thus an image need only be parsimonius, i.e., it only needs to show what is necessary to provide the expected image.

20. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Pixels T

21. BMS 524: Lecture 3 Purdue University Cytometry Laboratories

22. BMS 524: Lecture 3 Purdue University Cytometry Laboratories 320x240 x 24 Zoom x 2 Zoom x 8 Zoom x 4 Magnifying with inadequate information. This is known as “empty magnification” because there are insufficient data points. The final image appears to be very “boxy” this is known as “pixilation”.

23. BMS 524: Lecture 3 Purdue University Cytometry Laboratories 541x600x8 361x400x8 180x200x8 Magnifying with adequate information. Here, the original image was collected with many more pixels - so the magnified image looks better! Socrates?….well perhaps not...

24. BMS 524: Lecture 3 Purdue University Cytometry Laboratories 320x240 x 24 1500x1125x24 Originals collected at high resolution - compared to a low resolution image magnified

25. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Sampling Theory The Nyquist Theorem –Nyquest theory describes the sampling frequency required to represent the true identity of the sample. –i.e., how many times must you sample an image to know that your sample truly represents the image? Nyquist claimed that the rate was 2f. It has been determined that in reality the rate is 2.3f - in essence you must sample at least 2 times the highest frequency.

26. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Raman Scattering At an excitation line of 488 nm, Raman scatter will be at 584 nm or less with increased concentration of protein, etc. Is directly proportional to the power of the laser light

27. BMS 524: Lecture 3 Purdue University Cytometry Laboratories 3D Image Reconstruction z x y

28. BMS 524: Lecture 3 Purdue University Cytometry Laboratories z x y z y y 3D Image Reconstruction

29. BMS 524: Lecture 3 Purdue University Cytometry Laboratories x y z y y z 3D Image Reconstruction

30. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Fluorescent image of paper

31. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Pine Tree pollen - collected on a Bio-Rad MRC 1024 at Purdue University Cytometry Laboratories

32. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Fly eye! - collected on a Bio-Rad MRC 1024 at Purdue University Cytometry Laboratories

33. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Collagen fibers collected using transmitted light and fluorescence [ collected on a Bio-Rad MRC 1024 at Purdue University Cytometry Laboratories ]

34. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Collagen fibers collected using transmitted light [ collected on a Bio-Rad MRC 1024 at Purdue University Cytometry Laboratories ]

35. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Top: Endothelial cells (live) cultured on a coverslide chamber. The cells were stained with stain that identified superoxide production (hydroethidine) and were color coded (red =high stain, green =low stain) then a 3D reconstruction performed and a vertical slice of the culture shown. Here, the original image was collected with many more pixels - so the magnified image looks better! Left: Same endothelial cells with hydroethidine stain (live cells) showing a fluorescence reconstruction - note fluorescence is only in nuclear regions - no cytoplasm is stained. Imaged on Bio-Rad MRC 1024 system. Top: Endothelial cells (live) cultured on a coverslide chamber. The cells were stained with stain that identified superoxide production (hydroethidine) and were color coded (red =high stain, green =low stain) then a 3D reconstruction performed and a vertical slice of the culture shown. Here, the original image was collected with many more pixels - so the magnified image looks better! Left: Same endothelial cells with hydroethidine stain (live cells) showing a fluorescence reconstruction - note fluorescence is only in nuclear regions - no cytoplasm is stained. Imaged on Bio-Rad MRC 1024 system.

36. BMS 524: Lecture 3 Purdue University Cytometry Laboratories

37. BMS 524: Lecture 3 Purdue University Cytometry Laboratories Creating Stereo pairs z x y Pixel shifting -ive pixel shift for left +ive pixel shift for right

38. BMS 524: Lecture 3 Purdue University Cytometry Laboratories

39. BMS 524: Lecture 3 Purdue University Cytometry Laboratories SUMMARY SLIDE Components of a confocal system Optical pathways Optical filters Resolution 3D basics Light paths and lasers