Confocal and 2-photon Imaging. October 15, 2010

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1 Confocal and 2-photon Imaging October 15, 2010

2 Review Optical Elements Adapted from Sluder & Nordberg 2007

3 Review Optical Elements Collector Lens Adapted from Sluder & Nordberg 2007

4 Review Optical Elements Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

5 Review Optical Elements Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

6 Review Optical Elements Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

7 Review Optical Elements Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

8 Review Optical Elements Collector Lens Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

9 Review Optical Elements Aperture Diaphragm Collector Lens Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

10 Review Optical Elements Field diaphragm Aperture Diaphragm Collector Lens Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

11 Review Optical Elements Field diaphragm Aperture Diaphragm Collector Lens Filter Cube Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

12 Review Optical Elements Tube Lens Field diaphragm Aperture Diaphragm Collector Lens Filter Cube Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007

13 Why care about confocal? Confocal Widefield From Stephen Smith

14 Why care about confocal? Confocal Widefield From Stephen Smith

15 Other methods of getting confocal like images De-convolution (software solution) Sine-modulated Structured Illumination (hardware & software solution) Useful References: Conchello & Lichtman 2005, Optical Sectioning Microscopy, Nature Methods Handbook of Biological Confocal Microscopy FSU microscopy Website

16 What does confocal do? Optical Sectioning Getting rid of stray light See light coming from a thin section (deblur the image) Controlling depth of field Slightly better resolution

17 Remove stray light - Pinhole Illumination Condenser Objective From Stephen Smith

18 Remove image blur - Pinhole Detection From Stephen Smith

19 Control Depth of Field with Pinhole Size Optimal pinhole size? Airy Unit From Stephen Smith

20 Point Spread Function Image Plane Point Source

21 From Jeff Lichtman

22 From Jeff Lichtman

23 Adding Sources

24 From Jeff Lichtman

25 Why is the PSF worse in the 2 3

26

27 Effect of NA on Airy disk pattern Low NA Med NA High NA From Jeff Lichtman

28 Diffraction Limited Imaging From Jeff Lichtman

29 What is the best pinhole size to use? An Airy Disk Size! ie. 1 Airy Unit

30 What is the best pinhole size to use? An Airy Disk Size! ie. 1 Airy Unit What happens if I go lower than 1 Airy Unit?

31 Confocal enhances resolution What is resolution? How is it defined?

32 From Jeff Lichtman

33 From Jeff Lichtman

34 From Stephen Smith

35 From Stephen Smith

36 Limitations Bleaching out of focus regions High laser intensities killed live specimens Time it takes to scan Poor in deep scattering tissue

37 Bleaching out of focus regions Workaround: 2-photon From Stephen Smith

38 High intensities hurt live cells Work around: Spinning disk decrease light intensity 2-photon shift light to longer wavelengths Mainster MA 2006

39 Spinning Disk Scan multiple times w/ lower intensity EMCCD quantum efficiency Nipkow Disk Our CSU10 spinning disk spins at 1800 rpm, that s 3 rotations in 100ms (36 scans of the image). From Zeiss online campus

40 Quantum Efficiency Photometrics QuantEM 512sc

41 Multiplexing Time it takes to scan From Zeiss online campus

42 2-Photon Microscopy

43

44

45 From Stephen Smith

46 From Stephen Smith

47 From Stephen Smith

48 Depth Penetration Image depths 2-3 times that of single photon confocal microscopy

49 Lasers Light Amplification by Stimulated Emission of Radiation High intensity stimulation Monochromatic Coherent Stability

50 From Stephen Smith

51

52 END

53

54

55

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