1. Dave Piston May 18, 2014 Optical Sectioning 2: Confocal Designs and “Pseudo-confocals” Outline 1.PMT detectors 2.Laser Scanning Design Features 3.Pseudo-confocals 4.Signal and Background

2. Photomultiplier Tube (PMT) High-gain leads to high S/N Can detect single photons PMTs are very fast Drawback is relatively low QE

3. Newer PMTs (GaAsP) From Hamamatsu

4. CCD –QE Comparison

5. Non 90 ° Dichroic Mirror Alignment From Nikon Using laser excitation, it is no longer necessary to use a 90 ° orientation for the dichroic mirror. This has tremendous impact on multiband dichroic design and also for spectral imaging.

6. 2 Laser Synchronized Scanning One laser stimulates, the second laser simultaneously images. Coordination of stimulation and imaging is useful for FRAP, FLIP and photoactivation. From Olympus

7. Multi-Tracking: Rapid Laser Switching via AOTF FITC 488 nm Cy3 543 nm Overlay Both lasers ONMulti-Tracking: Lasers switching off and on From Zeiss Web Page

8. Spectral windows instead of filters: Leica AOBS Prism and slit system gives spectrometer type abilities No barrier filters No dichroic filters Up to 4 channels From Leica

9. AOBS Operation From Leica

10. 1. Order out (= ex in) 0. Order out (= em out) In (= ex in = em out) AOBS Operation From Leica

11. AOBS Operation From Leica

12. PMT array with 32 elements ZEISS LSM 510 META Contrast by Fluorescence Spectra

13. ZEISS LSM 710 Low-angle Spectral recycling

14. White Light Continuum Generation Laser From Leica

15. Excitation-Emission Contour Courtesy : Alberto Diaspro, Ph.D. University of Genoa From Leica

16. Spinning Disk with Microlens Array Only 1% of Nipkow disk is holes, this leads to low excitation rates Lens array directs >50% of the excitation light through the Nipkow Disk pinholes But, faster acquisition means faster photobleaching!

17. Laser Intensity Confocal Detection Confocal Microscopy

18. 2-D Array Scanning with Microlens Array is scanned and resulting fluorescence is descanned by a galvo Lens array directs many spots to the sample Again, faster acquisition means faster photobleaching!

19. Sweptfield Confocal (Prairie/Nikon) Prairie Website

20. Sweptfield Confocal (Prairie/Nikon) Prairie Website

21. Sweptfield Confocal (Prairie/Nikon) Prairie Website

22. Fast Confocal Line Scanning Illuminate whole line Detect through confocal slit Optical “trick” instead of a traditional dichroic mirror Uses a CCD-type detector Zeiss 5Live

23. Spinning Slit Microscope Olympus DSU

24. Laser Intensity Confocal Detection Confocal Microscopy

25. Pinhole Effect on Confocal Resolution Theory Experiment Widefield Pinhole 1 AU 0.2 AU Confocal Detection

26. t = 0.07umt = 0.75umt = 7.5um t = 75 um Confocal 55 21 2.9 2.6 Spinning disk 55 21 2.2 0.40 Line illumination 10 4.9 0.59 0.30 Conventional 3.4 1.8 0.084 0.0078 1.25-NA oil-immersion objective, and 488 nm. Signal-to-Background vs. Sample Thickness