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Imaging.

Published byEileen Carmel Mitchell Modified over 8 years ago

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Presentation on theme: "Imaging."— Presentation transcript:

1 Imaging

2 Out-line Microscopy Fluorescence Epifluorescence microscopy
Confocal microscopy Two-photon microscopy

3 Microscopy Light microscopy

4 Microscopy Light microscopy

5 Microscopy Light microscopy Bright field microscopy
Wide field microscopy

6 Fluorescense

7 Fluorescense S1 S0 (Simplified Jablonski diagram) Photo-bleaching….

8 microtubules (red), chromosomes (blue) and TPX2 (green).

9 Fluorescent proteins

10 Fluorescent proteins

11 Fluorescent proteins Osamu Shimomura Martin Chalfie Roger Y. Tsien

12 Fluorescent proteins Brainbow mice

13 Epiflourescent microscope
Confocal microscope Two photon microscope

14 Epi-fluorescent microscope
Wide field microscopy

15 Epi-fluorescent microscope
Two photon microscopy Epi-fluorescent microscope Limitations: Wide field = more photo-bleaching Tissue must be very thin

16 Epi-fluorescent microscope
Limitations: Wide field = more photo-bleaching Tissue must be very thin X20 Z

17 Epi-fluorescent microscope
Limitations: Wide field = more photo-bleaching Tissue must be very thin X20 Z

18 Confocal microscope Less photobleaching 2. Pinhole =
Dichroic mirror Sample Objective Detector Not wide field = Less photobleaching 2. Pinhole = Better Z resolution (pinhole size)

19 Confocal microscope Nikon tutorial – confocal VS. epifluorescent :

20 Confocal microscope Place for improvement:
Pinhole Dichroic mirror Sample Objective Detector Place for improvement: photobleaching Z resolution (High laser intensities are bad for the tissue)

21 The higher the energy the shorter the wave length
Two photon microscope So S1’ Energy S1 One photon excitation Two photon excitation S2’ S1 Excitation Excitation emission emission So (Simplified Jablonski diagram) The higher the energy the shorter the wave length

22 Two photon microscope Advantages:
Less photo-bleaching and tissue damage

23 Two photon microscopy Obj. Obj.
Zipfel et al. Nature Biotechnology (2003)

24 Two photon microscope Detector

25 Two photon microscope Advantages: Less photo-bleaching Thicker tissue

26 Two photon microscope optical slicing 40X Focal plane 1mm

27 Two photon microscope

28

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