Imaging.

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Presentation transcript:

Imaging

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

Microscopy Light microscopy

Microscopy Light microscopy

Microscopy Light microscopy Bright field microscopy Wide field microscopy

Fluorescense

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

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

Fluorescent proteins

Fluorescent proteins

Fluorescent proteins Osamu Shimomura Martin Chalfie Roger Y. Tsien

Fluorescent proteins Brainbow mice

Epiflourescent microscope Confocal microscope Two photon microscope

Epi-fluorescent microscope Wide field microscopy

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

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

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

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

Confocal microscope Nikon tutorial – confocal VS. epifluorescent : http://www.microscopyu.com/tutorials/java/virtual/confocal/index.html

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)

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

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

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

Two photon microscope Detector

Two photon microscope Advantages: Less photo-bleaching Thicker tissue

Two photon microscope optical slicing 40X Focal plane 1mm

Two photon microscope