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O-MUG

Comprehensive Course on Fluorescence Microscopy


Advanced Confocal/Multiphoton Techniques

Approximate Time:
Pre-requisite: Confocal/Multiphoton Microscopy Training
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This is an extension of the Confocal/Multiphoton Microscopy Training course. It is tailored to the user's needs for a specific technique such as deep two-photon imaging, intravital imaging, FRET, FRAP, and second harmonic generation. These techniques are not available on all microscopes. Please come talk to us well in advance to discuss you applications, so that we can help you choose the right instrument. The following is a list of the techniques and a description along with the microscopes that they can be performed on:

  • Deep two-photon imaging (Two-photon): This technique involves exciting the sample with about twice as long a wavelength as you would use in normal confocal excitation. The advantage of this is that you can penetrate deeper into samples since there is less light lost through scattering. This excitation is a highly improbable event except for in a small region of the focal plane, thus it eliminates the need to use a confocal pinhole to block out of focus light. For two-photon imaging we use a tunable Chameleon laser from 720-920nm.
  • Intravital imaging (Two-photon, Leica FCM): This involves imaging a live animal (usually a mouse) in confocal mode. There is usually alot of prep work involved, such as inserting a window chamber in the mouse (this is usually done at the beginning of the experiment) or surgically opening up the animal for observation, anesthesizing the animal, and finding a way to properly brace the animal on the microscope stage.
  • FRET (Two-photon)
  • FRAP (Two-photon)
  • Second Harmonic Imaging (Two-photon): This is a contrast imaging technique used to image cell and tissue structure and function. Biological materials, such as collagen, microtubules, and muscle myosin can produce second harmonic signals. This method requires intense laser light passing through the specimen. The light is altered by the molecular structure of the specimen, causing it to emerge with exactly half the wavelength (frequency doubled).

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