3.4 KiB
##Fluorescence Microscopy
- Fluorescent molecules absorb light at one wavelength and emit it at another-longer wavelength.
- Uses optical filters to allow only light of a given wavelength in and out.
- Can detect specific proteins or other molecules in cells and tissues.
- Fluorescein (emits green), rhodamine (deep red) are molecules that can be chemically coupled to proteins to detect their localization indirectly
- GFP, isolated from jellyfish is a protein (encoded by a gene) that has intrinsic fluorescence.
Note:
Fluorescent molecules can be detected with light in very small amounts. This lets us look at specific molecules within a cell if they are tagged with a probe. Remember that most animal cells are not fluorescent. The fluorophore absorbs light energy of a specific wavelength and re-emits light at a longer wavelength. The absorbed wavelengths, energy transfer efficiency, and time before emission depend on both the fluorophore structure and its chemical environment, as the molecule in its excited state interacts with surrounding molecules.
##Secondary antibodies recognize primary antibodies and are species specific.
Note:
Can couple many things to antibodies
##Fluorescence Microscopy
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Here is the maximum excitation and emission wavelengths of several common flourescent probes. The photon emitted is of lesser energy that the one absorbed. In a microscope a filter set is used to only allow a specfic wavelength in and out to the eye.
##Immunocytochemistry
If you want to look at a macromolecule inside a cell, but it is not fluorescent...use indirect immuno-fluorescence microscopy
Note:
##GFP is an intrinsically fluorescent protein
Note:
Freshly translated GFP is not fluorescent, but undergoes a self-catalyzed post-translational modification that generates a chromophore inside the barrel.
##GFP can be expressed under
##specific gene promoters
Figure 9-25 Molecular Biology of the Cell (© Garland Science 2008)
Here expressed in fly peripheral neurons
Note:
Remember that GFP is a gene that encodes a protein. You can put it behind the promoter to detect which cells express a given gene.
##GFP can be used to create designer pets
http://news.nationalgeographic.com/news/2009/05/photogalleries/
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##All kinds of fluorescent protein variants these days
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