biol125-lectures/methods.md

##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.

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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.

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##Secondary antibodies recognize primary antibodies and are species specific.

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Can couple many things to antibodies

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##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.

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##Immunocytochemistry

If you want to look at a macromolecule inside a cell, but it is not fluorescent...use indirect immuno-fluorescence microscopy

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##GFP is an intrinsically fluorescent protein

2008 Nobel prize site

2008 Nobel prize vid

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Freshly translated GFP is not fluorescent, but undergoes a self-catalyzed post-translational modification that generates a chromophore inside the barrel.

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##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

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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.

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##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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Brain imaging techniques

• Computerized tomography (CT) scan– uses X-rays in 3-dimensions to generate a brain image
• Can be digitally sectioned to show internal areas of the brain
• Can distinguish between grey matter and white matter, see the ventricles, has resolution of several millimeters

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Magnetic resonance imaging (MRI)

• Uses rotating magnets to generate image
• Non-invasive
• Can view images from any angle
• Resolution under 1 mm
• Can be adapted to do functional MRI imaging

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Tumor detection

MRI
CT-SCAN

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Magnetic resonance imaging (MRI)

Neuroscience 5e Animation 1.1

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functional magnetic resonance imaging (fMRI)

• Oxy-hemoglobin and deoxy-hemoglobin have different magnetic resonance signals
• Brain areas activated by a specific task utilize O2, then a pulse of O2 comes back and creates an influx of oxy-hemoglobin
• Can repeat task many times over
• Spatial resolution– millimeters
• Temporal resolution– seconds

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fMRI

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Mapping brain activity with fMRI

Neuroscience 5e Fig. 27.6

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Lie detection with fMRI

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build a new age lie detector

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