03 MICROSCOPY 2 2008 - D. Any color light What color of...

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Fluorescence Microscopy “fluorescence imaging has become the mainstay of microscopy” -- Nat. Methods, 2005 Objectives: 1. Understand the basics of fluorescence 2. Understand how fluorophores are used in microscopy 3. Understand how a microscope is set up to do fluorescence 4. Understand basics of immunofluorescence
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Have you used a fluorescence microscope before? A. Yes B. No
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Light is white
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Light is white But is composed of a mixture of colors.
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R O Y G B I V 700 650 600 550 500 450 400 E = h ν = h/f wavelengths in nm
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QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.
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FITC Fluorescein isothiocyanate
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A max = 490 nm E max = 520 nm FITC
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How fluorophores are used 1. Some bind to biological specimens 2. Others can be coupled (conjugated) to other non-fluorescent molecules that bind to specimens
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http://probes.invitrogen.com/
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http://fluorescence.nexus-solutions.net/frames6.htm
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What color of light do we want a fluorescein excitation filter to pass? A. Blue only B. Green only C. Red only
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Unformatted text preview: D. Any color light What color of light do we want a fluorescein emission filter to pass? A. Blue only B. Green only C. Red only D. Any color light What do we see when we look down a fluorescence microscope at a specimen stained with fluorescein? A. Blue background with green stain B. Green background with blue stain C. Red background with green stain D. Black background with green stain Epifluorescence Immunofluorescence 1. Direct 2. Indirect Imagine you made an antibody to protein Y by injecting a goat. Which of the following would be okay as a secondary antibody to localize protein Y? A. A goat anti-rabbit-immunoglobulin B. A horse anti-chicken-immunoglobulin C. A sheep anti-mouse-immunoglobulin D. A cow anti-goat-immunoglobulin GFP Green fluorescent protein J. W. Lichtman and J. A. Conchello, 2005. Fluorescence microscopy Nature Methods 2: 910-919. http://www.nature.com/nmeth/journal/v2/n12/full/nmeth817.html...
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This note was uploaded on 04/07/2008 for the course M 408c taught by Professor Mcadam during the Spring '06 term at University of Texas at Austin.

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03 MICROSCOPY 2 2008 - D. Any color light What color of...

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