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Fluorescence Microscopy“fluorescence imaging has become themainstay of microscopy” -- Nat. Methods, 2005Objectives: Understand the basics of fluorescence Understand how fluorophores are used in microscopy Understand how a microscope is set up to do fluorescence Understand basics of immunofluorescence Have you used a fluorescence microscope before? A. YesB. No Light is white Light is whiteBut is composed of a mixture of colors. R O Y G B I V 700 650 600 550 500 450 400 FITC Fluorescein isothiocyanate How fluorophores are used Some bind to biological specimens Others can be coupled (conjugated) to other non-fluorescent molecules that bind to specimens / What color of light do we want a fluorescein excitation filter to pass? A. Blue onlyB. Green onlyC. Red onlyD. Any color light What color of light do we want a fluorescein emission filter to pass? A. Blue onlyB. Green onlyC. Red onlyD. 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 stainB. Green background with blue stainC. Red background with green stainD. Black background with green stain Epifluorescence Immunofluorescence Direct 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-immunoglobulinB. A horse anti-chicken-immunoglobulinC. A sheep anti-mouse-immunoglobulinD. A cow anti-goat-immunoglobulin GFPGreen fluorescent protein J. W. Lichtman and J. A. Conchello, 2005. Fluorescence microscopy Nature Methods 2: 910-919./nmeth/journal/v2/n12/full/nmeth817.html * E = h????h/f wavelengths in nm Amax = 490 nm Emax = 520 nm FITC /frames6.htm *