Some lessons from last lab 1. Importance of illumination and proper

Some lessons from last lab 1. Importance of illumination and proper adjustment of the condenser 2. How to find objects, especially if they are transparent

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

Have you used a fluorescence microscope before? A. Yes B. No

Fluorescence microscopy uses substances called “fluorophores” or “fluorochromes” as stains. These substances have the property that they “fluoresce”, i.e. they absorb some wavelength of light, become excited, and decay to the ground state by emitting a slightly longer wavelength of light.

R O Y G B I V 700 650 600 550 500 450 400 wavelengths in nm E h

http://www.chroma.com/products/catalog

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

What color of light do we want a fluorescein excitation filter to pass? A. Blue only B. Green only C. Red only 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

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

Immunofluorescence 1. Direct 2. Indirect

Wikipedia

Immunofluorescence 1. Direct 2. Indirect

Variable region Constant region Wikipedia

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

To learn about fluorescence on the web: http://probes.invitrogen.com/ J. W. Lichtman and J. A. Conchello, 2005. Fluorescence microscopy Nature Methods 2: 910-919. http://www.nature.com/nmeth/journal/v2/n12/full/nmeth8 17.html

Laser pointer 532nm (green) 650nm (red)