Some lessons from last lab

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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

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Fluorescence Microscopyfluorescence imaging has
become themainstay of microscopy -- Nat.
Methods, 2005Objectives

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. YesB. No
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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.
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R O Y G B I V

• 700 650 600 550 500 450 400

wavelengths in nm
E h??
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http//www.chroma.com/products/catalog
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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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What color of light do we want a fluorescein
excitation filter to pass? A. Blue onlyB.
Green onlyC. Red onlyD. Any color light
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What color of light do we want a fluorescein
emission filter to pass? A. Blue onlyB. Green
onlyC. Red onlyD. Any color light
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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
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Epifluorescence
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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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Immunofluorescence

1. Direct
2. Indirect

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Wikipedia
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Immunofluorescence

1. Direct
2. Indirect

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Variable region
Constant region
Wikipedia
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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-immunoglobulinC. A
sheep anti-mouse-immunoglobulinD. A cow
anti-goat-immunoglobulin
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GFPGreen fluorescent protein
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http//probes.invitrogen.com/
To learn about fluorescence on the web
J. W. Lichtman and J. A. Conchello, 2005.
Fluorescence microscopy Nature Methods 2
910-919.http//www.nature.com/nmeth/journal/v2/n1
2/full/nmeth817.html
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Laser pointer 532nm (green)650nm (red)