Basics of fluorescence microscopy

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Basics of fluorescence microscopy
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Sources
Dr. J. Paul Robinson, Purdue University
Cytometry Laboratories http//www.cyto.purdue.edu/
flowcyt/educate/pptslide.htm Molecular
Expressions http//micro.magnet.fsu.edu Molecular
Probes http//www.molecularprobes.com
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Lecture map

• A quick primer on light.
• What is fluorescence?
• How does the fluorescence microscope work?

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A quick primer on light
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Visible light- memorize this!
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Visible light

Visible light 400-700 nm
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Light as a wave

• ? wavelength
• - measured from peak to peak, in nanometers (nm)
• - seen as color
• Amplitude
• from peak to valley
• seen as intensity/brightness

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Visible light
400 nm
700 nm
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What is fluorescence?
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Fluorescence
light source/objective
Incident/epi-illumination fluorescence
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Fluorescence

• Fluorophore a molecule which absorbs light (of
  a certain color) and then emits light (of another
  color).
• light -gt fluorophore -gt light

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Fluorescence

• light -gt fluorophore -gt light

EMISSION
EXCITATION
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Fluorescence
specimen
EXCITATION
EMISSION
light source/objective
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Fluorescence

• A specimen can be stained with fluorophores or
  we can cause the specimen to make fluorophores
  (such as GFP).
• Fluorochrome protein with a fluorophore attached
  to it.

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Jablonski diagram
www.molecularprobes.com/handbook/figures/0664.html
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Fluorescence demo
http//micro.magnet.fsu.edu/primer/java/jablonski/
lightandcolor/index.html
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Jablonski diagram
Excitation
Emission
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Common fluorophores
Name ex
em FITC (Fluorescein) blue
GREEN Alexa-488 blue
GREEN eGFP
blue GREEN TRITC green
RED Texas Red green
RED Propidium Iodide (PI) green
RED DAPI violet BLUE
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How does the fluorescence microscope work?
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Optical filters
A FILTER lets only certain wavelengths of light
pass through (i.e. it transmits certain ?).
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optical filters example
not a filter
white light
white light
red filter
blue/green light absorbed RED PASSES THROUGH
white light
modifications by GG
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Optical filters- examples
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Fluorescence microscope
Two filters per fluorophore Excitation filter
lets only excitation color pass through. Barrier
or emission filter lets only emission color pass
through.
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Fluorescence microscope
white light -gt excitation color -gt specimen
excitation filter
specimen -gt emission color -gt see it
barrier filter
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Filter cube
Often the excitation and barrier filters are
physically mounted a cube, along with a
dichromatic mirror. Dichromatic (or dichroic)
mirror reflects the excitation wavelength and
transmits the emission wavelength.
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Filter cube
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Filter cube
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Filter cube
You need a specific cube (or filter set) for each
fluorophore you want to see on a microscope.
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Summary

• A quick primer on light. Wavelength color.
• What is fluorescence? Fluorophores, excitation
  and emission.
• How does the fluorescence microscope work?
  Filter cubes.

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Fluorescence tutorial movie

• http//www.molecularprobes.com/resources/education