Confocal Microscopy

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Confocal Microscopy

• Aaron Derdowski
• Spearman Lab
• Aaron.derdowski_at_vanderbilt.edu
• D6201MCN

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Outline

• Fluorescence Microscopy
• Confocal Microscopy
• Basic Imaging
• Colocalization
• Live cell imaging
• FRET/FRAP
• Problem set

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Fluorescence
Luminescence
Photoluminescence
Fluorescence
Phosphorescence
Luminescence Family of processes in which
susceptible molecules emit light from
electronically excited states Photoluminescence
Generation of luminescence through excitation by
light Fluorescence is the property of some atoms
and molecules to absorb light at a particular
wavelength and to subsequently emit light of
longer wavelength after a brief interval, termed
the fluorescence lifetime. Phosphorescence
occurs in a manner similar to fluorescence, but
with a much longer excited state lifetime.
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Fluorescence Microscopes

• Generally use light sources of much lower flux
  than confocal systems
• Are cheaper than confocal systems
• Cannot view fluorescence emission in a single
  optical plane

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Fluorescent Microscope
Confocal Microscope
Arc Lamp
Excitation Diaphragm
Excitation Filter
Ocular
Objective
Emission Filter
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Fluorescent Microscope
Confocal Microscope
Arc Lamp
Laser
Excitation Diaphragm
Excitation Pinhole
Excitation Filter
Excitation Filter
Ocular
PMT
Objective
Emission Filter
Objective
Emission Filter
Emission Pinhole
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Benefits of Confocal Microscopy

• Control depth of field
• Elimination of Background
• Ability to collect optical sections

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Vanderbilt Cell Imaging Shared Resource (CISR)

• http//www.mc.vanderbilt.edu/cellimage/

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Basic Imaging
Protein A
Y
Y
Y
Protein A
Protein A
GFP
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Optical Slices

• The primary advantage of laser scanning confocal
  microscopy is the ability to serially produce
  thin optical sections.
• The image series is collected by coordinating
  incremental changes in the microscope fine focus
  mechanism with sequential image acquisition at
  each step.
• The non-invasive confocal optical sectioning
  technique enables the examination of both living
  and fixed specimens under a variety of conditions
  with enhanced clarity.

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Colocalization

• Two proteins at the same physical location in the
  cell
• Colocalization vs. protein-protein interaction
• Is it data, or just a picture?

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Quantitation

• Analyzed by comparing color values for each
  pixel, and plotting against each other
• Red-Green is most popular color pair

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Example
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Live Cell Imaging

• Able to image cells in real time
• Avoid problems with fixation
• Must use fusion proteins
• Difficult to quantitate movement

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FRETFluorescence Resonance Energy Transfer

• Overlap
• Orientation
• Distance

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FRETFluorescence Resonance Energy Transfer
YFP
514nm
527nm
CFP
CFP
YFP
458nm
501nm
458nm
501nm
527nm
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FRET Microscopy
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FRET at the Plasma Membrane
458nm
501nm
527nm
GagYFP
GagCFP
GagCFP
GagYFP
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Gag-Gag FRET at the Plasma Membrane
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FRAPFluorescence Recovery After Photobleaching
F
Intense laser Beam Bleaches Fluorescence
Time
Recovery of fluorescence
10 seconds
Zero time
30 seconds
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Summary

• Advantages and Disadvantages to Confocal vs.
  Fluorescence Microscopy
• Great tool for asking questions, must be sure to
  control properly to obtain actual data.
• Live cell imaging
• FRET can be used to measure protein-protein
  interactions
• FRAP can be used to measure protein movement
  within a cell

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Problem 1
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Problem 2