Fluorescence Microscopy

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Fluorescence Microscopy

• Physical Biochemistry, November 2006
• Dr Ardan Patwardhan, a.patwardhan_at_imperial.ac.uk,
  Faculty of Natural Sciences, Imperial College
  London

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Fluorescence

• Quantum Efficiency Q.E. Emitted photons/
  Absorbed photons
• The Molar Extinction Coefficient, e (M-1cm-1) is
  the absorbance of a 1 molar solution in a 1 cm
  lightpath at a specified wavelength.

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Confocal Microscopy

• Only one point in the specimen is imaged at any
  one given time
• By scanning the specimen in 3D, a 3D
  reconstruction of the specimen can be obtained

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Confocal Resolution

• Lateral resolution
• Axial resolution
• Ex l 400nm, n 1.5, NA 1.4 ? Dx 0.13
  mm, Dz 0.43 mm

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Case Study The glomerulus

• The glomerulus is the main filter of the nephron
  and is located within the Bowman's capsule. The
  glomerulus is semipermeable, allowing water and
  soluble wastes to pass through and be excreted
  out of the Bowman's capsule as urine
• The glomerulus is quite big (100-200 mm), which
  is why serial sectioning has to be used in
  combination with confocal microscopy to image the
  whole structure

Microsc. Microanal. 12, 262268, 2006
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Problems Speed

• Scanning a 3D specimen point by point takes some
  time
• Hybrid techniques have therefore been developed
  that compromize on confocality in order to gain
  speed
• On such technique involves scanning one line in
  the specimen at a time onto a CCD detector
• The loss of resolution in the z direction is not
  that pronounced
• Another technique involves scanning multiple
  points (well separated in space) in parrallel

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Problems Cross-talk
l
Detector 1
Detector 2
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Problems Fading

• Not well understood - Free radicals are thought
  to be an important culprit !
• Many anti-fading agent now available
• Anti-fading agents can be very cyto-toxic !

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Two photon microscopy

• Possible to use two photons of half the energy
  normally required if intensity is high enough
• Intensity high enough only in focal region 3D
  localization with the need for confocal!

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Advantages/Disadvantages

• Use of IR excitation allows penetration into
  deeper tissue
• Bleaching and phototoxic effects limited to focal
  region

• Resolution is slightly worse than confocal
• Requires lasers capable of generating intense
  short (100fs) pulses

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Deconvolution microscopy

• In a deconvolution microscope, several images of
  the specimen are taken while moving the specimen
  towards the objective
• No scanning in the x-y plane or pinholes is
  involved
• The smudging of information in the z-direction is
  mathematically compensated for computationally
• Simple, cheap and quick
• For many complicated specimens, not as good as
  confocal or two-photon

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FRET/ FRAP/anisotropy/liftetime imaging

• Most of the spectroscopic techiques that we have
  studied can be combined with microscopy in order
  to perform 3D/4D studies

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Antibody labeling

• Amplification of fluorescence signal with
  indirect immuno-labeling
• May however lead to greater background signal

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Example Direct antibody labelling

• Bovine pulmonary artery endothelial cells labeled
  with direct antibody probes for tubulin (green)
  and the mitochondria (red). Nonspecific stain for
  nuclei (blue)

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Example Indirect antibody labelling

• Formation of the cephalic furrow in the anterior
  end of a developing Drosophila melanogaster
  embryo. A primary antibody to neurotactin was
  visualized using a red-fluorescent Cy3 dye
  secondary antibody. Primary antibodies to plasma
  membranebound myosin and to nuclear-localized
  even-skipped (Eve) protein were visualized with
  green-fluorescent Alexa Fluor 488 goat antimouse
  IgG antibody and Alexa Fluor 488 goat antirabbit
  IgG antibody, respectively. The nuclei were
  stained with blue-fluorescent Hoechst 33342.

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FISH Fluorescence in-situ hybridization

• Useful for chromosomal studies
• Especially when combined with multiple labelling

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Example FISH

• Human metaphase chromosomes normal (left) and
  cancerous (right)
• Multicolored chromosomes on the right reveal
  chromosomal translocations and other aberrations
  in the cancerous cell line

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GFP Green Fluorescent Protein

• In vivo reporter from Aequorea victoria (jelly
  fish)
• The FP gene is fused to the gene of interest and
  as the fusion protein is expressed, one can
  localize or track the movement of the tagged
  protein in living cells
• GFP has a relatively high Q.E. and is quite
  robust, possibly due to the barrel-like
  structures protecting and isolating the buried
  chromophore from the environment
• An number of other fluorescent proteins are now
  available spanning the visible spectrum

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Concentration measurements

• Calcium gradients in neurons
• High temporal and spatial resolution incompatible
  with precise measurements

Indo-1
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Studies on living cells

• Inverted microscope for easy access to specimen
• Constant temperature stages and perfusion
  apparatus
• Non-cytotoxic wavelengths and anti-fading agents

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Main Points

• Confocal/two-photon and deconvolution
  microscopies make it possible to image specimens
  in 3D
• Most of the fluorescent techniques used in
  spectroscopy can be combined with microscopy to
  study phenomena in 4D
• Fluorescent proteins are a major tool in
  visualizing intracellular distributions and
  dynamics