Confocal Laser Scanning Microscopy: general considerations and techniques

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Confocal Laser Scanning Microscopy general
considerations and techniques
Simone Bossi
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Optical Microscopy

• Optical or light microscopy involves passing
  visible light transmitted through or reflected
  from the sample through a single or multiple
  lenses to allow a magnified view of the sample.
  The resulting image can be detected directly by
  the eye, imaged on a photographic plate or
  captured digitally.

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Optical microscopy techniques

• Bright field optical microscopy
• Oblique illumination
• Dark field optical microscopy
• Phase contrast optical microscopy
• Differential interference contrast microscopy
• Fluorescence microscopy
• Confocal laser scanning microscopy

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Confocal laser scanning microscopy

• Confocal laser scanning microscopy (CLSM) is a
  relatively new light microscopical imaging
  technique (introduced around 1980 by M. Petran
  and A. Boyde) which has found wide applications
  in the biological sciences c.f. Pawley,1990
  Boyde, 1994. The primary value of the CLSM to
  the biologist is its ability to produce optical
  sections through a 3-dimensional (3-D) specimen -
  e.g., an entire cell or a piece of tissue - that,
  to a good approximation, contain information from
  only one focal plane.

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LASER Light Amplification by the Stimulated
Emission of Radiation
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The Optic path
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3D Reconstruction
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3D reconstruction
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Auto fluorescence
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Fluorescent Probe Perfusion
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The fluorescent probe FLUO4-AM
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Sub localisation
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Quantisation of fluorescence
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GFP Green Fluorescent Protein
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GFP Fusion Protein
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Cameleon Construct
Amy Palmer in the Tsien laboratory started with
the original cameleon construct two fluorescent
proteins (cyan fluorescent protein (CFP) and
citrine) separated by calmodulin (CaM) and a
CaM-binding peptide. In the presence of Ca2, CaM
interacts with the CaM-binding peptide, and CFP
emission decreases as citrine emission increases,
which is indicative of increased fluorescence
resonance energy transfer (FRET).