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Confocal Laser Scanning Microscopy: general considerations and techniques

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

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Confocal Laser Scanning Microscopy: general considerations and techniques

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  1. Confocal Laser Scanning Microscopy: general considerations and techniques Simone Bossi

  2. 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.

  3. 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

  4. 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.

  5. LASER: Light Amplification by the Stimulated Emission of Radiation

  6. The Optic path

  7. 3D Reconstruction

  8. 3D reconstruction

  9. Auto fluorescence

  10. Fluorescent Probe Perfusion

  11. The fluorescent probe FLUO4-AM

  12. Sub localisation

  13. Quantisation of fluorescence

  14. GFP: Green Fluorescent Protein

  15. GFP Fusion Protein

  16. 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).

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