Confocal Laser Scanning Microscopy: general considerations and techniques Simone Bossi

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.

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

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.

LASER: Light Amplification by the Stimulated Emission of Radiation

The Optic path

3D Reconstruction

3D reconstruction

Auto fluorescence

Fluorescent Probe Perfusion

The fluorescent probe FLUO4-AM

Sub localisation

Quantisation of fluorescence

GFP: Green Fluorescent Protein

GFP Fusion Protein

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). Cameleon Construct