Confocal Laser Scanning Microscopy: general considerations and
techniques Simone Bossi Slide 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. Slide 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 Slide 4 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. Slide 5 LASER: Light Amplification by the
Stimulated Emission of Radiation Slide 6 The Optic path Slide 7
Slide 8 3D Reconstruction Slide 9 3D reconstruction Slide 10 Auto
fluorescence Slide 11 Slide 12 Fluorescent Probe Perfusion Slide 13
The fluorescent probe FLUO4-AM Slide 14 Sub localisation Slide 15
Quantisation of fluorescence Slide 16 GFP: Green Fluorescent
Protein Slide 17 GFP Fusion Protein Slide 18 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
