By Peter J. Verveer
This quantity offers an summary of complex fluorescence microscopy, masking a huge variety of equipment. each one bankruptcy specializes in a special procedure and offers a realistic advisor for program in organic platforms. Written within the hugely winning Methods in Molecular Biology sequence structure, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, easily reproducible laboratory protocols, and pointers on troubleshooting and keeping off recognized pitfalls.
Authoritative and state-of-the-art, Advanced Fluorescence Microscopy: equipment and Protocols seeks to supply scientists with tools for organic platforms which are of curiosity.
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Extra info for Advanced Fluorescence Microscopy: Methods and Protocols
Burridge K, Fath K, Kelly T et al (1988) Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 4:487–525 52. Burridge K, Chrzanowska-Wodnicka M (1996) Focal adhesions, contractility, and signaling. Webb DJ, Parsons JT, Horwitz AF (2002) Adhesion assembly, disassembly and turnover in migrating cells-over and over and over again. Ezratty EJ, Bertaux C, Marcantonio EE et al (2009) Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells.
Neubauer cell counting chamber. 35 mm glass-bottomed imaging dishes (MatTek Corporation, USA). 49, Nikon). l) set at 37 °C for live-cell imaging. 3. Green diode 561 nm laser. 4. 12-bit iXon 1 M EM-CCD camera. 5. 2 software. Trypsinise a T75 flask of MDA-MB-231 cells and count the number of cells per 1 ml using a counting chamber. Seed 6 × 105 cells onto each 35 mm glass bottom dish and incubate in complete medium at 37 °C, 5 % CO2 for 24 h. On day 2, transiently transfect cells with paxillin-RFP using Lipofectamine 2000 according to the manufacturer’s instructions using a ratio of 4 μg DNA to 10 μl Lipofectamine 2000 per 35 mm dish of cells.
Compared to confocal microscopy, 2PE microscopy improves depth penetration, owing to the longer excitation wavelength required and to the ability to collect scattered emission photons as a useful signal. It also minimizes photodamage because lower energy photons are used and because fluorescence is confined to the geometrical focus of the laser spot. 2PE is therefore ideal for high-resolution, deep-tissue, time-lapse imaging of dynamic processes in cell biology. Here, we provide examples of important applications of 2PE for in vivo imaging of neuronal structure and signals; we also describe how it can be combined with optogenetics or photolysis of caged molecules to simultaneously probe and control neuronal activity.