Read e-book online Advanced Fluorescence Microscopy: Methods and Protocols PDF

By Peter J. Verveer

ISBN-10: 1493920790

ISBN-13: 9781493920792

ISBN-10: 1493920804

ISBN-13: 9781493920808

This quantity offers an outline of complex fluorescence microscopy, masking a huge variety of equipment. every one bankruptcy specializes in a unique technique and offers a realistic consultant for software in organic structures. Written within the hugely winning Methods in Molecular Biology sequence structure, chapters contain introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and pointers on troubleshooting and warding off identified pitfalls.

Authoritative and state-of-the-art, Advanced Fluorescence Microscopy: equipment and Protocols seeks to supply scientists with tools for organic structures which are of curiosity.

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Extra info for Advanced Fluorescence Microscopy: Methods and Protocols

Example text

Sund SE, Swanson JA, Axelrod D (1999) Cell membrane orientation visualized by polarized total internal reflection fluorescence. Axelrod D (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence. Stock K, Sailer R, Strauss W et al (2003) Variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM): realization and application of a compact illumination device. Fish KN (2009) Total internal reflection fluorescence (TIRF) microscopy. Olveczky BP, Periasamy N, Verkman A (1997) Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy.

For calcium imaging this is a critical issue, because when faster scanning is implemented (see below), the pixel dwell time of the laser will be reduced, resulting in less signal from the indicator. Major efforts are underway to design better genetically encoded calcium indicators to solve these problems [28]. 2 Faster Imaging A major problem with calcium imaging is that it is hard to relate changes in fluorescence of the indicator molecule to neuronal spiking both in terms of determining the exact numbers of action potentials and especially the timing between them.

FLaSh, VSFP2, SPARC, Flare, O ­ pto-patch) have been designed that work well with 2PE microscopy and could in theory be useful to monitor the activity of thousands of individual neurons simultaneously [67–69]. These sensors are usually FRET based and can report both subthreshold changes in membrane potential and spiking activity of neurons. Unfortunately, most current approaches for voltage sensing with genetically encoded or synthetic indicators have poor spatial resolution and very low signal-to-­noise ratio, requiring the averaging of many stimuli to detect responses.

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Advanced Fluorescence Microscopy: Methods and Protocols by Peter J. Verveer


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