Handbook of Single Molecule Fluorescence Spectroscopy

by Gell, Christopher; Brockwell, David; Smith, Alastair

• ISBN13: 9780198529422
• ISBN10: 0198529422
• Edition: 1st
• Format: Hardcover
• Copyright: 2006-10-12
• Publisher: Oxford University Press

Summary

Analytical measurements at the single molecule level under ambient conditions have become almost routine in the past few years. The application of this technology to fundamental studies of heterogeneity in biomolecular structure and dynamics, chemical and biological reaction kinetics and photophysics provides a rich playground for molecular scientists. The potential use of single molecule detection for nanotechnology and quantum information processing is a new and almost unexplored area. This handbook is intended for those interested in a practical introduction to single molecule investigations using fluorescence techniques and places special emphasis on the practicalities of achieving single molecule resolution, analyzing the resulting data and exploration of the applications in biophysics. It is ideal for graduate research students and others embarking on work in this exciting field.

Author Biography

Chris Gell is Laser Facilities Manager at the Institute of Molecular Biophysics, University of Leeds David Brockwell is a Lecturer at the School of Biochemistry and Microbiology, University of Leeds Alastair Smith is the Director of the Institute of Molecular Biophysics, University of Leeds

Table of Contents

Acknowledgements	xi
Glossary of Terms and Symbols	xiii
Introduction	1	(9)
Motivation	1	(1)
A historical perspective	2	(1)
This book	3	(2)
Single molecule measurements	5	(5)
References	8	(2)
Single molecule fluorescence techniques	10	(87)
Introduction	10	(1)
Burst analysis	10	(2)
Photon counting histograms	12	(12)
Fluorescence correlation spectroscopy	24	(20)
Fluorescence resonance energy transfer	44	(22)
Measurements of immobilized single molecules	66	(14)
Other related techniques	80	(17)
References	89	(8)
Single molecule fluorescence instrumentation	97	(62)
Introduction	97	(5)
Optical arrangements for single molecule detection	102	(17)
Methods for discriminating signal from noise	119	(3)
Wavelength or polarization selection optics	122	(2)
Excitation sources	124	(3)
Microscope objectives for single molecule fluorescence detection	127	(6)
Detectors for single molecule fluorescence experiments	133	(7)
Acquisition cards and software	140	(2)
Realizing single molecule instrumentation	142	(17)
References	155	(4)
Preparation of samples for single molecule fluorescence spectroscopy	159	(42)
Introduction	159	(1)
Dye selection	160	(12)
Labelling of biomolecules	172	(8)
Doubly labelling single protein molecules for FRET studies	180	(6)
Optimizing biochemical systems for single molecule fluorescence studies	186	(3)
Immobilization methods	189	(12)
References	196	(5)
Fluorescence spectroscopy of freely diffusing single molecules: examples	201	(24)
Introduction	201	(1)
Single molecule studies of freely diffusing molecules	201	(24)
References	224	(1)
Fluorescence spectroscopy of immobilized single molecules: examples	225	(24)
Introduction	225	(1)
Single molecule studies of immobilized molecules	226	(23)
References	247	(2)
The outlook for single molecule fluorescence measurements	249	(6)
Outlook	249	(6)
References	252	(3)
Index	255