Single molecule fluorescence detection

Fig. 18.9 Single molecule fluorescence detection in LC ARROW chip, (a) Top view of experi mental beam geometry of dye molecule in sub picoliter excitation volume (dotted ellipse) (2exc excitation beam, dF fluorescence signal) (b) fluorescence signal as function of molecules in excitation volume symbols, different experimental runs, dashed line linear fit...

Rigneault H., Capoulade J., Dintinger J., Wenger J., Bonod N., Popov E., Ebbesen T. W., and Lenne P.-F. (2005). Enhancement of single-molecule fluorescence detection in subwavelength apertures. Phys. Rev. Lett. 95 117401. 

Single-molecule fluorescence detection was subsequently demonstrated at room temperature, first by detecting the burst of light as a molecule passes through the focus of a laser beam [67,68], but each molecule could be detected only once in this way. Correlation analysis of many such bursts provides a window into a variety of dynamical effects ranging from diffusion to intersystem crossing to rotational correlation [69], and this area termed fluorescence correlation spectroscopy (FCS, ([70-72]) has been reviewed in [73]. 

To achieve single-molecule fluorescence detection, there are three common experimental practices. First, experiments are done at low concentrations (10 9-l() 12 mol I ) to spatially separate molecules, so each of them can be studied without interference from surrounding molecules. Second, fluorescence signal detection is confined to a small volume (<10-151) to minimize background noises for single-molecule sensitivity. Third, biomolecules are often immobilized, so a single molecule can be studied over time. 

For the real-time single-turnover detection of fluorogenic reactions, catalytic reactions with fast turnover rates (or fast product dissociation) pose a time resolution challenge, as single-molecule fluorescence detection often requires detection of hundreds of photons to obtain statistically significant information. For these fast enzymes, one can vary the substrates, use enzyme variants from different organisms, or use mutants to slow the catalysis down. 

Peck K, Stryer L, Glazer A N and Mathles R A 1989 Single-molecule fluorescence detection autocorrelation criterion and experimental realization with phycoerythrin Proc. Natl Acad. Scl. USA 86 4087-91... 

Dittrich P.S., Manz A., Single-molecule fluorescence detection in microfluidic channels—The Holy Grail in mu TAS Anal. Bioanal. Chem., 382, 1771-1782 (2005). 

Peck, K., Stryer, L., Glazer, A. N., and Mathies, R. A., Single-molecule fluorescence detection Autocorrelation criterion and experimental realization with phycoerythrin, Pmc. Natl Acad. Sci. USA, 86, 4087,1989. 

Dittrich PS, Manz A (2005) Single-molecule fluorescence detection in mitaofluidic channels-the Holy Grail in [iTAS Anal Bioanal Chtan 382(8) 1771—1782... 

A convenient method is to use a concentrated suspension of powdered milk and remove any emission filters so that the scattered excitation light is detected. It may be necessary to insert attenuating filters to reduce the scattered light intensity to a level similar to that encountered in single molecule fluorescence detection. 

Microscope objectives for single molecule fluorescence detection... 

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