Optically biased diffusion of single molecules studied by confocal fluorescence microscopy

Journal of Physical Chemistry B

Volumn 102, Issue 17, 1998, Pages 3160-3167

  Osborne, M A ^a   Balasubramanian, S ^a   Furey, W S ^a   Klenerman, D ^a  

^a UNIVERSITY OF CAMBRIDGE   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 0000465693     PISSN: 15206106     EISSN: None     Source Type: Journal    
DOI: 10.1021/jp9715078     Document Type: Article

References (37)

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26. -2 for a 528 nm diameter focus. This is only two-thirds of the saturation power and only 28% of the ground state is depopulated. Saturation effects will be even less significant at powers below this maximum. For a discussion of the effects of saturation on single-molecule fluorescence, see ref 14.
27. -1, taking into account the overall detection efficiency of about 1%. Under these conditions a molecule will cycle for up to 20 ms before photobleaching, which is longer than the typical transit time of a molecule (1-15 ms) through the laser focus. Furthermore, we have measured the bleaching times of single Rhodamine molecules bound to immobilized single-stranded DNA in solution at incident power of 0.5 mW and find an average time of 160 ms (this work will be published later). If photobleaching was a dominant effect, then a decrease in the transit time with increasing laser power would be expected. The increase in transit time indicates that the optical biasing effect dominates and that photodestruction of the fluorophore is less significant.
28. The discrimination level is set at the greatest count level that contributes more than one-half of the spectrum. In the case of R6G the blank is simply a sample of MilliQ water which gives a threshold of 2 counts/ms. For the D* and D*/KF, equivalent samples of unlabeled nonfluorescent DNA are used as blanks to determined the threshold count levels. In both these cases a discrimination level of 4 counts was determined. Large background signals are observed in the spectra of the fluorescent microspheres. In water a threshold of 8 counts is deemed suitable while in the water-glycerol mixture the threshold increases to 10 counts when the small contribution of Raman scatter from a blank sample of glycerol is taken into account.
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