Fluorescence bursts

Keiier R A 1998 Singie-moiecuie identification in flowing sampie streams by fluorescence burst size and intraburst fluorescence decay rate Anal. Chem. 70 1444-51... 

Fluorescence Correlation Spectroscopy and Fluorescence Burst Analysis. Several nanoscopic chemical imaging approaches work very well for measurements of chemical kinetics, interactions, and mobility in solution. Fluorescence correlation spectroscopy (FCS) measures the temporal fluctuations of fluorescent markers as molecules diffuse or flow in solution through a femtoliter focal volume.54 Their average diffusive dwell times reveal their diffusion coefficients, and additional faster fluctuations can reveal chemical reactions and their kinetics if the reaction provides fluorescence modulation. Cross-correlation of the fluorescence of two distinguishable fluorophore types can very effectively reveal chemical binding kinetics and equilibria at nanomolar concentrations. 

Fluorescence burst analysis, a variation of FCS procedures that has an optimum configuration for simple presentation, uses a uniform nanoscopic flow channel with an optically perfect ceiling, uniform cross section, and periodic electrodes that can now be constructed by careful electron lithography techniques.55 By application of controlled electric fields, uniform plug flow of solution through... 

Fig. 12. Fluorescence burst transients originating from individual YOYO-1 stained A-DNA passing through a focused laser beam molecules in a molded PDMS electrophoresis chip. DNA concentration 82 fM, E = 200 V/cm...

Time-resolved fluorescence was used to detect rhodamine 6G (R6G), sulfor-hodamine 101 (SR101), and rhodamine B (RB). A Ti-sapphire laser (800 nm, 50 fs) was used, but the excitation wavelength has been converted to 400 nm (for R6G) or 532 nm (for SR 101 or RB) by an optical parametric amplifier or by second harmonic generation [676], In another report, fluorescence burst detection was used for detection of single chromophore molecules [677]. 

J. Muller, J. M. Lupton, A. L. Rogach, and J. Feldmann, Air-induced fluorescence bursts from single semiconductor nanocrystals. Appl. Phys. Lett. 85(3) 381—3S3 (2004). 

Figure 17.21 Single nanometric aperture for real time single molecule DNA sequencing. A DNA polymerase molecule attached to the bottom surface is used to successively Incorporate fluorescent nucleotides complementary to the DNA strand, causing fluorescence bursts for each incorporation process (see text for details). Image copyright of Pacific Biosciences Inc., reprinted with permission.

The proof that single molecules could be measured by confocal illumination was given in a study where fluorescence bursts of Rhodamine molecules passing through a sharply focused laser beam were analyzed. From the burst... 

The reaction must lead to a change of the diffusion properties of the fluorescent species under investigation (Fig. 8.1a). Thereby, the t3q)ical duration of the fluorescence bursts due to the passage of fluorescent molecules through the excitation volume is changed. This has been exploited in particular for ligand-receptor interaction studies [17]. 

Van den Bogaart G, Krasnikov V, Poolman B (2007) Dual-colo fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel. MscL Biophys J 92 1233-1240... 

Figure 7. Fluorescence decay curves due to single molecule transits in a 10-1,M aqueous solution of R-6G. The 3D-plot shows the fluorescence bursts on the macroscopic time scale (bin width 10 ms) and the respective time-resolved decays on the nanosecond time scale. (Adopted from [49].)...

If the fluorescent analyte is allowed to flow or diffuse into and out of a small excitation/collection volume defined, in part, by a focused laser beam, then this gives rise to a stochastic series of short-lived fluorescence bursts detected above the background noise level (see Figure 2.1(a)). This type of experiment was one of the first used to demonstrate the feasibility of fluorescence detection of single... 

---