Single molecule fluorescence measurement

One such approach has recently been developed and shown to enable high-resolution NSOM fluorescence and force measurements on viable cultured human arterial smooth muscle (HASM) cells under buffered conditions [28,29], This approach takes advantage of the nanofabrication capabilities of focused ion beam (FIB) milling to sculpt a light delivery structure into the end of a conventional AFM probe. The FIB technique, which utilizes a focused beam of gallium ions to mill samples with nanometer resolution, was first used by van Hulst and co-workers to modify conventional NSOM probes [30]. They demonstrated an improvement in single molecule fluorescence measurements using... 

Table 3.5 Some examples of data acquisition schemes for single molecule fluorescence measurements...

Some Advantages and Problems of Fluorescent Dyes in Single Molecule Fluorescence Measurements... 

Weston, K D., Dyck, M., Tinnefeld, P., Muller, C., Herten, D. P. and Sauer, M. (2002) Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons. Anal. Chem., 74, 5342-5347. 

In the ensemble-averaged measurements described in Sec. IV, the biexponential fluorescence decays of MG molecules were tentatively ascribed to bimodal structure of the host matrices [11,12], By virtue of single-molecule fluorescence lifetime measurements we for the first time found evidence for bimodal distribution of sites for individual CV molecules on a thin film of PMMA [15], Recently, a theoretical study appeared foreseeing bimodality of a supercooled liquid [91],... 

Weiss, S. 2000. Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nat. Struct. Biol. 7 724-729. 

Figure 2.6 Application of splinted RNA ligation procedure single molecule FRET. (A) Diagram of prism-type total internal reflection fluorescence microscope (TIRFM) for single molecule FRET measurements. (B) Distribution of single-molecule FRET values for dye-labeled telomerase RNA molecules generated by splinted RNA ligation. (C) Dye intensity and FRET traces of a single telomerase RNA molecule Cy3 emission (green), Cy5 emission (red), FRET ratio (blue).

The dye-loaded dendrimer 110 can be detected by means of single-molecule fluorescence.48,57,281,282 This multichromophoric system was embedded into a thin polymeric film at very low concentrations so that individual dendrimer molecules could be studied by far-field fluorescence microscopy at room temperature. Temporal fluorescence intensity fluctuations, fluorescence spectra, and decay times were measured separately or simultaneously. Collective on/off jumps of the fluorescence intensity as well as different emissive levels were observed for single dendrimer molecules with eight perylenemonoimide chromo-... 

Two probes fluorescently labeled to undergo FRET can hybridize to areas flanking the splice sites of an RNA molecule and demonstrate a difference in ERET efficiency between spliced and unspliced mRNA as shown by both bulk solution and single-molecule spectroscopy measurements (33). 

To characterize the binding and the motions of the placed single T4 lysozyme molecules on cell walls, we used single-molecule fluorescence polarization measurements. The orientation of the single-molecule transition dipole can be probed by either linear polarized excitation or linear polarized emission. In this work, the excitation light was unpolarized. The emission was split into orthogonal (s polarized /i and p polarized I2) polarizations and detected by two photon detectors [22]. The intensity trajectories probed at the two orthogonal polarizations are shown in Fig. 24.9c (upper panel). The polarization P is defined as ... 

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