A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane: 1. Theory and a first application.
Just because there is an average of one molecule in the observation volume of a solution or membrane (single-phase), one cannot say that this is an individual molecule since many different single molecules measured one by one or the same single, individual molecule not leaving the detection volume on time average can cause a single-molecule event. The latter case is of interest and allows the continuous observation of one and the same single molecule without averaging over many 'different' single molecules. For the first time a universal theoretical and experimental framework is presented for the continuous observation of the same single, individual molecule without immobilization, hydrodynamic flow, or burst size histograms of fluorescence intensity traces. In this original article, the stochastic approach is derived and its main characteristics are demonstrated with the free fluorophore rhodamine-green in solution for simpler experimental realization. Single (solution)-phase single-molecule fluorescence auto- (or two-color cross-) correlation spectroscopy (SPSM-FCS) is used as a specific application in order to count the absolute number of molecules in the observation volume. The absolute number of molecules, the diffusion coefficient of the single fluorescent molecule, the lower limit of distance, and the molar concentration of the bulk phase (solution) were directly obtained from the measured auto- or (cross)-correlation curves of the SPSM-FCS experiments. For this purpose, the detection volume that was measured was less then 1 fl (10(-15) l). Then, a concentration of the bulk solution was chosen in such a way that the probability of detecting more than one molecule in the detection volume was very small. The Poisson probability was experimentally determined for the absolute number of molecules depending upon a specified bulk concentration. From the diffusion coefficient of the molecule, it was found that the probability of the molecule diffusing out of the probe volume during the measurements was negligibly small.
Földes-Papp
Zeno
Z
Clinical Immunology and Jean Dausset Laboratory, Graz University Medical School and Hospital, Auenbruggerplatz 8, A-8036 Graz, LKH, Austria. Zeno.Foldes-Papp@uni-graz.at
Demel
Ulrike
U
Tilz
Gernot P
GP
D005456:Fluorescent Dyes / Q000737:chemistry*; D008956:Models, Chemical; D012235:Rhodamines / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D012235:Rhodamines