Data registration and selective single-molecule analysis using multi-parameter fluorescence detection.
A general strategy to identify and quantify sample molecules in dilute solution employing a new spectroscopic method for data registration and specific burst analysis denoted as multi-parameter fluorescence detection (MFD) was recently developed. While keeping the experimental advantage of monitoring single molecules diffusing through the microscopic open volume element of a confocal epi-illuminated set-up as in experiments of fluorescence correlation spectroscopy, MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the four-dimensional fluorescence information (intensity, F; lifetime, tau; anisotropy, r; and spectral range, lambda(r)) in real time and allows for exclusion of extraneous events for subsequent analysis. In this review, the versatility of this technique in confocal fluorescence spectroscopy will be presented by identifying freely diffusing single dyes via their characteristic fluorescence properties in homogenous assays, resulting in significantly reduced misclassification probabilities. Major improvements in background suppression are demonstrated by time-gated autocorrelation analysis of fluorescence intensity traces extracted from MFD data. Finally, applications of MFD to real-time conformational dynamics studies of fluorescence labeled oligonucleotides will be presented.
Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, D-37077 Gottingen, Germany.
C
C
Eggeling
S
S
Berger
L
L
Brand
J R
JR
Fries
J
J
Schaffer
A
A
Volkmer
C A
CA
Seidel
D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis / Q000737:chemistry; D008956:Models, Chemical; D009841:Oligonucleotides / Q000032:analysis*; D020112:Rhodamine 123 / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins; D020112:Rhodamine 123