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SYBP: Biophotonik

SYBP 2: Einzelmolekülmethoden II

SYBP 2.3: Hauptvortrag

Donnerstag, 7. März 2002, 15:00–15:30, HS 11/215

Multilifetime and multicolor colocalization of single molecules with nanometer precision — •Markus Sauer, M. Heileman, C. Müller, P. Tinnefeld, and K.D. Weston — Physikalisch-Chemisches Institut, Universität Heidelberg, Germany

We present a technique for ultrahigh-resolution colocalization of conventional single fluorescent dyes with nanometer accuracy using multi-lifetime, and multi-color detection, i.e. spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM). The method takes advantage of single fluorescent dyes that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their emission maxima, and in their fluorescence lifetime. A combined analysis of the fractional intensities and fluorescence lifetimes recorded on two spectrally-separated detectors enables the classification of the portion of each dye per pixel in a point-spread-function (PSF) image with high accuracy, even though only a limited number (generally a few thousand) photons are detected per single dye. From these portions two separate PSF images are calculated and fitted to two-dimensional (2D) Gaussian functions to localize their centers with a precision of a few nanometers. To circumvent patchy PSF images and subsequent lower precision in localization, the off times of the dyes used (triplet lifetime) should be shorter than the integration time per pixel. Furthermore, the dyes should exhibit constant fluorescence emission maxima and lifetimes. We demonstrate that by the use of appropriately selected dyes, the presented technique permits the determination of the distance between two single dye molecules down to   30 nm with a precision of   10 nm without any chromatic aberrations.