&lt;title&gt;Lifetime-based identification of single molecules&lt;/title&gt;

Enderlein, Joerg; Krahl, Rolf; Ortmann, Uwe; Wahl, Michael; Erdmann, Rainer; Klose, E.

doi:10.1117/12.208465

Cited by 3 publications

(3 citation statements)

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“…These demands have triggered new developments towards complete and compact, turn-key solutions for fluorescence sensing instrumentation. Especially, lifetime analysis of laser induced fluorescence by means of Time Correlated Single Photon Counting (TCSPC) has gained importance for Single Molecule Detection (SMD) and other areas in the life sciences, where sensitivity is critical [2][3][4][5][8][9][10][11][12][13] . The fluorescence decay times of appropriate fluorescent dyes provide a powerful additional discriminative feature to distinguish the molecules of interest from the background or other species 3,5,10,20 .…”

Section: Motivationmentioning

confidence: 99%

Two-channel fluorescence lifetime microscope with two colour laser excitation, single-molecule sensitivity, and submicrometer resolution

et al. 2003

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We present results from a two channel confocal microscope set-up allowing one to efficiently record two-colour as well as polarization resolved time-correlated single molecule fluorescence data. In addition to their spectral characteristics, single molecules can be distinguished by their fluorescence lifetime and polarization. This provides independent distinctive information and results in enhanced detection sensitivity. The set-up we present uses two picosecond diode lasers (440nm and 635 nm) for fluorescence excitation and a piezo scanner for sample movement. A learning scan algorithm permits very fast piezo scanner movement and offers a superior positioning accuracy on single molecules. The time-correlated photon counting system uses Time-Tagged Time-Resolved (TTTR) data aquisition, in which each photon is recorded individually. This method allows for the reconstruction not only fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function on a single spot of interest. Cross-correlation between two channels can be used to eliminate detector artifacts. Finally, fluorescence antibunching can also be analyzed. We show results obtained with immobilized and diffusing red and blue excited fluorescently labelled latex microspheres, as well as from single fluorophore molecules.

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Section: Motivationmentioning

confidence: 99%

Two-channel fluorescence lifetime microscope with two colour laser excitation, single-molecule sensitivity, and submicrometer resolution

et al. 2003

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“…This kind of information is not available from conventional histograms from TCSPC data collected over minutes or hours. To solve the problem one has to continuously collect histograms over very short intervals [4][5][6][7] . Alternatively one can record each fluorescence photon together with its arrival time relative to the start of the experiment or to its predecessor 8 .…”

Section: Introductionmentioning

confidence: 99%

“…The fluorescence decay times of appropriate fluorescent dyes provide a powerful additional discrimination feature to distinguish the molecules of interest from the background or other species [1][2][3][4][5] .…”

Section: Introductionmentioning

confidence: 99%

Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

et al. 2002

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Fluorescence lifetime measurements of organic fluorophores (upon laser excitation) provide a powerful additional method for distinguishing molecules of interest from the background or other species. This is of particular interest for ultra sensitive analysis and most notably for Single Molecule Detection (SMD). Another demand, in many of these applications, is to provide 2-D information (imaging) together with lifetime information. The method of choice for collection of such data is Time-Correlated Single Photon Counting (TCSPC). We have developed a compact TCSPC system on a single PC board that can perform TCSPC at high throughput, while synchronously driving a piezo scanner table that holds the sample immobilized on a surface. The TCSPC board allows measurement rates up to 3 MHz and provides a time bin resolution down to 30 ps. The detector input has a programmable Constant Fraction Discriminator (CFD). These features qualify the system for use with all common single photon detectors such as Photomultiplier Tubes and Single Photon Avalanche Photodiodes. An overall Instrument Response Function (IRF) width as low as 250 ps FWHM can be achieved with inexpensive PMTs and fast diode lasers. The board is designed for the Peripheral Component Interconnect (PCI) bus, permitting data throughput without loss of counts, sustained over a virtually unlimited measurement time. The board is software reconfigurable to operate in different modes. Here we emphasize the Time-Tagged Time-Resolved (TTTR) measurement mode, which permits recording all photon events with a realtime tag, to allow single molecule photon burst detection and subsequent offline data analysis with unlimited flexibility, e.g. for burst detection and selective histogramming. The Time-Tag clock is used to trigger an external piezo scanner driver that moves the sample. Since the clock source is common for both scanning and time tagging, the individual photons can be matched to scan pixels. We have studied single molecule solutions of Ja167 on a cover slide, to demonstrate the capabilities of the system. Fluorescence Lifetime Imaging can be performed at high resolution with as few as 100 photons per pixel.

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