Time-Resolved Fluorescence: Novel Technical Solutions

Ortmann, Uwe; Wahl, Michael; Kapusta, P.

doi:10.1007/4243_2008_048

Cited by 4 publications

(2 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ultimate time resolution of this setup is better than 10 ps. We evaluated the timing response of Perkin-Elmer SPCM-AQR-14 single photon sensitive APDs incorporated in the MicroTime200 time-resolved confocal microscope (PicoQuant GmbH) [23,24]. An LDH-P-C-470B pulsed laser diode (similar to the laser of the FluoTime200 fluorometer) was used as an excitation source in these experiments.…”

Section: Time-resolved Fluorescence Spectroscopy and Microscopymentioning

confidence: 99%

Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation

Luchowski

Kapusta

Szabelski

et al. 2009

Meas. Sci. Technol.

View full text Add to dashboard Cite

Förster resonance energy transfer (FRET) can be utilized to achieve ultrashort fluorescence responses in time-domain fluorometry. In a poly(vinyl) alcohol matrix, the presence of 60 mM Rhodamine 800 acceptor shortens the fluorescence lifetime of a pyridine 1 donor to about 20 ps. Such a fast fluorescence response is very similar to the instrument response function (IRF) obtained using scattered excitation light. A solid fluorescent sample (e.g a film) with picosecond lifetime is ideal for IRF measurements and particularly useful for time-resolved microscopy. Avalanche photodiode detectors, commonly used in this field, feature color- dependent-timing responses. We demonstrate that recording the fluorescence decay of the proposed FRET-based reference sample yields a better IRF approximation than the conventional light-scattering method and therefore avoids systematic errors in decay curve analysis.

show abstract

Section: Time-resolved Fluorescence Spectroscopy and Microscopymentioning

confidence: 99%

Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation

Luchowski

Kapusta

Szabelski

et al. 2009

Meas. Sci. Technol.

View full text Add to dashboard Cite

show abstract

“…Specifically, the external TCSPC equipment consisted of picosecond pulsed laser diodes of wavelength of 485 nm for excitation of Cy2 or 640 nm for excitation of Cy5 (PDL 800-D with LDH-P-C-485 or LDH-P-C-640B; PicoQuant GmbH, Berlin) with a pulse rate of 40 MHz, and the TimeHarp 200 TCSPC board (PicoQuant GmbH). Synchronization of the data acquisition with the scanner movement was controlled by SymPhoTime software using the time-tagged time-resolved (TTTR) measurement mode in which each photon is stored separately. − A PMA-182-N-M photomultiplier tube (PMT) detector (PicoQuant GmbH) with a low photocathode quantum efficiency (15% at 450 nm and 9% at 550 nm) along with band pass emission filters HQ520/35 for Cy2, HQ580/70 for Cy3, or HQ685/70 for Cy5 (AHF Analysentechnik, Tübingen, Germany) were used for measuring the fluorescent emission. The instrument response function (IRF) of the whole setup was measured to be approximately 250 ps.…”

Section: Methodsmentioning

confidence: 99%

Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics

et al. 2016

Self Cite

View full text Add to dashboard Cite

Limitations in the sensitivity and dynamic range of two-dimensional gel electrophoresis (2-DE) are currently hampering its utility in global proteomics and biomarker discovery applications. In the current study, we present proof-of-concept analyses showing that introducing time-resolved fluorescence in the image acquisition step of in-gel protein quantification provides a sensitive and accurate method for subtracting confounding background fluorescence at the photon level. In-gel protein detection using the minimal difference gel electrophoresis workflow showed improvements in lowest limit of quantification in terms of CyDye molecules per pixel of 330-fold in the blue-green region (Cy2) and 8000-fold in the red region (Cy5) over conventional state-of-the-art image acquisition instrumentation, here represented by the Typhoon 9400 instrument. These improvements make possible the detection of low-abundance proteins present at sub-attomolar levels, thereby representing a quantum leap for the use of gel-based proteomics in biomarker discovery. These improvements were achieved using significantly lower laser powers and overall excitation times, thereby drastically decreasing photobleaching during repeated scanning. The single-fluorochrome detection limits achieved by the cumulative time-resolved emission two-dimensional electrophoresis (CuTEDGE) technology facilitates in-depth proteomics characterization of very scarce samples, for example, primary human tissue materials collected in clinical studies. The unique information provided by high-sensitivity 2-DE, including positional shifts due to post-translational modifications, may increase the chance to detect biomarker signatures of relevance for identification of disease subphenotypes.

show abstract

Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

2014

View full text Add to dashboard Cite

We review the principles and applications of statistical filtering in multichannel fluorescence microscopy. This alternative approach to separation of signals from individual fluorophore populations has many important advantages, especially when spectral and/or temporal overlap, or the complicated nature of those signals, makes their discrimination or sorting impossible by means of hardware. This situation is typically encountered for biological samples. This review of well established statistical filtering techniques and of emerging, very promising new methods of analysis reveals remarkable progress in bioanalytical applications of fluorescence microscopy.

show abstract

Time-Resolved Fluorescence: Novel Technical Solutions

Cited by 4 publications

References 14 publications

Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation

Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation

Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics

Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

Contact Info

Product

Resources

About