rapidFLIM – a Novel and Fast Fluorescence Lifetime Imaging Technique
Redefining the Standards for Dynamic FLIM Imaging
- Diffusion of two types of dye-labeled beads in water, imaged with rapidFLIM at a speed of three frames per second. The two dye species can be distinguished based on their lifetime difference of about 700 ps. The beads form a random structure at the edge of the water droplet. A selection of full FLIM images from the video is shown. Acquisition of each image took about 333 ms.
The rapidFLIM technique exploits recent advances in Time-Correlated Single Photon Counting electronics, where ultra short dead times allows for imaging dynamic processes via Fluorescence Lifetime Imaging. Fast FLIM image acquisition with up to several frames per second enables to study dynamic processes (e.g., protein interaction, chemical reaction, or ion flux), highly mobile species (e.g., mobility of cell organelles or particles, cell migration), or investigating FRET dynamics.
Fluorescence Lifetime Imaging (FLIM) is a very versatile microscopy method where fluorescence lifetime information is combined with spatial localization in the sample, allowing investigating, for example, biochemical and physical processes, detecting changes in the local environment of the sample, molecular interactions, or conformational changes via Förster Resonance Energy Transfer (FRET).
FLIM data acquisition is typically based on Time-Correlated Single Photon Counting (TCSPC) electronics, pulsed diode lasers, and highly sensitive single photon counting detectors. Up to now TCSPC data acquisition is considered a somewhat slow process, due to the time required to collect a sufficient number of photons per pixel for reliable data analysis.
The novel rapidFLIM method allows acquiring several FLIM images per second by exploiting recent hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies. These improved hardware components enable higher detection count rates, making it possible to achieve much better photon statistics in shorter time spans.
Thus, FLIM imaging can be performed for dynamic processes (e.g., transient protein interaction in living cells, chemical reaction, or ion flux) as well as highly mobile species in a precise manner and with high optical resolution. As an additional benefit, the high accuracy of the data analysis is comparable to that of conventional TCSPC measurements.
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