Redefining the standards for dynamic FLIM imaging with rapidFLIM
rapidFLIM measurements enable the imaging of dynamic processes via fluorescence lifetime imaging (FLIM). This new approach allows for fast FLIM acquisition up to several frames per second for imaging of dynamic processes (e.g., protein interaction, chemical reaction, or ion flux), highly mobile species (e.g., mobility of cell organelles or particles, cell migration), and investigating FRET dynamics. More than 10 frames per second can be acquired, depending on sample brightness and image size.
Time-Correlated Single Photon Counting (TCSPC) is used to determine the fluorescence lifetime. In TCSPC, one measures the time between sample excitation by a pulsed laser and the arrival of the emitted photon at the detector very precisely with picosecond accuracy. Classical TCSPC electronics as well as single photon counting detectors have dead times on the order of 80 to 100 ns. Within this time the system is busy with data processing and can not detect any other photon. The dead time is typically longer than the repetition rate of the excitation pulses. Thus, if more than a photon is emitted per excitation cycle, it can not be detected. To avoid this situation that would lead to measurement artefacts, it is necessary to work in conditions in which most of the excitation pulses lead to no emission, i.e. when only receiving one fluorescence photon out of every 50-100 laser pulses. This requirement slows down the FLIM acquisition time.
In order to acquire a FLIM image with a confocal microscope, one typically scans a pulsed laser beam accross the sample. In case of a fast scanning system, this very often leads to only a few detected photons per pixel, which will not allow proper FLIM analysis. It is therefore necessary to increase the number of detected photons per pixel either by extending the pixel dwell time or by accumulating several frames to sum up the photons. This is another reason why it typically takes several seconds up to half a minute to get a good FLIM image.
To speed up the data acquisition, a TCSPC unit with a very short dead time like the TimeHarp 260 NANO must be used. The short dead time of < 1 ns permits to detect several photons within one excitation cycle. Although the temporal resolution of the TimeHarp 260 is only 250 ps, it is still sufficient for most FLIM applications. With this TCSPC unit and the much lower dead time one can now detect many more photons within the same time which speeds up FLIM acquisition significantly. Learn More...