Wide-Field Multi-Parameter FLIM: Long-Term Minimal Invasive Observation of Proteins in Living Cells.

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes

T Cell Activation Results in Conformational Changes in the Src Family Kinase Lck to Induce Its Activation

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Time domain diffuse Raman spectrometer based on a TCSPC camera for the depth analysis of diffusive media

We present a time domain diffuse Raman spectrometer for depth probing of highly scattering media. The system is based on, to the best of our knowledge, a novel time-correlated single-photon counting (TCSPC) camera that simultaneously acquires both spectral and temporal information of Raman photons. A dedicated non-contact probe was built, and time domain Raman measurements were performed on a tissue mimicking bilayer phantom. The fluorescence contamination of the Raman signal was eliminated by early time gating (0-212 ps) the Raman photons. Depth sensitivity is achieved by time gating Raman photons at different delays with a gate width of 106 ps. Importantly, the time domain can provide time-dependent depth sensitivity leading to a high contrast between two layers of Raman signal. As a result, an enhancement factor of 2170 was found for our bilayer phantom which is much higher than the values obtained by spatial offset Raman spectroscopy (SORS), frequency offset Raman spectroscopy (FORS), or hybrid FORS-SORS on a similar phantom

Time domain diffuse Raman spectrometer based on a TCSPC camera for the depth analysis of diffusive media

We present a time domain diffuse Raman spectrometer for depth probing of highly scattering media. The system is based on, to the best of our knowledge, a novel time-correlated single-photon counting (TCSPC) camera that simultaneously acquires both spectral and temporal information of Raman photons. A dedicated non-contact probe was built, and time domain Raman measurements were performed on a tissue mimicking bilayer phantom. The fluorescence contamination of the Raman signal was eliminated by early time gating (0–212 ps) the Raman photons. Depth sensitivity is achieved by time gating Raman photons at different delays with a gate width of 106 ps. Importantly, the time domain can provide time-dependent depth sensitivity leading to a high contrast between two layers of Raman signal. As a result, an enhancement factor of 2170 was found for our bilayer phantom which is much higher than the values obtained by spatial offset Raman spectroscopy (SORS), frequency offset Raman spectroscopy (FORS), or hybrid FORS-SORS on a similar phantom

Observation of the Nucleation of an Aggregation Induced Emission (AIE) Molecule in a Microfluidic System by Fluorescence Lifetime Video (FLIM)

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