Exact(5)
For our baseline in vivo measurements, we performed rough multi-exponential lifetime fits with high spatial resolution.
We therefore consider 5000 as the minimum number of photons required to perform our multiple-component lifetime fits, containing 2 constrained lifetimes and 2 free lifetimes.
We consider 5000 to be the minimum number of photons required to perform rough lifetime fits using our method of constraining the first 2 of 4 lifetime values.
We regard this value as the approximate minimum number of photons required to perform our multiple-component lifetime fits, containing 2 constrained lifetimes and 2 free lifetimes and described in Subsections 2.3 and 3.2, at each pixel [ 30].
For our measurements of in vivo metabolic transients, we performed precise multi-exponential lifetime fits with lower spatial resolution, a suitable approach for investigating global phenomena such as anoxia.
Similar(55)
Our baseline measurements consisted of multi-exponential lifetime fitting at each pixel using fluorescence profiles consisting of approximately 5000 photons.
Manually sampled pixels are subject to selection bias and obtaining sufficient numbers of photons in a single pixel for an accurate lifetime fit using a double exponential decay models is difficult.
Multi-exponential fluorescence lifetime fitting requires higher photon count per particle due to increased parameter uncertainty, which could be achieved by extending the particle dwell time by decreasing the flow rate or enlarge the illumination volume, either of which would reduce the device throughput.
Lhcas were linked for all experimental conditions except for Full PSI LHCI upon 475 nm whose lifetime was fitted independently to improve significantly the residuals at long time scale.
Fluorescence from the collagen and elastin fibers can also be seen in the deepest image at 100 µm, with potential contributions from SHG in the blue channel A double exponential lifetime was fitted to each ROI (cell) for each spectral channel for all images containing cells.
For rapid multiwell plate imaging, we typically record each image with seven time gates and, for an average excitation power of less than 1 mW at the sample, acquisition times are typically <10 s per image, permitting us to read a 96-well plate in less than 20 minutes and deliver fluorescence lifetime images fitted pixelwise to monoexponential decay profiles.
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