Lifetime-based analysis of binary fluorophores mixtures in the low photon count limit

Abstract

Summary Single biomolecule sensing often requires the quantification of multiple fluorescent species. Here, we theoretically and experimentally use time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify fluorescent species with similar chromatic signatures. A modified maximum likelihood estimator is introduced to include two fluorophore species, with compensation of the instrument response function. We apply this algorithm to simulated data of a simplified two-fluorescent species model, as well as to experimental data of fluorophores' mixtures and to a model protein, doubly labeled with different fluorophores' ratio. We show that 100 to 200 photons per fluorophore, in a 10-ms timescale, are sufficient to provide an accurate estimation of the dyes' ratio on the model protein. Our results provide estimation for the desired photon integration time toward implementation of TCSPC in systems with fast occurring events, such as translocation of biomolecules through nanopores or single-molecule burst analyses experiments.

Autofluorescence lifetime flow cytometry with time‐correlated single photon counting

“…The acquisition speed of this system is inversely proportional to the target number of photons per cell. At our target for accurate two-component lifetime analysis (10,000 photons or 5 ms interrogation per cell [27,28]), the upper limit of acquisition speed is 100 cells per second, assuming a 50% cell transit duty cycle. This throughput is one or two orders of magnitude lower than conventional flow cytometry.…”

“…The flow velocity determines the transit time of the cells through the Ø12 µm laser illumination spot. Given the empirical autofluorescence photon count rate of 2 million photons per second observed from T cells with this setup, and a target photon count of 10,000 photons per cell for accurate two-component lifetime estimation [27,28], an integration time of 5 ms per cell was required. Therefore, a flow velocity of 2.4 mm/s ( i.e ., 12µm/5ms) or slower was necessary.…”

Near Infrared Autofluorescence Lifetime Imaging of Human Retinal Pigment Epithelium Using Adaptive Optics Scanning Light Ophthalmoscopy

“…A threshold of 300 photons within each kernel was used as a limit to reduce the background noise. 37 An instrument response function (IRF) was measured in the system and deconvolved during the decay curve fitting for an accurate fit of the lifetime components. 38 For all young human subjects’ data, a kernel of 15 × 15 pixels was used that corresponds to about 11.13 µm in the retina.…”

Fluorescence Lifetime Imaging of Human Retinal Pigment Epithelium in Pentosan Polysulfate Toxicity Using Adaptive Optics Scanning Light Ophthalmoscopy

“…Thresholding was based on previously determined threshold limits. 29 A kernel size of 17 × 17 pixels is within the range of an RPE cell; 14 to 28 pixel diameters. Individual lifetime parameters were exported for additional analysis in MATLAB (Mathworks, Natick, MA, USA) and SigmaPlot (Systat version 14.5; Palo Alto, CA, USA).…”