Noise‐Corrected Principal Component Analysis of fluorescence lifetime imaging data

Abstract

Fluorescence Lifetime Imaging (FLIM) is an attractive microscopy method in the life sciences, yielding information on the sample otherwise unavailable through intensity-based techniques. A novel Noise-Corrected Principal Component Analysis (NC-PCA) method for time-domain FLIM data is presented here. The presence and distribution of distinct microenvironments are identified at lower photon counts than previously reported, without requiring prior knowledge of their number or of the dye's decay kinetics. A noise correction based on the Poisson statistics inherent to Time-Correlated Single Photon Counting is incorporated. The approach is validated using simulated data, and further applied to experimental FLIM data of HeLa cells stained with membrane dye di-4-ANEPPDHQ. Two distinct lipid phases were resolved in the cell membranes, and the modification of the order parameters of the plasma membrane during cholesterol depletion was also detected. Noise-corrected Principal Component Analysis of FLIM data resolves distinct microenvironments in cell membranes of live HeLa cells.

The squared distance approach to frequency domain time‐resolved fluorescence analysis

“…However currently, as mentioned in Section 1.2, these methods do not utilize most of the FRD, which as proved in this paper, contain valuable information. In addition, the NC‐PCA is inherent to single‐photon detection such as the time‐correlated single photon counting (TCSPC) technique .…”

“…However currently, as mentioned in Section 1.2, these methods do not utilize most of the FRD, which as proved in this paper, contain valuable information. In addition, the NC-PCA is inherent to singlephoton detection such as the time-correlated single photon counting (TCSPC) technique [19][20][21][22]. The discussion of this paper is limited to the FD FLT method; however, D 2 is also configurable for FD anisotropy decay measurements.…”

Fast fluorescence lifetime imaging techniques: A review on challenge and development

“…[135][136][137][138][139][140][141][142][143][144][145][146][147][148][149][150][151][152][153][154] Algorithms Notes: Ã single exponential decay, ÃÃ bi-exponential decay. 158 on the photon rate which can safely be obtained from the sample, on the e±ciency at which the technique obtains°uorescence lifetimes or decay curves from these photons, on the number of pixels of the image, on the demands for the lifetime accuracy, for the time resolution, and for the image quality. It also matters whether a technique records a full°uorescence decay curve in each pixel, a phasor, or just intensities in a few time gates after the excitation pulse.…”

“…Le Marois et al 158 have proposed a noisecorrected principal component analysis (NCPCA) based on Poisson statistics to correct the noise error in TCSPC-FLIM data acquisition, and the system is able to detect the distribution of the microenvironment at low photon count. The number of photons required to determine the unknown components is even less than that required in phasor analysis, thus increasing the execution speed.…”

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

“…1,4 There are a number of reviews and quite a few books that contain detailed description of basic principles of FLIM, as well as recent technical achievements and data analysis that allow to perform lifetime measurements with high accuracy in various samples, including live cells and tissues. 4,29,[32][33][34] Therefore, this paper does not contain this kind of information and is mainly focused on FP applications for studies of cellular molecular processes by FLIM.…”