Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

Abstract

We review the principles and applications of statistical filtering in multichannel fluorescence microscopy. This alternative approach to separation of signals from individual fluorophore populations has many important advantages, especially when spectral and/or temporal overlap, or the complicated nature of those signals, makes their discrimination or sorting impossible by means of hardware. This situation is typically encountered for biological samples. This review of well established statistical filtering techniques and of emerging, very promising new methods of analysis reveals remarkable progress in bioanalytical applications of fluorescence microscopy.

Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy

“…thermal noise and afterpulses) and other signal components (e.g. elastic scattering and Raman signal) that occur at the millisecond or lower timescale can be greatly eliminated (Supplementary Figure S3)( 36 , 43 ). For live cell FLCS measurement, different from in standard lipofectamine transfection, we used a much less plasmid DNA (10-fold lower) to decrease the expression of redundant MBD3-GFP in cell nucleus.…”

“…Enabled by recording of the fluorescence fluctuation owing to the molecular diffusion in a sub-femtoliter detection volume, FCS can provide a robust measure of dynamics at the microsecond level, from which subtle changes in biomolecular dynamics and association can be inferred ( 32 – 34 ). In this study, we applied fluorescence lifetime correlation spectroscopy (FLCS), an elegant derivative of FCS ( 35 , 36 ), to extract the real-time diffusion characteristics of MBD3 in different cell phases and found that MBD3 might have distinct DNA binding behaviors in the G1-phase and the G2-phase. Further, we analyzed the in vivo binding stoichiometry of MBD3 with photon counting histogram (PCH) and validated the salt-dependent ‘two-site sequential binding’ mode in the G1-phase nucleus.…”

A practical guide to time-resolved fluorescence microscopy and spectroscopy

“…Figure 2A shows a typical intensity trace for fluorescent liposomes diffusing through the confocal volume. An autocorrelation is performed on the data using equation 1 (65):…”

Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications

“…In particular, 2D FLCS allows us to detect the inhomogeneity of the system and to quantitatively analyze the diffusion and the interconversion dynamics of all species with distinct lifetimes in a species-specific manner as described below. The excellent reviews of FLCS can be found in the literature [ 26 , 27 , 28 , 29 ]. However, a comprehensive review of 2D FLCS has not yet been published, except for a book [ 30 ].…”