Autofluorescence Correction Research Articles

Practical 10-Color T-Cell Panel for Phenotyping Diverse Populations Using Spectral Flow Cytometry: A Beginner's Guide.

Flow cytometry stands as the most employed high-throughput single-cell analysis technique, facilitating the profiling of remarkably diverse samples, such as blood, bone marrow and body fluids. In addition, it allows for the discrimination of diverse immune cell subsets, including infrequently encountered types like T regulatory cells and exhausted CD28Null T cells. However, analyzing rare immune cell subsets with conventional flow cytometry poses challenges stemming from factors like fluorophore overlap, compensation issues, and limited flexibility in fluorophore selection. Therefore, spectral flow cytometry offers advantages over traditional flow cytometry. It measures the full emission spectrum and then separates it to identify different fluorochromes. This enables the use of fluorochromes with significant overlap in a single test, allowing for the analysis of more protein markers. Following this, spectral technology employs precise calculations to separate individual fluorochromes, thereby enabling the detection and elimination of autofluorescent signals originating from cells within the entire emission spectrum. This capability is pivotal in achieving deep phenotyping of immune cells with the requisite sensitivity and resolution essential for monitoring the immune systems of patients with compromised immunity, such as cancer and autoimmune disorders. Additionally, it allows for the exploration of interactions between distinct immune subsets. In this context, we introduce an optimized protocol utilizing spectral flow cytometry for precise T-cell characterization and differentiation, encompassing the assessment of their activation states. Furthermore, this protocol extends its applicability to the identification of less common circulating T-cell populations, notably T-regulatory and CD28Null T cells, following autofluorescence correction within the spectrum. This protocol provides a set of steps and reagents for the surface and intracellular staining of human T cells using whole peripheral blood. The spectral-based design of this panel allows for its applicability to other spectral machines, providing a versatile and efficient tool for T-cell analysis. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Achieving optimal staining through effective antibody titration Basic Protocol 2: Single-cell staining Basic Protocol 3: Comprehensive panel staining post-titration and spectral library integration.

Pixel-by-pixel autofluorescence corrected FRET in fluorescence microscopy improves accuracy for samples with spatially varied autofluorescence to signal ratio

The actual interaction between signaling species in cellular processes is often more important than their expression levels. Förster resonance energy transfer (FRET) is a popular tool for studying molecular interactions, since it is highly sensitive to proximity in the range of 2–10 nm. Spectral spillover-corrected quantitative (3-cube) FRET is a cost effective and versatile approach, which can be applied in flow cytometry and various modalities of fluorescence microscopy, but may be hampered by varying levels of autofluorescence. Here, we have implemented pixel-by-pixel autofluorescence correction in microscopy FRET measurements, exploiting cell-free calibration standards void of autofluorescence that allow the correct determination of all spectral spillover factors. We also present an ImageJ/Fiji plugin for interactive analysis of single images as well as automatic creation of quantitative FRET efficiency maps from large image sets. For validation, we used bead and cell based FRET models covering a range of signal to autofluorescence ratios and FRET efficiencies and compared the approach with conventional average autofluorescence/background correction. Pixel-by-pixel autofluorescence correction proved to be superior in the accuracy of results, particularly for samples with spatially varying autofluorescence and low fluorescence to autofluorescence ratios, the latter often being the case for physiological expression levels.

A flexible open-source processing workflow for multiplexed fluorescence imaging based on cycles.

Background Multiplexing tissue imaging is developing as a complement for single cell analysis, bringing the spatial information of cells in tissue in addition to multiple parameters measurements. More and more commercial or home-made systems are available. These techniques allow the imaging of tens of fluorescent reporters, where the spectral overlap is solved by imaging by cycles the fluorophores using microfluidics to change the reporters between each cycle. Methods For several systems, the acquisition system coupled to the microfluidic system is a wide field microscope, and the acquisition process is done by mosaicking to cover a large field of view, relying on image processing to obtain the data set to be analysed in intensity. The processed data set allows the identification of different populations, quite similarly to cytometry analysis, but with spatial information in addition. To obtain the final image for analysis from the raw acquisitions, several preprocessing steps are needed for inter-cycle registration, tissue autofluorescence correction or mosaicking. We propose a workflow for this preprocessing, implemented as an open source software (as a library, command line tool and standalone). Results We exemplify the workflow on the commercial system PhenoCycler TM (formerly named CODEX®) and provide a reduced size data set for testing. Conclusions We compare our processor with the commercially provided processor and show that we solve some problems also reported by other users.

SAIBR: a simple, platform-independent method for spectral autofluorescence correction.

ABSTRACTBiological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, owing to typically lower expression levels, experiments using endogenously tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image Correction By Regression (SAIBR), a simple platform-independent protocol and FIJI plug-in to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source; it achieves accurate quantitation of fluorophore signal, and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible low-barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

Flow Cytometric FRET Analysis of Protein Interactions.

In the past decades, investigation of protein-protein interactions in situ in living or intact cells has gained expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments required confirmation at the cellular level. Förster (fluorescence) resonance energy transfer (FRET)-based methods are excellent tools for determining proximity and supramolecular organization of biomolecules at the cell surface or inside the cell. This could well be the basis for the increasing popularity of FRET. In fact, the number of publications exploiting FRET has exploded since the turn of the millennium. Interestingly, most applications are microscope-based, and only a fraction employs flow cytometry, even though the latter offers great statistical power owed to the potentially huge number of individually measured cells. However, with the increased availability of multi-laser flow cytometers, strategies to obtain absolute FRET efficiencies can now be relatively facilely implemented. In this chapter, we intend to provide generally useable protocols for measuring FRET in flow cytometry. After a concise theoretical introduction, recipes are provided for successful labeling techniques and measurement approaches. The simple, quenching-based population-level measurement, the classic ratiometric, intensity-based technique providing cell-by-cell actual FRET efficiencies, and a more advanced version of the latter, allowing for cell-by-cell autofluorescence correction are described. An Excel macro pre-loaded with spectral data of the most commonly used fluorophores is also provided for easy calculation of average FRET efficiencies. Finally, points of caution are given to help design proper experiments and critically interpret the results.

Green autofluorescence, a double edged monitoring tool for bacterial growth and activity in micro-plates

The intrinsic green autofluorescence of an Escherichia coli culture has long been overlooked and empirically corrected in green fluorescent protein (GFP) reporter experiments. We show here, by using complementary methods of fluorescence analysis and HPLC, that this autofluorescence, principally arise from the secreted flavins in the external media. The cells secrete roughly 10 times more than what they keep inside. We show next that the secreted flavin fluorescence can be used as a complementary method in measuring the cell concentration particularly when the classical method, based on optical density measure, starts to fail. We also demonstrate that the same external flavins limit the dynamical range of GFP quantification and can lead to a false interpretation of lower global dynamic range of expression than what really happens. In the end we evaluate different autofluorescence correction methods to extract the real GFP signal.

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population.

Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population.

A two-channel detection method for autofluorescence correction and efficient on-bead screening of one-bead one-compound combinatorial libraries using the COPAS fluorescence activated bead sorting system

One-bead one-compound combinatorial library beads exhibit varying levels of autofluorescence after solid phase combinatorial synthesis. Very often this causes significant problems for automated on-bead screening using TentaGel beads and fluorescently labeled target proteins. Herein, we present a method to overcome this limitation when fluorescence activated bead sorting is used as the screening method. We have equipped the COPAS bead sorting instrument with a high-speed profiling unit and developed a spectral autofluorescence correction method. The correction method is based on a simple algebraic operation using the fluorescence data from two detection channels and is applied on-the-fly in order to reliably identify hit beads by COPAS bead sorting. Our method provides a practical tool for the fast and efficient isolation of hit beads from one-bead one-compound library screens using either fluorescently labeled target proteins or biotinylated target proteins. This method makes hit bead identification easier and more reliable. It reduces false positives and eliminates the need for time-consuming pre-sorting of library beads in order to remove autofluorescent beads.

Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice

The CXCR4 antagonist Plerixafor (AMD3100) induces the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. AMD3100 similarly induces the mobilization of human acute lymphoblastic leukemia (ALL) cells into the blood in mice. In this study, the temporal response of pre-B ALL cells to AMD3100 was compared with that of normal hematopoietic progenitor cells (HPC) using an NOD/SCID xenograft model of ALL and BALB/c mice, respectively. ALL cells remained in the circulation up to 6 hours after AMD3100 administration, by which time normal HPCs were no longer detectable. AMD3100 also increased the proportion of actively cycling ALL cells in the peripheral blood. Together, these data suggest that ALL cells are more sensitive to the effects of bone marrow disruption than normal progenitors. Using the NOD/SCID xenograft model, we demonstrated that AMD3100 increased the efficacy of the cell cycle specific drug vincristine, resulting in reduced disease levels in the blood and spleens of animals over 3 weeks and extended the survival of NOD/SCID mice with ALL. These data demonstrate that mobilizing agents can increase the therapeutic effect of cell cycle dependent chemotherapeutic agents.

Changes in fluorescence intensity of selected leukocyte surface markers following fixation

Immunophenotyping of blood leukocytes often involves fixation with paraformaldehyde prior to cytometry analysis. However, the influence of cell type and marker specificity on the stability of fluorescence intensity after fixation has not been well studied. Human whole blood was stained using a panel of fluorescein isothiocyanate-labeled antibodies to surface markers. Unfixed and fixed samples were analyzed by flow cytometry at 0, 2, 4, 6, 24, 48, and 96 h after staining. Fluorescence measurements were converted to molecules of equivalent soluble fluorochrome for comparison. Fixation caused a significant decrease in both forward and side scatter at 48 h which required gating adjustments to achieve resolution of cell populations. The autofluorescence increased progressively in fixed samples (ninefold at 96 h for monocytes). Variable decreases in marker-associated fluorescence became apparent after correction for autofluorescence. The magnitude of the decrease at 96 h varied with cell type and marker, from 5% for CD32 on monocytes to 39% for CD16 on neutrophils. The change in fluorescence intensity following staining and fixation of leukocytes varies with cell type and surface marker. Fluorescence stability should be determined for each cell type and marker used, and the confounding effects of fixation on cell autofluorescence should be considered.

Measuring FRET in Flow Cytometry and Microscopy

This unit presents protocols describing the measurement of protein associations using FRET as determined by flow and image cytometry. The proteins under investigation can be labeled by fluorescent antibodies or fluorescent protein (FP) variants. The flow cytometry protocols determine FRET based on the measurement of donor quenching, which provides a FRET value on a population basis, or based on the measurement of fluorescence intensities in the donor, FRET, and acceptor channels, which provides cell-by-cell FRET values. An extension of this protocol is based on cell-by-cell correction for autofluorescence and requires the measurement of four fluorescence intensities. The algorithm described can be applied in image cytometric FRET as well. The image protocol determines FRET resolved by donor photobleaching. The authors provide extensive discussion of pitfalls, limitations, and interpretation.

A simple correction for cell autofluorescence for multiparameter cell‐based analysis of human solid tumors

Corrections that have been proposed to minimize the unwanted contribution of cell autofluorescence to the total fluorescence signal often require either specialized instrumentation or the sacrifice of a data channel so as to perform a measurement that can be used to correct for autofluorescence in individual cells. Here we propose a simple cell by cell correction for autofluorescence that is suitable for multiparameter laser scanning cytometry (LSC) studies in human solid tumors that relies on the ratio of mean autofluorescence to mean total cell fluorescence (mean Flauto/mean Fltotal). This approach assumes a correlation between the autofluorescence component and the total signal in individual cells. This correction does not require specialized instrumentation, and does not sacrifice a data channel in multiparameter studies. A potential disadvantage is that errors may be introduced by the assumption of a correlation between the two components of the total fluorescence signal in individual cells in samples in which no such correlation exists. Distributions of cell autofluorescence and total Her-2/neu cell fluorescence were obtained separately by LSC in three human breast cancer cell lines and in three samples of primary human lung cancer. In the breast cancer cell lines, autofluorescence measurements and Her-2/neu measurements were also obtained on the same cells. We show that there is a partial correlation between autofluorescence and total Her-2/neu/FITC fluorescence in individual cells in the three breast cancer cell lines. We also show that the results of a ratio-based autofluorescence correction agree with those based on a true cell by cell correction. Computer simulation studies suggest that in samples with no correlation between the autofluorescence component and the true probe/dye fluorescence component, the ratio correction produces robust estimates of the mean true fluorescence signal, with relatively small but systematic underestimates of the coefficient of variation of such measurements under conditions commonly encountered in the measurement of human solid tumors. A simple cell by cell correction for autofluorescence based on the ratio of mean Flauto to mean Fltotal can be applied in cell samples in which there is a correlation between cell autofluorescence and true probe/dye fluorescence in individual cells. In cell samples that lack this correlation, or in which it is not known whether such a correlation exists, this correction can be used with the reservation that there is a systematic but relatively small underestimation of the degree of variability of the measurements.

Computer program for determining fluorescence resonance energy transfer efficiency from flow cytometric data on a cell-by-cell basis.

The determination of fluorescence resonance energy transfer (FRET) with flow cytometry (FCET) is one of the most efficient tools to study the proximity relationships of cell membrane components in cell populations on a cell-by-cell basis. Because of the high amount of data and the relatively tedious calculations, this procedure should be assisted by powerful data processing software. The currently available programs are not able to fulfill this requirement. We developed a Windows-based program to calculate fluorescence resonance energy transfer efficiency values from list mode flow cytometry standard (FCS) files. This program displays the measured data in standard plots by generating one- and two-parameter histograms on linear or logarithmic scales. A graphical gating tool allows the user to select the desired cell population according to any combination of the parameter values. The program performs several statistical calculations, including mean, S.D., percent of the gated data. We have implemented two types of data sheet for FRET calculations to aid and guide the user during the analysis: one with population-mean-based autofluorescence correction and the other with spectrum-based cell-by-cell autofluorescence correction. In this paper, we describe the gating algorithms, the file opening procedure and the rules of gating. The structure of the program and a short description of the graphical user-interface (GUI) are also presented in this article.

Computer program for determining fluorescence resonance energy transfer efficiency from flow cytometric data on a cell-by-cell basis

The determination of fluorescence resonance energy transfer (FRET) with flow cytometry (FCET) is one of the most efficient tools to study the proximity relationships of cell membrane components in cell populations on a cell-by-cell basis. Because of the high amount of data and the relatively tedious calculations, this procedure should be assisted by powerful data processing software. The currently available programs are not able to fulfill this requirement. We developed a Windows-based program to calculate fluorescence resonance energy transfer efficiency values from list mode flow cytometry standard (FCS) files. This program displays the measured data in standard plots by generating one- and two-parameter histograms on linear or logarithmic scales. A graphical gating tool allows the user to select the desired cell population according to any combination of the parameter values. The program performs several statistical calculations, including mean, S.D., percent of the gated data. We have implemented two types of data sheet for FRET calculations to aid and guide the user during the analysis: one with population-mean-based autofluorescence correction and the other with spectrum-based cell-by-cell autofluorescence correction. In this paper, we describe the gating algorithms, the file opening procedure and the rules of gating. The structure of the program and a short description of the graphical user-interface (GUI) are also presented in this article.

A ratiometric method of autofluorescence correction used for the quantification of Evans blue dye fluorescence in rabbit arterial tissues.

Evans blue dye (EBD) conjugates with albumin in the circulation and is frequently used to measure vascular protein leakage. The fluorescence of the dye from tissue sections can be used to measure its uptake at very specific anatomical locations, but problems arise with dye quantification because tissue components also fluoresce; so-called autofluorescence. We have measured uptake of EBD by blood vessel walls at various points around the aorto-renal branch of rabbits. High resolution, digitised, fluorescence images of histological sections of artery wall allowed detailed microscopic analysis of EBD accumulation; and a ratiometric method was developed to enable autofluorescence to be separated from EBD fluorescence. When EBD-free tissue sections were illuminated with blue light, the ratio of red to green fluorescence was constant throughout the tissue (0.59 +/- 0.03, mean +/- S.D., n = 32). Therefore, at each individual pixel, the level of red autofluorescence could be determined by multiplying the green intensity at that pixel by the calculated red to green ratio. Since EBD fluorescence was detected only in the red region of the spectrum, intensity values of the dye alone were obtained from EBD-exposed tissue by subtracting the red autofluorescence estimated by this ratiometric method. In such cases the red to green fluorescence ratio was measured from adjacent sites known to be free of EDB (0.59 +/- 0.02, mean +/- S.D., n = 56). We were therefore able to increase the sensitivity of tracer quantification by complete elimination of background autofluorescence on a pixel-by-pixel basis. Use of EBD standards allowed calibration of corrected fluorescence intensities and calculation of mass transfer coefficients for albumin into the artery wall. Spatial variations in the permeability of the artery wall around the renal ostium were detected with the present high resolution technique, with an average mass transfer coefficient of (6.8 +/- 0.9) x 10(-8) cm s(-1) for all sites combined (n = 56). The present ratiometric method could potentially be applied to other quantitative fluorescence-based techniques.

Microspectroscopic imaging of nodulation factor-binding sites on living Vicia sativa roots using a novel bioactive fluorescent nodulation factor

A novel bioactive fluorescent nodulation (Nod) factor, NodRlv-IV(BODIPY FL-C16), has been synthesized by attaching a BODIPY FL-C16 acyl chain to the primary amino group of chitotetraose deacetylated at the nonreducing terminus by recombinant NodB. The binding of the fluorescent Nod factor to root systems of Vicia sativa was investigated with fluorescence spectral imaging microscopy (FSPIM) and fluorescence ratio imaging microscopy (FRIM). Spatially resolved fluorescence spectra of living and labeled Vicia sativa root systems were measured by FSPIM. Strong autofluorescence, inherent to many plant systems when excited at 488 nm, was corrected for by utilizing the difference in fluorescence emission spectra of the autofluorescence and NodRlv-IV(BODIPY FL-C16). A methodology is presented to break down the in situ fluorescence emission spectra into spatially resolved autofluorescence and BODIPY FL fluorescence spectra. Furthermore, an FRIM method was developed for correcting autofluorescence in fluorescence micrographs for this system. After autofluorescence correction it was shown that NodRlv-IV(BODIPY FL-C16) was concentrated in the root hairs, but was also bound to other parts of the root surface.

Autofluorescence correction for fluorescence in situ hybridization.

Optimal sensitivity of fluorescence in situ hybridization (FISH) requires bright signals and low background fluorescence. Use of locus-specific probes is especially dependent on high sensitivity. Some tissue preparations show high autofluorescence, masking small or dim signals. We have developed a new method for subtracting autofluorescence from digital images on a pixel-by-pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emission spectra than the chemical components responsible for autofluorescence. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in each individual image. By subtracting autofluorescence components, we were able to enhance centromeric signals and make previously indistinguishable cosmid signals clearly visible. This image-processing approach to autofluorescence correction may widen the applicability of gene-specific probes in FISH analysis of tumor material.

Effect of morphological abnormalities on blood retinal barrier permeability in diabetic retinopathy.

The morphological base for the impaired function of the blood retinal barrier was studied in 50 eyes of 10 insulin dependent and 21 non-insulin dependent patients with various levels of diabetic retinopathy. The permeability of the blood retinal barrier (PBRB) was determined by vitreous fluorophotometry with correction for autofluorescence, lens transmission and non-protein bound plasma fluorescein concentration. Morphological abnormalities of diabetic retinopathy assessed by fundus photography and fluorescein angiography were individually scored on a decimal scale and related to the PBRB by multiple regression analysis. The PBRB was not correlated to morphological abnormalities of non-proliferative retinopathy [(1) microaneurysms, (2) hard exudates, (3) soft exudates, (4) intraretinal hemorrhages, (5) fluorescein leakage, and (6) capillary closure, p greater than 0.3]. The PBRB was correlated to morphological abnormalities of (pre)proliferative retinopathy [(1) intraretinal microvascular abnormalities (SIRMA) and (2) new vessels (Sneo): PBRB = A+B.SIRMA + C.Sneo with PBRB in nm/sec, A = 1.5 +/- 0.5, B = 0.9 +/- 0.2 and C = 1.7 +/- 0.4, R2 = 0.65, p less than 0.0001]. It can be concluded that the increased blood retinal barrier permeability in diabetic patients is mainly due to (pre)proliferative abnormalities and not to non-proliferative abnormalities.

Cell‐by‐cell autofluorescence correction for low signal‐to‐noise systems: Application to epidermal growth factor endocytosis by 3T3 fibroblasts

Autofluorescence of cells can be a major portion of the fluorescence signal in many systems, especially when fluorescent conjugates are used to study receptor-ligand systems for which there are less than 70,000 receptors per cell. We have devised a method for the cell-by-cell correction of autofluorescence for flow cytometric data by using an additional parameter to measure and correct for autofluorescence in the fluorescence channel. The principle has been extended to allow simultaneous correction for autofluorescence and dual-fluorescence spillover compensation in samples labeled with two different fluorochromes; all corrections were done in software, making them applicable to any flow cytometer. The autofluorescence correction method was used to analyze the acidification of epidermal growth factor (EGF) by Swiss 3T3 cells. EGF is acidified to pH 6.2 starting two min after labeling, with a half-time for acidification of 45 s.