Dual‐laser, differential fluorescence correction method for reducing cellular background autofluorescence

Steinkamp, John A.; Stewart, Carleton C.

doi:10.1002/cyto.990070611

Cited by 84 publications

(49 citation statements)

References 18 publications

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“…In addition, several tools have been developed to correct for autofluorescence in flow cytometric data to increase sensitivity and thus the ability to detect weakly positive cell populations. Steinkamp and Stewart described a method utilizing electronic circuitry that subtracts autofluorescence-derived signals from the total signal measured by means of dual, specific and nonspecific, laser excitation (17). Alberti et al developed an alternative method requiring only single laser excitation (18).…”

Section: Background Caused By Autofluorescencementioning

confidence: 99%

Considerations for the control of background fluorescence in clinical flow cytometry

Hulspas¹,

O’Gorman

Wood

et al. 2009

Cytometry Part B Clinical

219

190

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Section: Background Caused By Autofluorescencementioning

confidence: 99%

Considerations for the control of background fluorescence in clinical flow cytometry

Hulspas¹,

O’Gorman

Wood

et al. 2009

Cytometry Part B Clinical

219

190

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“…CuSO 4 in ammonium acetate buffer, Sudan black B in 700 mL/L ethanol (10 ), NaBH 4 (11 ), and pontamine sky blue (12 ) are some examples. It is also possible to use mathematical models to reduce autofluorescence by exploiting its broader excitation spectrum compared with the spectra of fluorescent labels (13,14 ). Additionally, 2-photon excitation and time-resolved fluorescence can be used to diminish or eliminate autofluorescence (15 ).…”

mentioning

confidence: 99%

Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection

et al. 2010

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Background: The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research. Methods: We used horseradish peroxidase (HRP)-conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event. Results: We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both in cultured cells and in tissue samples. Conclusions: The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.

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“…The latter approach includes methods that rely on a separate autofluorescence-related measurement obtained simultaneously on each cell (4)(5)(6). Analysis of spectral imaging data can resolve the contributions to an image of individual fluorogenic or chromogenic components (7)(8)(9), and this approach can be used to identify and reduce background autofluorescence (9).…”

mentioning

confidence: 99%

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

Smith

Pollice

Emlet

et al. 2006

Cytometry Part B Clinical

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Background: 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 Fl auto /mean Fl total ). 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.Methods: 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.Results: 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.Conclusions: A simple cell by cell correction for autofluorescence based on the ratio of mean Fl auto to mean Fl total 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. q

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Dual‐laser, differential fluorescence correction method for reducing cellular background autofluorescence

Cited by 84 publications

References 18 publications

Considerations for the control of background fluorescence in clinical flow cytometry

Considerations for the control of background fluorescence in clinical flow cytometry

Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection

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

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