Autofluorescence Can Interfere with Flow Cytometry Imaging
April 28, 2023
What is autofluorescence?
The first observations of autofluorescence were reported over 100 years ago, as a spontaneously occurring phenomenon caused by endogenous molecules with fluorophore-like properties accumulating within cytoplasm.1,2
When excited with radiation of a suitable wavelength, fluorophores will pass to an excited state and decay to the ground state, with a loss of energy.3 Part of this energy loss consists of fluorescence emission.3
In flow cytometry, this occurs when fluorescently labelled cells flow through the light path, or channel, of an integration point, most commonly a laser, whilst suspended in a buffered salt-based solution.4
Fluorescent emission is read by detectors, converted into electronic signals and analysed by a computer.3 Fluorescence emission can also occur when some unlabelled cell and tissues components are excited by radiation, behaving as endogenous fluorophores.3
Autofluorescence differentiates endogenous fluorescence from the fluorescence obtained when specimens are treated with exogenous fluorescent markers that bind to cell and tissue structures.3 Endogenous fluorophores, such as proteins containing aromatic amino acids, NAD(P)H, flavins and lipopigments, are widely distributed in cells and tissues.3
Autofluorescence is cell type dependent, with larger and more granular cells producing relatively higher autofluorescence.5 Autofluorescence has also been shown to provide estimates of cellular metabolic activity, as changes in emission properties are influenced by the nature, amount, physico-chemical state, intra-tissue distribution and microenvironment of endogenous fluorophores.1,6
How does it impact flow cytometry analysis?
When cells and tissues are labelled with exogenous fluorochromes, autofluorescence presents a complication, as its signal results in a background that can hinder the specific detection and analysis of exogenous marker emissions.1,7 The accuracy of flow cytometry relies on distinguishing true-positive from false-positive cell populations.8
How to deal with autofluorescence
Analysis of autofluorescence poses a challenge to conventional flow cytometry as it interferes with other fluorophores (diminishing the resolution of dim signals) and compromises the accurate definition of cellular phenotypes.9,10 Proper controls must be used to consider the fraction of fluorescence signal attributable to autofluorescence rather than the target protein marker.11
The inclusion of empty cytometer channels containing no fluorescent dye - allows for autofluorescence in the empty channel to be visualised on one axis in an XY dot plot format, against the cytometer channel for the target antigen on the other axis.11
Autofluorescence in conventional flow cytometry can also be addressed by using fluorophores showing lower autofluorescence interferences.12 Typically, far-red wavelength fluorophores that emit in the far-red or near-infrared region are best for this, as fewer biological components emit in this spectra range.12,13
Autofluorescence measurement in spectral flow cytometry
Spectral flow cytometry allows the separate measurement and analysis of autofluorescence.14 This technique measures as much of the emission spectrum of a fluorophore as possible, across a large number of detectors, creating a detailed fluorescent ‘signature’ for each fluorophore.14
In the analysis of flow cytometry data, the process of compensation transforms measurement values to estimates of cell-associated fluorescence.15 Measurements from a single detector comprise contribution from multiple fluorescences because of spectral spillover, which can be transformed into values estimating individual “pure” fluorescences using standard linear algebra.15
This avoidance of fluorophore-specific detector design offers the opportunity to identify and characterise autofluorescence as a full-fledged parameter.9 An in-silico model of multiparameter fluorescence measurements can be used to create a value for intrinsic autofluorescence and for the cell-associated dye fluorescence for each cell.15
When intrinsic dye fluorescence values multiplied by respective spillover coefficients are added to intrinsic autofluorescence, the “true signal” is computed.15
Autofluorescence removal can improve resolution
The ‘signature’ created by spectral flow cytometry not only functions as a measurement of autofluorescence: it can also be used to separate fluorophores and ‘unmix’ them from one another, reducing autofluorescent signals.14
Also known as extraction, unmixing the autofluorescent signal can improve resolution, particularly in cells from highly autofluorescent organs.14 Using full- spectrum unmixing algorithms, pure autofluorescence references can be removed from polychromatic measurements, improving the definition of rare markers and cellular phenotypes.10
Watch the video on autofluorescence unmixing.
