Tackling Challenges from Autofluorescence Interference in Flow Cytometry
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 the cytoplasm.1,2 When excited with radiation of a suitable wavelength, fluorophores 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 labeled cells flow through the light path or channel of an integration point, most commonly a laser, while suspended in a buffered salt-based solution.4 Fluorescent emission is read by detectors, converted into electronic signals and analyzed by a computer.3 Fluorescence emission can also occur when unlabeled cell and tissue 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, physio-chemical state, intra-tissue distribution and microenvironment of endogenous fluorophores.1,6
How does autofluorescence impact flow cytometry analysis?
When cells and tissues are labeled with exogenous fluorochromes, autofluorescence presents a complication as its signal results in 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 tackle challenges from autofluorescence in conventional flow cytometry experiments
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 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 uses more detectors than fluorochromes to measure the signature of a fluorochrome across the full spectrum. Acquisition of unstained cells further offers the unprecedented ability to measure the cell autofluorescence signature and use it as a fluorescent parameter. Autofluorescence unmixing can effectively improve resolution by extracting the background introduced by autofluorescence. This can be particularly advantageous for the analysis of highly autofluorescent cells such as cell lines, tissue-derived cells and granulocytes.
Watch the educational video to learn more about autofluorescence in spectral flow cytometry and find answers to questions such as:
- What is autofluorescence unmixing?
- How can autofluorescence impact biological resolution?
- What are cell-specific autofluorescence signatures?
