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Updated: May 28, 2025
Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence
Published on: April 12, 2024
Multispectral autofluorescence for label free classification of immune cell type and activation/polarization status
Abbas Habibalahi1,2, Ayad G Anwer1,2, Aline Knab1,2
1Graduate School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney, New South Wales, Australia.
Insights
Multispectral imaging of cell autofluorescence offers a rapid, label-free method to assess immune cell types and activation states. This technique accurately characterizes immune status through a single, non-invasive assay.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Immunology
Background:
- Assessing immune status typically requires time-consuming analysis of multiple biomarkers.
- Current methods often involve numerous complex assays.
- A need exists for efficient, non-invasive techniques to evaluate immune cell populations and functions.
Purpose of the Study:
- To evaluate the efficacy of multispectral imaging of cell autofluorescence for characterizing immune cell types and activation states.
- To establish a label-free, single-assay method for immune status assessment.
- To identify distinct spectral signatures for major immune cell populations.
Main Methods:
- Utilized multispectral microscopy with 56 spectral channels (excitation 345-476 nm, emission 414-675 nm).
- Analyzed autofluorescence signatures of NAD(P)H and flavins, which correlate with cellular metabolism.
- Cultured and stimulated immune cell lines (Jurkat, Ramos, THP-1, HL-60) and macrophages (M0, M1, M2).
Main Results:
- Identified distinct spectral signatures corresponding to T cells, B cells, monocytes, and neutrophils.
- Successfully differentiated between activated and resting immune cells.
- Achieved classification accuracy between 92% and 100% (ROC AUC) for immune cell characterization.
- Demonstrated discrimination of M0, M1, and M2 macrophage polarization states.
Conclusions:
- Multispectral imaging of cell autofluorescence is a viable, sensitive, and label-free technique for immune status evaluation.
- This method enables the characterization of immune cell types and their activation status in a single, non-invasive assay.
- The findings support the application of this technique for streamlined immune diagnostics and research.
Abstract:
Evaluating immune status is a challenging and time-consuming process that involves analysing various biomarkers through numerous assays. The sensitive label-free technique of multispectral imaging of cell autofluorescence involves directly assessing the molecular composition of cells to gather biological information. Cells were cultured in RPMI 1640 modified media supplemented with penicillin-streptomycin and 10% foetal bovine serum at 37°C, with 5% CO2 and 95% humidity. Activation and differentiation was confirmed using immunofluorophores against relevant markers. Multispectral microscopy utilized defined spectral regions, which spanned the excitation (345-476 nm) and emission (414-675 nm) wavelength ranges. In total, 56 distinct spectral channels were applied. These channels cover the spectrum of several fluorophores notably NAD(P)H and flavins, whose concentrations depend on cellular metabolism. We identified distinct spectral signatures for characterizing cells from the Jurkat, Ramos, THP-1, and HL-60 immune cell lines. These signatures correspond to four major immune cell types: T cells (Lymphocytes), B cells (Lymphocytes), monocytes and neutrophils. Moreover, our investigation explored the potential identification of both activated and resting forms of these cells, including the discrimination of M0, M1 and M2 polarized macrophages. Classification accuracy ranged from 92% to 100% based on receiver operator characteristic area under the curve (ROC AUC) assessment. These results indicate that the multispectral evaluation of cell autofluorescence is applicable for characterization of immune status. This includes the assessment of cell types and their activation status, all achievable through a single non-invasive assay.
