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Nondestructive, High-Resolution T Cell Characterization and Subtyping via Deep-UV Microscopy
Viswanath Gorti1, Caroline E Serafini1,2, Aaron D Silva Trenkle1
1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.
Insights
Deep-ultraviolet (UV) microscopy offers a fast, label-free method for T cell analysis. This technique accurately assesses T cell viability, activation, and subtypes, advancing immune cell characterization.
Area of Science:
- Immunology
- Microscopy
- Cell Biology
Background:
- T cell characterization is vital for immune function, disease monitoring, and cell therapies.
- Current methods like flow cytometry require labeling and are destructive.
- Existing label-free imaging methods have limitations in speed, specificity, and complexity.
Purpose of the Study:
- To establish deep-ultraviolet (UV) microscopy as a rapid, label-free imaging technique for T cell assessment.
- To evaluate the accuracy of deep-UV microscopy in determining T cell viability, activation state, and subtypes.
- To demonstrate the potential of deep-UV microscopy for high-throughput immune cell analysis.
Main Methods:
- Utilized static deep-UV images to assess T cell viability and activation.
- Employed dynamic deep-UV time-series imaging to quantify intracellular activity for T cell subtyping (CD4+ and CD8+).
- Compared deep-UV microscopy results with flow cytometry for validation.
Main Results:
- Static deep-UV images showed high correlation (R² > 0.97) with flow cytometry for T cell viability and activation.
- Dynamic deep-UV imaging accurately subtyped CD4+ and CD8+ T cells with ~90% sensitivity and specificity.
- Intracellular activity patterns in deep-UV images corresponded to known metabolic differences between T cell subtypes.
Conclusions:
- Deep-ultraviolet (UV) microscopy provides a fast, label-free, and accurate method for T cell characterization.
- This technique has broad applications in immunology research, immune monitoring, and cell-based therapy development.
- Deep-UV microscopy overcomes limitations of current T cell analysis methods, offering a powerful alternative.
Abstract:
Objective and Impact Statement: We establish deep-ultraviolet (UV) microscopy as a fast, label-free, and simple imaging approach for assessing T cell viability, activation state, and subtype with high accuracy. Introduction: T cell characterization is critical for understanding immune function, monitoring disease progression, and optimizing cell-based therapies. Current technologies to characterize T cells, such as flow cytometry, require fluorescent labeling and are typically destructive endpoint measurements. Nondestructive, label-free imaging methods have been proposed but face limitations with throughput, specificity, and system complexity. Methods: In this work, we use static deep-UV images to characterize T cell viability and activation state and dynamic deep-UV time series to quantify intracellular activity for assessment of T cell subtype (CD4+ and CD8+). Results: T cell viability and activation state predicted from static deep-UV images showed strong agreement with flow cytometry, with a correlation of R 2 > 0.97. Dynamic deep-UV images revealed unique intracellular activity that enabled accurate subtyping of CD4+ and CD8+ T cells, with a sensitivity and specificity of ~90%, corroborating recent studies on metabolic activity differences between these subtypes. Conclusion: Together, deep-UV microscopy offers a powerful tool for high-throughput immune cell characterization, with broad applications in immunology research, immune monitoring, and development of emerging cell-based therapies.
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