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Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized
Rosy Favicchio1, Giannis Zacharakis, Katerina Oikonomaki
1Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, 71110 Heraklion, Crete, Greece. rosy@imbb.forth.gr
Insights
A new noninvasive imaging method, Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS), enables simultaneous detection of multiple fluorescent T cells. This breakthrough allows researchers to track T-cell activation and expansion in vivo with unprecedented accuracy.
Area of Science:
- Immunology
- Optical Imaging
- Biotechnology
Background:
- Detecting multiple fluorophores in low signal conditions limits in vivo optical imaging in immunology.
- Fluorescent labels are crucial for reporting T-cell functional characteristics.
Purpose of the Study:
- To develop a noninvasive method for simultaneous, quantitative detection of multiple fluorophores in low signal-to-noise ratios.
- To apply this method for tracking T-cell activation and clonal expansion in vivo.
Main Methods:
- Developed Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) for in vivo imaging.
- Utilized spectral unmixing to differentiate overlapping fluorescent emissions from DsRed- and GFP-labeled T cells.
- Validated M-SNELS results with in vitro and in vivo flow cytometry.
Main Results:
- M-SNELS successfully quantified two colocalized fluorescent T-cell populations simultaneously.
- Demonstrated the method's ability to track T-cell activation and antigen-driven proliferation longitudinally.
- Achieved significant correlation between M-SNELS, flow cytometry, and in vitro/in vivo validation.
Conclusions:
- Noninvasive M-SNELS provides a valid alternative to traditional invasive methods for detecting T-cell dynamics.
- The developed technique overcomes limitations in detecting multiple fluorophores under low signal conditions.
- Enables precise monitoring of T-cell activation and clonal expansion in immunological studies.
Abstract:
Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.
