Visible and near infrared fluorescence spectral flow cytometry

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• Measuring complete emission spectra from individual cells in flow cytometry has long been of interest.
• Existing methods often face limitations in routine application and robustness.

Purpose of the Study:

• To develop and validate novel flow cytometry instruments and analysis approaches for routine, robust spectral emission measurement from single cells.
• To evaluate the performance of spectral unmixing algorithms and characterize near-infrared fluorophores for spectral flow cytometry.

Main Methods:

• Development of spectral flow cytometers utilizing holographic gratings and CCD detectors for high-speed, wavelength-resolved detection.
• Customized software for displaying and analyzing single-cell spectral data to derive new parameters.
• Utilized reference beads, quantum dot-stained microspheres, and antibody capture beads for instrument calibration, spectral unmixing comparison (virtual bandpass vs. classic least squares), and immunophenotyping.

Main Results:

• Demonstrated quantitative assessment of instrument performance using calibration beads.
• Compared virtual bandpass filtering with classic least squares (CLS) spectral unmixing using quantum dot-stained microspheres.
• Successfully performed immunophenotyping of peripheral blood mononuclear cells using antibody capture beads and CLS unmixing.
• Characterized and evaluated near-infrared (NIR) emitting fluorophores for spectral flow cytometry applications.

Conclusions:

• Spectral flow cytometry provides a simplified optical path, high spectral resolution, and streamlined quantitative multiparameter measurements for single-cell analysis.
• The developed instrumentation, software, and analysis approaches enable routine and robust spectral measurements, facilitating broader application development.