Integrated photothermal flow cytometry in vivo.

Area of Science:

• Biomedical Optics
• Cellular Biology
• Microcirculation Research

Background:

• In vivo detection of circulating cells in their natural states is challenging.
• Existing methods lack real-time, high-resolution capabilities for intravital analysis.
• Photothermal (PT) techniques offer potential for label-free cellular analysis.

Purpose of the Study:

• To demonstrate the capability of integrated flow cytometry for real-time, in vivo detection of moving cells.
• To showcase a novel system combining photothermal techniques with high-speed digital microscopy.
• To explore the potential applications of this new optical tool, termed PT flow cytometry (PTFC).

Main Methods:

• Integration of dual pump-probe photothermal (PT) techniques (PT imaging, thermolens, velocimetry).
• Utilized high-resolution (0.3 µm), high-speed (1000 fps) transmission digital microscopy (TDM) and fluorescence imaging.
• Irradiation of cells in rat mesenteric microvessels with a tunable laser pulse and detection of refractive index variations with a probe laser beam.

Main Results:

• Demonstrated real-time, in vivo detection of circulating red and white blood cells in rat mesentery.
• Successfully monitored the integral PT response from single, moving, unlabeled cells (100-500 cells per measurement).
• Achieved high-resolution and high-speed imaging of cellular dynamics in microvessels.

Conclusions:

• Photothermal flow cytometry (PTFC) is a viable tool for in vivo cellular analysis.
• PTFC enables identification of cells based on absorptive properties and size, assessment of laser-induced damage, and flow velocity estimation.
• This technology holds significant potential for monitoring circulating cells and understanding microvascular dynamics.