Photothermal flow cytometry in vitro for detection and imaging of individual moving cells

Abstract

Photothermal (PT) cytometry has recently demonstrated great potential for the label-free detection of nonfluorescent cells under static conditions. The goal of our investigation was to expand this technique to the detection of flowing cells in vitro. Cells in flow were irradiated with short, tunable laser pulses (420-2,300 nm, 8 ns), and the absorbed energy was detected by monitoring of the temperature-dependent variations in the refractive index in the cells with a second, collinear probe beam in two modes: (a) PT imaging of single cells with a pulsed probe beam (639 nm, 13 ns) and (b) thermolens monitoring of the integral PT responses from individual cells as whole with a continuous-wave probe beam (633 nm, 2 mW). PT flow cytometry at the current speed of analysis of 10 cell/s, with the capability to image selected cells of interest flowing at velocities up to 2 m/s, demonstrated the capability for (a) label-free detection of flowing single cells (e.g., blood and cancer cells) on the basis of the differences in their endogenous absorption properties, (b) identification of cells labeled with gold nanoparticles, (c) rapid cell viability testing, (d) aggregation immunoassay, and (e) optimization of selective nanophotothermolysis. PT cytometry can be extended to the study of cells in flow. This new technique increases the speed of cell analysis approximately 10(2) times over that of conventional PT technique, with the potential to achieve a rate of 10(4)-10(5) cells/s in specific PT applications, which has previously been realized only with cells under static conditions.