Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological
parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and
protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for
detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified.
Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance
measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous
measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2
m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous
excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme
allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors.
In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line
pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this
system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and
assessment of cell viability using a conventional fluorescent apoptosis assays.