Two-photon flow cytometry with laser scanning two-dimensional airy beams

Flow cytometry is an essential technique in biomedical discovery for cell counting, cell sorting, and biomarker detection. In vivo flow cytometers based on one-photon or two-photon excited fluorescence have been developed for over a decade. One drawback of the laser beam scanning two-photon flow cytometer is that the two-photon excitation volume is limited to the focal spot due to the short Rayleigh range of focused Gaussian beams. Hence, the sampling volume is much smaller than in one-photon flow cytometers, making it challenging to count or detect rare circulating cells in vivo. Non-diffracting light waves like Bessel beams and Airy beams have narrow intensity profiles with an effective spot size as small as several wavelengths, making them comparable to Gaussian beams. More significantly, the theoretical depth of field (propagation distance without diffraction) can be infinite, making them an ideal solution as a light source for scanning beam flow cytometry. The trade-off of using Airy beams rather than Gaussian beams is side lobes Airy beams have, which contribute to background noise. Two-photon excitation can reduce this noise, as the excitation efficiency is proportional to intensity squared. Therefore, we developed a two-photon flow cytometer using 2D Airy beams to form a light-sheet that intersects the blood vessel a microfluidic channel, which was used to model a blood vessel. The setup can successfully detect and count flowing fluorescent microspheres in a microchannel.