Spatially confined green-to-red photoconversion of fluorescent proteins with high-power, pulsed laser illumination is negligible, thus precluding optical selection of single cells in vivo. We report primed conversion, in which low-power, dual-wavelength, continuous-wave illumination results in pronounced photoconversion. With a straightforward addition to a conventional confocal microscope, we show confined primed conversion in living zebrafish and reveal the complex anatomy of individual neurons packed between neighboring cells.

No abstract

Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO 4 and BaTiO 3 nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow.

While nanoparticles are an increasingly popular choice for labeling and tracking stem cells in biomedical applications such as cell therapy, their intracellular fate and subsequent effect on stem cell differentiation remain elusive. To establish an effective stem cell labeling strategy, the intracellular nanocrystal concentration should be minimized to avoid adverse effects, without compromising the intensity and persistence of the signal necessary for long-term tracking. Here, the use of second-harmonic generating barium titanate nanocrystals is reported, whose achievable brightness allows for high contrast stem cell labeling with at least one order of magnitude lower intracellular nanocrystals than previously reported. Their long-term photostability enables to investigate quantitatively at the single cell level their cellular fate in hematopoietic stem cells (HSCs) using both multiphoton and electron microscopy. It is found that the concentration of nanocrystals in proliferative multipotent progenitors is over 2.5-fold greater compared to quiescent stem cells; this difference vanishes when HSCs enter a nonquiescent, proliferative state, while their potency remains unaffected. Understanding the nanoparticle stem cell interaction allows to establish an effective and safe nanoparticle labeling strategy into somatic stem cells that can critically contribute to an understanding of their in vivo therapeutic potential.