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.

Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination - converting two photons into one photon of half the incident wavelength - and can be detected by conventional two-photon microscopy. Because the optical signal of SHG nanoprobes is based on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long-term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution.

To address the need for a bright, photostable labeling tool that allows long-term in vivo imaging in whole organisms, we recently introduced second harmonic generating (SHG) nanoprobes. Here we present a protocol for the preparation and use of a particular SHG nanoprobe label, barium titanate (BT), for in vivo imaging in living zebrafish embryos. Chemical treatment of the BT nanoparticles results in surface coating with amine-terminal groups, which act as a platform for a variety of chemical modifications for biological applications. Here we describe cross-linking of BT to a biotin-linked moiety using click chemistry methods and coating of BT with nonreactive poly(ethylene glycol) (PEG). We also provide details for injecting PEG-coated SHG nanoprobes into zygote-stage zebrafish embryos, and in vivo imaging of SHG nanoprobes during gastrulation and segmentation. Implementing the PROCEDURE requires a basic understanding of laser-scanning microscopy, experience with handling zebrafish embryos and chemistry laboratory experience. Functionalization of the SHG nanoprobes takes ∼3 d, whereas zebrafish preparation, injection and imaging setup should take approximately 2-4 h.

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1–2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.