Person:

Kwok, Sheldon

The advent of phototransformable fluorescent proteins has led to significant advances in optical imaging, including the unambiguous tracking of cells over large spatiotemporal scales. However, these proteins typically require activating light in the UV-blue spectrum, which limits their in vivo applicability due to poor light penetration and associated phototoxicity on cells and tissue. We report that cyanine-based, organic dyes can be efficiently photoconverted by nonlinear excitation at the near infrared (NIR) window. Photoconversion likely involves singlet-oxygen mediated photochemical cleavage, yielding blue-shifted fluorescent products. Using SYTO62, a biocompatible and cell-permeable dye, we demonstrate photoconversion in a variety of cell lines, including depth-resolved labeling of cells in 3D culture. Two-photon photoconversion of cyanine-based dyes offer several advantages over existing photoconvertible proteins, including use of minimally toxic NIR light, labeling without need for genetic intervention, rapid kinetics, remote subsurface targeting, and long persistence of photoconverted signal. These findings are expected to be useful for applications involving rapid labeling of cells deep in tissue.

Purpose Scleral cross-linking (SXL) with a photosensitizer and light is a potential strategy to mechanically reinforce the sclera and prevent progressive axial elongation responsible for severe myopia. Current approaches for light delivery to the sclera are cumbersome, do not provide uniform illumination, and only treat a limited area of sclera. To overcome these challenges, we developed flexible optical waveguides optimized for efficient, homogeneous light delivery. Methods: Waveguides were fabricated from polydimethylsiloxane elastomer. Blue light (445 nm) is coupled into the waveguide with an input fiber. Light delivery efficiency from the waveguide to scleral tissue was measured and fit to a theoretical model. SXL was performed on fresh porcine eyes stained with 0.5% riboflavin, using irradiances of 0, 25, and 50 mW/cm2 around the entire equator of the eye. Stiffness of scleral strips was characterized with tensiometry. Results: Light delivery with a waveguide of tapered thickness (1.4–0.5 mm) enhanced the uniformity of light delivery, compared to a flat waveguide, achieving a coefficient of variation of less than 10%. At 8% strain, sclera cross-linked with the waveguides at 50 mW/cm2 for 30 minutes had a Young's modulus of 10.7 ± 1.0 MPa, compared to 5.9 ± 0.5 MPa for no irradiation, with no difference in stiffness between proximally and distally treated halves. The stiffness of waveguide-irradiated samples did not differ from direct irradiation at the same irradiance. Conclusions: We developed flexible waveguides for periscleral cross-linking. We demonstrated efficient and uniform stiffening of a 5-mm-wide equatorial band of scleral tissue.

Purpose To investigate how corneal hydration affects the Brillouin frequency of corneal stroma. Methods: From a simple analytical model considering the volume fraction of water in corneal stroma, we derived the dependence of Brillouin frequency on hydration and hydration-induced corneal thickness variation. The Brillouin frequencies of fresh ex vivo porcine corneas were measured as their hydration was varied in dextran solution and water. Healthy volunteers (8 eyes) were scanned in vivo repeatedly over the course of 9 hours, and the diurnal variations of Brillouin frequency and central corneal thickness (CCT) were measured. Results: The measured dependence of Brillouin frequency on hydration, both ex vivo and in vivo, agreed well with the theoretical prediction. The Brillouin frequencies of human corneas scanned immediately after waking were on average ∼25 MHz lower than their daytime average values. For stabilized corneas, the typical variation of Brillouin frequency was ± 7.2 MHz. With respect to CCT increase or swelling, the Brillouin frequency decreased with a slope of −1.06 MHz/μm in vivo. Conclusions: The ex vivo and in vivo data agree with our theoretical model and support that the effect of corneal hydration on Brillouin frequency comes predominantly from the dependence of the tissue compressibility on the water. Corneal hydration correlates negatively with the Brillouin frequency. During daytime activities, the influence of physiological hydration changes in human corneas is < ± 10 MHz. The sensitivity to hydration may potentially be useful in detecting abnormal hydration change in patients with endothelial disorders.

Large-scale single-cell analyses have become increasingly important given the role of cellular heterogeneity in complex biological systems. However, no current techniques enable optical imaging of uniquely-tagged individual cells. Fluorescence-based approaches can only distinguish a handful of distinct cells or cell groups at a time because of spectral crosstalk between conventional fluorophores. Here we show a novel class of imaging probes emitting coherent laser light, called laser particles. Made of silica-coated semiconductor microcavities, these laser particles have single-mode emission over a broad range from 1170 to 1580 nm with sub-nm linewidths, enabling massive spectral multiplexing. We demonstrate the stability and biocompatibility of these probes in vitro and their utility for wavelength-multiplexed cell tagging and imaging. We demonstrate real-time tracking of thousands of individual cells in a 3D tumor model for several days showing different behavioral phenotypes. We expect laser particles will enable new approaches for single-cell analyses.

Large-scale single-cell analyses have become increasingly important given the role of cellular heterogeneity in complex biological systems. However, no current techniques enable optical imaging of uniquely-tagged individual cells. Fluorescence-based approaches can only distinguish a small number of distinct cells or cell groups at a time because of spectral crosstalk between conventional fluorophores. Here we investigate large-scale cell tracking using intracellular laser particles as imaging probes that emit coherent laser light with a characteristic wavelength. Made of silica-coated semiconductor microcavities, these laser particles have single-mode emission over a broad range from 1170 to 1580 nm with sub-nm linewidths, enabling massive spectral multiplexing. We explore the stability and biocompatibility of these probes in vitro and their utility for wavelength-multiplexed cell tagging and imaging. We demonstrate real-time tracking of thousands of individual cells in a 3D tumour model over several days showing different behavioural phenotypes.