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The evolution of laser technologies and the invention of ultrashort laser pulses have resulted in a sharp jump in laser applications in life sciences. Developmental biology is no exception. The unique ability of ultrashort laser pulses to deposit energy into a microscopic volume in the bulk of transparent material without disrupting the surrounding tissues makes ultrashort lasers a versatile tool for precise microsurgery of cells and subcellular components within structurally complex and fragile specimens like embryos as well as for high-resolution imaging of embryonic processes and developmental mechanisms. Here, we present an overview of recent applications of ultrashort lasers in developmental biology, including techniques of noncontact laser-assisted microsurgery of preimplantation mammalian embryos for oocyte/blastomere enucleation and embryonic cell fusion, as well as techniques of optical transfection and injection for targeted delivery of biomolecules into living embryos and laser-mediated microsurgery of externally developing embryos. Possible applications of ultrashort laser pulses for use in Assisted Reproductive Technologies are also highlighted. Moreover, we discuss various nonlinear optical microscopy techniques (two-photon excited fluorescence, second and third harmonic generation, and coherent Raman scattering) and their application for label-free non-invasive imaging of embryos in their unperturbed state or post-laser-induced modifications. Full article

Single cell microinjection provides precise tuning of the volume and timing of delivery into the treated cells; however, it also introduces workflow complexity that requires highly skilled operators and specialized equipment. Laser-based microinjection provides an alternative method for targeting a single cell using a common laser and a workflow that may be readily standardized. This paper presents experiments using a 1550 nm, 100 fs pulse duration laser with a repetition rate of 20 ns for laser-based microinjection and calculations of the hypothesized physical mechanism responsible for the experimentally observed permeabilization. Chinese Hamster Ovarian (CHO) cells exposed to this laser underwent propidium iodide uptake, demonstrating the potential for selective cell permeabilization. The agreement between the experimental conditions and the electropermeabilization threshold based on estimated changes in the transmembrane potential induced by a laser-induced plasma membrane temperature gradient, even without accounting for enhancement due to traditional electroporation, strengthens the hypothesis of this mechanism for the experimental observations. Compared to standard 800 nm lasers, 1550 nm fs lasers may ultimately provide a lower cost microinjection method that readily interfaces with a microscope and is agnostic to operator skill, while inducing fewer deleterious effects (e.g., temperature rise, shockwaves, and cavitation bubbles). Full article