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Femtosecond laser microsurgery is a powerful method for studying cellular function, neural circuits, neuronal injury and neuronal regeneration because of its capability to selectively ablate sub-micron targets in vitro and in vivo with minimal damage to the surrounding tissue. Here, we present a step-by-step protocol for constructing a femtosecond laser microsurgery setup for use with a widely available compound fluorescence microscope. The protocol begins with the assembly and alignment of beam-conditioning optics at the output of a femtosecond laser. Then a dichroic mount is assembled and installed to direct the laser beam into the objective lens of a standard inverted microscope. Finally, the laser is focused on the image plane of the microscope to allow simultaneous surgery and fluorescence imaging. We illustrate the use of this setup by presenting axotomy in Caenorhabditis elegans as an example. This protocol can be completed in 2 d.

The zebrafish larva is an optically-transparent vertebrate model with complex organs that is widely used to study genetics, developmental biology, and to model various human diseases. In this article, we present a set of novel technologies that significantly increase the throughput and capabilities of our previously described vertebrate automated screening technology (VAST). We developed a robust multi-thread system that can simultaneously process multiple animals. System throughput is limited only by the image acquisition speed rather than by the fluidic or mechanical processes. We developed image recognition algorithms that fully automate manipulation of animals, including orienting and positioning regions of interest within the microscope's field of view. We also identified the optimal capillary materials for high-resolution, distortion-free, low-background imaging of zebrafish larvae., National Institutes of Health (U.S.) (Director's New Innovator Award DP2 OD002989), National Institutes of Health (U.S.) (Transformative Research Award R01 NS073127), David & Lucile Packard Foundation (Award in Science and Engineering), Broad Institute of MIT and Harvard (SPARC Award), Foxconn International Holdings Ltd., Athinoula A. Martinos Center for Biomedical Imaging (Training Grant)

In this paper, we explore the use of anatomical information as a guide in the image formation process of fluorescence molecular tomography (FMT). Namely, anatomical knowledge obtained from high resolution computed tomography (micro-CT) is used to construct a model for the diffusion of light and to constrain the reconstruction to areas candidate to contain fluorescent volumes. Moreover, a sparse regularization term is added to the state-of-the-art least square solution to contribute to the sparsity of the localization. We present results showing the increase in accuracy of the combined system over conventional FMT, for a simulated experiment of lung cancer detection in mice.

Analytically predicting photon paths in real-high scattering-anisotropic tissues is extremely complex, due to the significant random scattering events that photons suffer as they traverse the tissue, especially at boundaries between areas with different optical properties. An statistically correct optical and anatomical model of photon trajectories inside laboratory animals will therefore improve considerably our understanding about how light diffuses within the animals, and therefore help us designing efficient experimental setups and reconstruction algorithms for fluorescence mediated tomography (FMT). Here, we present new simulations of photon propagation and fluorescence emission in anisotropic media using realistic models of laboratory animals and a Monte Carlo (MC) based approach. We compare the MC simulation results with an approximation of the solution of the diffusion equation using finite differences and discuss the different behaviour of the two methods.