Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-μm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12-to 350-μm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca 2+ signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5-20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation.laser surgery | calcium imaging | Drosophila melanogaster I ntravital imaging of small model organisms is a staple technique in many fields of biomedicine. Gaining optical access to the body's interior usually requires removal or thinning of a highly scattering, protective exterior. Unfortunately, surgical creation of an optical window is generally time-consuming, requires considerable skill and dexterity, and often represents an unwanted source of experimental variability. To address these challenges, we created laser surgical means to open optical windows in the bodies of live animals.Our approach is adaptable to many species and applications, but we mainly focused on fluorescence brain imaging in alert fruit flies, a pursuit of growing importance for the study of neural systems (1, 2). Most prior studies that have monitored flies' neural activity have relied on immobilized fly preparations suitable for optical microscopy (3, 4) or electrophysiology (5, 6). Recent improvements in genetically encoded Ca 2+ indicators and imaging methods now allow in vivo fluorescence imaging of neural activity in behaving flies (2). The fly brain's small size, <250 μm deep in the adult, is conducive to two-photon imaging of a variety of neural populations after opening of the cuticle (2, 4).Multiple laboratories have developed protoco...
