19–21 The technique is a two-step fabrication process that consists of: (1) permanent material modification following nonlinear absorption of focused femtosecond laser pulses (2) etching of the laser modified zone by a hydrofluoric acid (HF) solution. The effective integration of the cylindrical lens with the microfluidic channel is enabled by the unique 3D processing capabilities and the high level of precision and control of femtosecond laser micromachining (FLM). In our optofluidic device, the light sheet is created across a microfluidic channel by an embedded cylindrical lens and the scanning is performed by letting the sample flow at a constant speed through the light sheet. Scanning is then normally achieved by moving the sample with a motorized translation stage. SPIM employs a cylindrical lens to form a light sheet in a single section of the specimen. We demonstrate high-throughput LSFM, segmentation and quantification in thick, three-dimensional cellular tissues. We use this device to automatically scan biological samples under a conventional microscope, without the need of any motorized stage. The device, fully fabricated by femtosecond laser micromachining, includes an optofluidic cylindrical lens with an aspherical profile to create an aberration-free light sheet illumination across the channel where the sample is circulated by a microfluidic pump. Here we present a millimeter-scaled optofluidic lab-on-a-chip that integrates SPIM illumination and continuous sample delivery in a microfluidic channel. 13,17,18 These limitations have hindered high-throughput, three dimensional imaging of biological samples. 14 However, these approaches are limited to small specimen imaging 14–16 or require the mechanical translation of the whole device with respect to the imaging system, to record the entire samples in 3D. An intense effort has been recently devoted to this topic: 12,13 the preferred methods consist of the combination of fluidic capillaries with LSFM setups, or in the integration of light sheet illumination in lab on chips. The mounting procedure is time consuming, taking much longer time compared to the acquisition itself and is not compatible with multi-specimen imaging, limiting experimental throughput and statistics.Īpplication of LSFM to toxicology, drug screening and gene expression studies on a large scale requires new systems able to perform massive recording and analysis of multiple samples. 11 These samples, normally embedded in a gel, are imaged individually by mounting them one at a time on a microscope. tissue spheroids), 9,10 and to entire embryos ( e.g. 2–7 LSFM has been used to image biological systems of various spatial scales, from single cells 8 to artificial tissues ( e.g. In contrast with wide-field and confocal microscopy, they provide fast, high-resolution optical sectioning over large tissue volumes with low photo-toxicity. Light sheet fluorescence microscopy (LSFM) methods, such as Selective Plane Illumination Microscopy (SPIM), 1 have revolutionized the field of biological imaging. Using femtosecond laser micromachining, we created an integrated optofluidic device that allows obtaining continuous flow imaging, three-dimensional reconstruction and high-throughput analysis of large multicellular spheroids at a subcellular resolution. Complex sample preparation and system alignment normally limit the throughput of the method. Selective plane illumination microscopy can image biological samples at a high spatiotemporal resolution.
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