Abstract
Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.
Highlights
Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues
We develop an easy-to-use multi-immersion open-top light-sheet (OTLS) microscope that overcomes the limitations of previous light-sheet fluorescence microscopy (LSFM) systems, enabling high-throughput 3D imaging of one or more specimens prepared with any published clearing protocol[5,13,15]
The point spread function (PSF) of the system is shown in Fig. 1c, in which sub-micron resolution is achieved lateral to the collection axis
Summary
Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. Many early LSFM systems that were designed to image cleared tissues, such as ultramicroscopy (UM) and CLARITY-optimized light-sheet microscopy (COLM), imaged specimens from above, with either air objectives or immersion objectives oriented downward toward (or into) a large liquid-filled chamber[9,10] While this geometry is suitable for imaging moderately sized specimens such as mouse brains with dual-sided illumination for increased light penetration, the specimens are laterally constrained by the chamber and the arrangement of the objectives. With LSTM, a high-magnification collection objective is dipped downward in the vertical direction into a liquid reservoir, with a pair of longer working distance objectives (oriented at 60 ° with respect to the vertical axis) to generate the light sheet While both diSPIM and LSTM enable laterally unconstrained imaging, there are several drawbacks:[1] When fully immersed, cleared tissues equilibrate to a similar density as the surrounding medium, which often causes the tissues to float and drift (Supplementary Fig. 2)[2]. Specimens are imaged from above, which makes it difficult to rely upon gravity or gentle pressure to flatten the top surfaces of the specimens (if so desired)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
