Abstract
The combination of biological tissue clearing methods with light-sheet fluorescence microscopy (LSFM) allows acquiring images of specific biological structures of interest at whole organ scale and microscopic resolution. Differently to classical epifluorescence techniques, where the sample is cut into slices, LSFM preserves the whole organ architecture, which is of particular relevance for investigations of long-range neuronal circuits. This imaging modality comes with the need of new protocols for sample mounting. Gel matrix, hooks, tips, glues, and quartz cuvettes have been used to keep whole rodent organs in place during image acquisitions. The last one has the advantage of avoiding sample damage and optical aberrations when using a quartz refractive index (RI) matching solution. However, commercially available quartz cuvettes for such large samples are expensive. We propose the use of polydimethylsiloxane (PDMS) for creating tailor-made cuvettes for sample holding. For validation, we compared PDMS and quartz cuvettes by measuring light transmittance and performing whole mouse-brain imaging with LSFM. Moreover, imaging can be performed using an inexpensive RI matching solution, which further reduces the cost of the imaging process. Worth of note, the RI matching solution used in combination with PDMS leads to a moderate expansion of the sample with respect to its original size, which may represent an advantage when investigating small components, such as neuronal processes. Overall, we found the use of custom-made PDMS cuvettes advantageous in term of cost, image quality, or preservation of sample integrity with respect to other whole-mouse brain mounting strategies adopted for LSFM.
Highlights
Light-sheet fluorescence microscopy (LSFM) has allowed high-throughput imaging of large intact biological samples at subcellular resolution (Voie et al, 1993; Engelbrecht and Stelzer, 2006; Dodt et al, 2007)
For larger specimens, such as mouse brain, the agarose concentration needed for creating a stable matrix has a detrimental impact on the light sheet quality, because of optical aberrations caused by the refractive index (RI) inhomogeneity between the agarose sample holder and the mounting medium
Customized PDMS cuvettes for LSFM samples holding were successfully obtained by replicating a negative aluminum (Al) mold realized with tailored geometry by standard machining processes (Figures 1A,B)
Summary
Light-sheet fluorescence microscopy (LSFM) has allowed high-throughput imaging of large intact biological samples at subcellular resolution (Voie et al, 1993; Engelbrecht and Stelzer, 2006; Dodt et al, 2007). Noted by Reynaud et al (2015): ‘‘Light-sheet microscopy offers the possibility of designing microscopes for specific, often threedimensional (3D), samples, but this can mean doing away with the coverslip and, with it, many well-established protocols for sample mounting.’’ Ranging from custom-made to commercially available light-sheet microscopes, different mounting techniques for imaging of large samples have been designed (Silvestri et al, 2013; Reynaud et al, 2015) One of these consists in embedding the sample in a matrix gel, such as agarose. High volume shrinkage occurs after curing, resulting in a possible build-up of internal stresses, the formation of defects as well as dimensional changes Because of these constraints, we ruled out UV-curable resins and focused instead on thermocurable compounds. The PDMS cuvette was realized combining standard micromachining with polymer casting molding processes, which involve casting of material against a mold having the negative shape required for obtaining the final object
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