Tailored Sample Mounting for Light-Sheet Fluorescence Microscopy of Clarified Specimens by Polydimethylsiloxane Casting

Anno: 2019

Autori: Di Giovanna AP., Credi C., Franceschini A., Muellenbroich MC., Silvestri L., Pavone FS.

Affiliazione autori: Univ Florence, European Lab Nonlinear Spect, Sesto Fiorentino, Italy; Univ Florence, Dept Informat Engn DINFO, Florence, Italy; Univ Glasgow, Sch Phys & Astron, Glasgow, Lanark, Scotland; CNR, Natl Inst Opt, Florence, Italy; Univ Florence, Dept Phys & Astron, Sesto Fiorentino, Italy

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.

Giornale/Rivista: FRONTIERS IN NEUROANATOMY

Volume: 13      Da Pagina: 35-1  A: 35-10

Maggiori informazioni: This project received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreements No. 720270 (Human Brain Project) and 654148 (Laserlab-Europe), and from the EU programme H2020 EXCELLENT SCIENCE-European Research Council (ERC) under grant agreement ID No. 692943 (BrainBIT). The project has also been supported by the Italian Ministry for Education, University, and Research (Ministero dell’Istruzione, dell’Universita e della Ricerca) in the framework of the Flagship Project NanoMAX and of Eurobioimaging Italian Nodes (ESFRI research infrastructure), and by Ente Cassa di Risparmio di Firenze (private foundation).
Parole chiavi: light-sheet microscopy; tissue clearing; brain; sample mounting; PDMS
DOI: 10.3389/fnana.2019.00035

Citazioni: 8
dati da “WEB OF SCIENCE” (of Thomson Reuters) aggiornati al: 2025-05-18
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