A versatile sample holder for single plane illumination microscopy

Abstract

Single Plane Illumination Microscopy is an emerging and powerful technology for live imaging of whole living organisms. However, sample handling that relies on specimen embedding in agarose or gel is often a key limitation, especially for time-lapse monitoring. To address this issue, we developed a new concept for a holder device allowing us to prepare a sample container made of hydrogel. The production process of this holder is based on 3D printing of both a frame and casting devices. The simplicity of production and the advantages of this versatile new sample holder are shown with time-lapse recording of multicellular tumour spheroid growth. More importantly, we also show that cell division is not impaired in contrast to what is observed with gel embedding. The benefit of this new holder for other sample types, applications and experiments remains to be evaluated, but this innovative concept of fully customizable sample holder preparation potentially represents a major step forward to facilitate the large diffusion of single plane illumination microscopy technology.

Light-sheet microscopy: a tutorial

“…This is a timely technique as biomedical sciences are currently experiencing a strong need to visualize samples in 3D as biological processes occur in volumes. Therefore, despite the fact that 2D cells placed in petri dishes have been widely used to study biological process, LSFM allows the use of agarose or fluorinated ethylene propylene (FEP) tubes [17,42,43] for mounting 3D living samples. Thus, 2D models have been constantly replaced by 3D cell cultures [44][45][46][47], spheroids [48][49][50][51][52], organs and organoids [26,[53][54][55], and model organisms such as Drosophila melanogaster, zebrafish and C. elegans [2,30,33,56].…”

“…To keep the sample hydrated and to allow the flux of nutrients toward the living sample inside the chamber, samples are embedded in a transparent porous substrate. Depending on the sample and on the application, this substrate can be made up of agarose gels, molten phytagel [43], and collagen fibrous microenvironments [121], among others. More recently, FEP materials have proven to be useful since their refractive index is close to that of water, helping to minimize aberrations.…”

3D print customized sample holders for live light sheet microscopy

“…[8] with an HCT116 cell line stably expressing the H2B-mcherry fusion protein [9]. A spheroid was placed in each chamber of a multichamber sample holder using a micropipette.…”

“…This procedure is simple and enables multiview imaging [3,6]; however, it is not always adapted for time-lapse acquisitions of living samples, because the mechanical forces exerted by the gel are known to interfere with biological processes [7]. We previously reported the preparation of a hydrogel-based sample holder that provides the living sample with a constraintless and controllable environment [8]. Although a significant improvement over embedding samples, this type of hydrogel holder suffers from a lack of versatility and above all does not allow for the simultaneous imaging of multiple samples.…”

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

“…The sample is confined in the rigid agarose and can be imaged well for a short period of time, but morphogenetic changes and growth of the embryo are strongly impaired when imaging for several hours 7,8 . Although solutions for other applications have been developed 9 , the noninvasive long-term time-lapse zebrafish imaging potential of light sheet microscopy cannot be exploited using the conventional 1.5% agarose embedding.…”