A polymer gel index-matched to water enables diverse applications in fluorescence microscopy

Abstract

We demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

Volumetric imaging of human mesenchymal stem cells (hMSCs) for non-destructive quantification of 3D cell culture growth

“…Furthermore, single objective lightsheet systems or oblique plane microscopy could better enable on-line imaging of microfluidic samples as there is only a single samplefacing objective and more space for sample mounting and translation below or above the objective [81,82]. Additional motivation is presented with development of microfluidic chips composed of a polymer with a refractive-index matched to water which is compatible with the presented ELIAS method [83]. These systems and methods are more complex than traditional sampling and imaging methods, but would enable non-destructive, robust, and automated analysis of microcarrier-based and other suspension cultures.…”

Multiview super-resolution microscopy

“…In a second example, we imaged the densely labeled nerve ring region in a fixed, 28 m thick L4 stage C. elegans larva embedded in a polymer gel with the same refractive index as water 56 (Fig. 6d), first comparing the raw View C confocal input and the associated 2D SIM output derived from deep learning.…”

“…Our results show that the expansion factor for whole embryos is 3.29 ± 0.14, with nearly isotropic expansion (3.24 ± 0.14, 3.39 ± 0.14, 3.26 ± 0.17 for x, y and z dimensions, respectively). Point scanning confocal imaging for comparison with triple-view line confocal imaging A DCR8528 L4 stage larval worm was transferred from an agar plate into a drop (~10 µL) of UVcurable polymer BIO-133 56 (MY Polymers Ltd.) on a #1.5 cover glass (24 mm x mm, VWR, 48393-241). The droplet was positioned between two µm spacers (Precision Brand, 44910), and was compressed by a glass slide followed by 2-minute polymerization under a UV lamp (365nm, Spectroline ENB-280C).…”