Optimization of GFP Fluorescence Preservation by a Modified uDISCO Clearing Protocol

Abstract

Tissue optical clearing techniques provide alternative approaches for imaging large-volume specimens. uDISCO, an organic-solvent-based method, stands out from the enormous array of available optical clearing methods by achieving whole-brain imaging with high transparency, size reduction and fluorescence preservation. In this study, we aimed to modify the uDISCO protocol to achieve better fluorescence preservation and to thereby further improve its optical imaging quality. First, we determined the optimal pH value for optimized uDISCO, termed “a-uDISCO” (alkaline pH-based uDISCO). Then, we compared fluorescence preservation between a-uDISCO and uDISCO. In addition, we validated the clearing performance of the optimized method according to several parameters, including tissue transparency, size changes, and the maintenance of cell morphology. Finally, we demonstrated that a-uDISCO enabled the high-quality brain-wide visualization of neuronal structures. This method potentially provides a better alternative for high-throughput imaging of samples with low-level fluorescence protein expression or for archiving and repetitive revisiting of rare samples.

Fast, 3D isotropic imaging of whole mouse brain using multi-angle-resolved subvoxel SPIM

“…An 8-week-old whole mouse brain ( Tg : thy1-GFP-M ) was optically cleared using the a-uDISCO method 42 , before being imaged by the Mars-SPIM. The brain shrank in size from ~9.3 × 14 × 7.1 mm to ~7 × 9.5 × 5 mm after clearing (Figure 3a).…”

A new pipeline for pathophysiological analysis of the mammary gland based on organoid transplantation and organ clearing

“…However, although this technique is compatible with immunodetection of endogenous and tagged proteins, we noticed that some level of autofluorescence in the GFP channel could disturb the visualization of weak signals. In this respect, it might be useful to implement and adapt the protocol recently designed for decreasing autofluorescence in the brain (Li et al, 2018). A summary of the main advantages and limitations of the present method is provided in Table 1.…”

Advances in Ex Situ Tissue Optical Clearing

“…Two prominent features of uDISCO are: 1) significant, up to 65% reduction of organ volume, which is essential for whole‐body LSFM imaging, and 2) robust preservation of transgenically expressed fluorescent proteins (XFPs), achieved not by alkalization of solutions (as in original FluoClearBABB), but by replacing standard BABB with BABB‐D, a mix of BABB, diphenyl ether and scavenger of peroxides—tocopherol. A group led by Dan Zhu has recently reported that application of alkaline dehydrating agents (THF or tert ‐butanol) as well as alkaline RI‐matching BABB‐D, along with incubations and sample storage performed at 4 °C, might increase fluorescent signal preservation even further . Orderly screening resulted in description of FDISCO—a protocol that relies on sample dehydration with alkaline (pH 9.0) THF and DBE‐mediated RI‐matching.…”

“…This was first linked to the tissue deprivation of water molecules, the presence of which was suspected obligatory for the maintenance of XFPs emission . Nevertheless, data obtained by several research groups clearly demonstrated that alkalization of dehydrating solutions (to pH of 9.0–9.5) prevents rapid loss of XFPs stability regardless of chemical used ( tert ‐butanol in case of FluoClearBABB and PEGASOS, tert ‐butanol and BABB‐D in a‐uDISCO, THF in FDISCO, ethanol in ECi, and 1‐propanol in 2nd generation of ECi). Based on these, it seems apparent that it is acidic pH of dehydrating solution, not loss of water molecules, that should be attributed for rapid quenching of XFPs.…”