A method for ultrafast tissue clearing that preserves fluorescence for multimodal and longitudinal brain imaging

Abstract

Background Tissue-clearing techniques have recently been developed to make tissues transparent for three-dimensional (3D) imaging at different scales, including single-cell resolution. However, current tissue-clearing workflows have several disadvantages, including complex protocols, time-consuming application, and fluorescence quenching. Additionally, they can be used mainly for clearing larger-volume samples, preventing wide and easy applicability in conventional experimental approaches. In this study, we aimed to develop a versatile, fast, and convenient method for clearing thin and semi-thick samples, which can be used for three-dimensional imaging of experimental or even clinical samples. Results We developed an alkaline solution (AKS) containing a combination of 2,2′-thiodiethanol (TDE), DMSO, D-sorbitol, and Tris for tissue clearing, as the alkaline environment is suitable for maintaining the fluorescence of most commonly used fluorescence protein GFP and its variants, and tested its clearing effect on samples from mice and human brains. We assessed the clearing speed, the preservation of fluorescence protein and dyes, and the imaging depth and quality. The results showed that AKS treatment rapidly cleared 300-μm-thick brain slices and 1-mm-thick slices from different organs within 5 min and 1 h, respectively. Moreover, AKS was compatible with a variety of fluorescence proteins and dyes. Most importantly, AKS enhanced the fluorescence of YFP, in contrast to the majority of existing tissue-clearing methods which reduce the fluorescence intensity of fluorescent proteins. Using AKS, we performed long-time high-resolution imaging of weak fluorescent protein-labelled tissues, long-distance fibre tracking, larger-scale 3D imaging and cell counting of the entire brain area, neural circuit tracing, 3D neuromorphic reconstruction, and 3D histopathology imaging. Conclusions AKS can be used for simple and rapid clearing of samples from mice and human brains and is widely compatible with a variety of fluorescent dyes. Therefore, AKS has great potential to be used as a broad tissue-clearing reagent for biological optical imaging, especially for time-sensitive experiments.

Temporal tracking of microglial and monocyte single-cell transcriptomics in lethal flavivirus infection

“…After, sections were washed in PBS and then incubated with anti-goat-CF488A (SAB4600032, Sigma-Aldrich), streptavidin-AF594 (Invitrogen) and anti-rabbit-CF647 (SAB4600352, Sigma-Aldrich) for 3 days. Sections were washed and then cleared in 20% DMSO, 40% 2,2'-thiodiethanol, 20% d-sorbitol and 0.5 M TRIS for 1 h before imaging [ 71 ]. Images were captured on the Zeiss LSM800 (Sydney Microscopy & Microanalysis, USYD) using Zen Blue software.…”

Acute Rift Valley fever virus infection induces inflammatory cytokines and cell death in ex vivo rat brain slice culture

“…pathogenic RVFV were fixed overnight in 4 % paraformaldehyde then free-float stained for viral antigen (anti-RVFV N pAb) and counterstained with DAPI. Slices were cleared using alkaline solution (AKS) [23] and imaged at 2.5X on a Leica DMI8. At 24 h p.i.…”

“…Slices were counterstained with Hoechst, washed again with BPS and transferred to glass slides with bridge mounts. Slides were cleared by adding alkaline solution and incubating 5 min at RT in the dark [23]. Cover glass was then affixed over the bridge-mounted slices.…”

Selective modification of ascending spinal outputs in acute and neuropathic pain states

“…Spinal cords were dissected and placed into 4% PFA overnight at 4 °C then transferred to 1X PBS. Clearing solution was made by dissolving 20% (vol/vol) DMSO, 40% (vol/vol) TDE, 20% (wt/vol) sorbitol, and 6% (wt/vol) Tris base into ddH2) 67 . Spinal cords were removed from vertebral columns and cut into 1.5 cm sections.…”