A method for ultrafast tissue clearing that preserves fluorescence for multimodal and longitudinal brain imaging.
3D histopathology imaging
Fluorescence enhancement
Neuro-circuit tracing
Three-dimensional visualisation
Ultrafast tissue clearing
Journal
BMC biology
ISSN: 1741-7007
Titre abrégé: BMC Biol
Pays: England
ID NLM: 101190720
Informations de publication
Date de publication:
29 03 2022
29 03 2022
Historique:
received:
12
05
2021
accepted:
09
03
2022
entrez:
30
3
2022
pubmed:
31
3
2022
medline:
6
4
2022
Statut:
epublish
Résumé
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. 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. 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.
Sections du résumé
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.
Identifiants
pubmed: 35351101
doi: 10.1186/s12915-022-01275-6
pii: 10.1186/s12915-022-01275-6
pmc: PMC8966190
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
77Informations de copyright
© 2022. The Author(s).
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