干旱区生态环境知识资源中心(Arid): Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

Arid

DOI	10.3389/fnana.2020.599282
	Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index
	Bekkouche, Bo M. B.; Fritz, Helena K. M.; Rigosi, Elisa; O'Carroll, David C.
通讯作者	Bekkouche, BMB
来源期刊	FRONTIERS IN NEUROANATOMY
ISSN	1662-5129
出版年	2020
卷号	14
英文摘要	Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount (MS/P) with several more recently applied clearing media that offer tunable refractive index (n): 2,2 '-thiodiethanol (TDE), SeeDB2 (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, n = 1.52 and 1.47, respectively) and Rapiclear (also with n = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.
英文关键词	transparency shrinkage insect brain brain imaging super-resolution microscopy digital light-sheet neuron imaging optical tissue clearing
类型	Article
语种	英语
开放获取类型	Green Published, gold
收录类别	SCI-E
WOS记录号	WOS:000595444100001
WOS关键词	DETECTING NEURONS ; CENTRAL COMPLEX ; DESERT LOCUST ; TISSUE ; WATER ; DROSOPHILA ; FIXATION ; CLARITY ; ETHANOL
WOS类目	Anatomy & Morphology ; Neurosciences
WOS研究方向	Anatomy & Morphology ; Neurosciences & Neurology
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/327830
作者单位	[Bekkouche, Bo M. B.; Fritz, Helena K. M.; Rigosi, Elisa; O'Carroll, David C.] Lund Univ, Dept Biol, Lund, Sweden
推荐引用方式 GB/T 7714	Bekkouche, Bo M. B.,Fritz, Helena K. M.,Rigosi, Elisa,et al. Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index[J],2020,14.
APA	Bekkouche, Bo M. B.,Fritz, Helena K. M.,Rigosi, Elisa,&O'Carroll, David C..(2020).Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index.FRONTIERS IN NEUROANATOMY,14.
MLA	Bekkouche, Bo M. B.,et al."Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index".FRONTIERS IN NEUROANATOMY 14(2020).