Evaluation of seven optical clearing methods in mouse brain

Abstract

Recently, a variety of tissue optical clearing techniques have been developed to reduce light scattering for imaging deeper and three-dimensional reconstruction of tissue structures. Combined with optical imaging techniques and diverse labeling methods, these clearing methods have significantly promoted the development of neuroscience. Each of them has its own characteristics with certain advantages and disadvantages. Though there are some comparison results, the clearing methods covered are limited and the evaluation indices lack uniformity, which made it difficult to select a best-fit protocol from numerous methods for clearing in practical applications. Hence, it is necessary to systematically assess and compare these clearing methods. We evaluated the performance of seven typical clearing methods, including 3-D imaging of solvent-cleared organs (3DISCO), ultimate DISCO (uDISCO), see deep brain (SeeDB), ScaeS, , clear, unobstructed brain imaging cocktails and computational analysis, and passive CLARITY technique (PACT), on mouse brain samples. First, we compared the clearing effect and clearing time as well as size deformation on brain tissues. Further, we evaluated the fluorescence preservation and the increase of imaging depth induced by different methods. The results showed that 3DISCO, uDISCO, and PACT possessed excellent clearing capability on mouse brains, ScaeS and SeeDB rendered moderate transparency, whereas performed the worst. uDISCO and 3DISCO induced substantial size reduction on brain sections, and PACT expanded the mouse brain most seriously. Among those methods, ScaeS performed best on fluorescence retention, 3DISCO induced the biggest decline of the fluorescence. PACT achieved the highest increase of imaging depth, and SeeDB and possessed the shallowest imaging depth. This study is expected to provide important reference for users in choosing the most suitable brain optical clearing method.

An optimized tissue clearing protocol for rat brain labeling, imaging, and high throughput analysis

“…We began by applying the solvent based iDISCO+ (Renier, Adams et al 2016) methodology, which offers several advantages for application to the larger brain of the common Long Evans outbred rat strain. iDISCO+ delipidation and clearing causes minimal distortion of tissue size as compared to aqueous based protocols which tend to enlarge samples (Wan, Zhu et al 2018) and was developed to optimize the penetration of antibodies for immunolabeling of endogenous proteins. In addition, the inherent autofluorescence present in the 488nm wavelength spectrum of iDISCO+ cleared brains provides highly detailed structural information which can be used to register resulting image data sets to standard MRI based brain atlases.…”

Confocal Microscopy: Principles and Modern Practices

“…Several of these techniques are reviewed in Wan et al. (). Multiphoton lasers are another available tool for deep imaging and are characterized by the use of pulsed near‐infrared illumination that penetrates tissues deeper than visible wavelengths (Denk, Strickler, & Webb, ).…”

“…Recent advances in sample preparation include methods to "clear" tissue with readily available chemicals that remove lipids and other highly scattering cellular components. Several of these techniques are reviewed in Wan et al (2018). Multiphoton lasers are another available tool for deep imaging and are characterized by the use of pulsed near-infrared illumination that penetrates tissues deeper than visible wavelengths (Denk, Strickler, & Webb, 1990).…”

Optimization and quantitative evaluation of fluorescence in situ hybridization chain reaction in clarified fresh-frozen brain tissues

“…The advent of the HCR FISH method alongside the recent development of intact tissue clearing and high-throughput microscopic imaging methods has broadened the path to obtain unprecedented information about the molecular identity, interactions, and activity-dependent functions in the brain. To date, several tissue clearing approaches have been used to achieve rodent brain transparency but only a few have proved to be versatile and successful across a wide range of applications (Silvestri et al 2016;Azaripour et al 2016;Wan et al 2018). Based on factors like clearing capability, target biomolecule fixation, ability to perform ex-vivo labeling, fluorescence retention and imaging depth, we chose to explore two methods 1) tissue lipid matrix replacementbased CLARITY (Chung et al 2013) with modifications (Tomer et al 2014;Yang et al 2014;Zheng and Rinaman 2016;Krolewski et al 2018) and 2) non-aqueous solvent-based iDISCO+ (Renier et al 2014).…”