New Approaches of 3D Imaging and Image Analysis of Neural Tissues in Connectomics Researches

New and improved methods are urgently needed for the simultaneous evaluation of large populations of cells in three dimensions, with a focus on fine details of their cytoarchitecture and their structural contacts with surrounding cells (Lo and Chiang, 2016). Several methods have been developed for the large-scale imaging of transparent and intact tissues, including BABB (Dodt et al., 2007), Scale (Hama et al., 2011), 3DISCO (Erturk et al., 2012), ClearT (Kuwajima et al., 2013), SeeDB (Ke et al., 2013), CLARITY (Chung et al., 2013), passive CLARITY (Tomer et al., 2014), PACT (Yang et al., 2014), CUBIC (Susaki et al., 2014; Tainaka et al., 2014) FASTClear (Liu et al., 2016), and FACT (Xu et al., 2017). Of these approaches, the ones that clear tissue by replacing the water in the tissue with organic solvents, such as BABB and 3DISCO, cannot prevent the quenching of fluorescent protein signals for longer than two days (Dodt et al., 2007; Erturk et al., 2012; Ke et al., 2013). Therefore, these approaches are limited in their usefulness for long-term tissue preservation or prolonged imaging applications. To overcome this serious limitation, aqueous-based clearing approaches such as Scale, SeeDB, and ClearT have been developed, and these can prevent fluorescent quenching for approximately one week without any changes in tissue size (Hama et al., 2011; Ke et al., 2013; Kuwajima et al., 2013). However, these powerful approaches are restricted to transgenic labels in animal models. To address these issues, hydrogel-based clearing methods, including CLARITY and PACT, have been introduced (Chung and Deisseroth, 2013; Yang et al., 2014). These approaches provide conditions for antibody labeling of tissue markers, and they can also be used with transgenic labels in animal models. However, CLARITY uses electrophoretic tissue clearing (ETC) to extract lipids from large samples, and these results in the destruction of fine cellular structures (Chung et al., 2013). The PACT (Yang et al., 2014) and passive CLARITY (Tomer et al., 2014) methods have faster clearing speed and preserve the tissue structure by avoiding the use of ETC. However, for long-term imaging, the deformation of tissues caused by hydrogel expansion during clearing limits the usefulness of these powerful methods for evaluating fine structures such as microglia branches and neuronal processes. As a further improvement, the FASTClear (Liu et al., 2016) method avoids the use of hydrogel and is performed at 50°C to increase the clearing speed compared to PACT. However, the FASTClear approach has been limited to antibody labeling (Liu et al., 2016). Thus, it was necessary to develop an optimized method to clear thick fluorescent tissue by reducing the clearing time while optimizing the reagents and temperature so as to preserve the fluorochrome signal. A new method has been developed by merging and modifying the PACT and FASTClear approaches (Xu et al., 2017). Removing the hydrogel perfusion and embedding steps from the PACT method improved the speed of clearing, and decreasing the temperature in the FASTClear method to 37°C and optimizing the clearing solution pH to 7.5 decreased the quenching of fluorescent transgenic labels. Thus, Fast Free-of-Acrylamide Clearing Tissue (FACT) is a simple and rapid approach which provides optimal conditions for visualizing transgenic fluorescent proteins and antibody labeling of tissue markers. The FACT protocol is original and distinct from other protocols in that it improves the signal to noise ratio, depth of tissue penetration, speed of processing, long-term retention of fluorescent signal, and preservation of cytoarchitecture

Keywords:

3D Imaging , Neural tissues , Connectomics Researches