Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Abstract

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion

“…However, as observed with the classical histological paraffin inclusion process (Gerdes et al, 1982;Iwadare et al, 1984), the 3DISCO method induces TG shrinkage (due to dehydration). Tissue shrinkage has recently been estimated at about 20% for the spinal cord (Erturk and Bradke, 2013;Erturk et al, 2014). However, the proportion and spatial organization of the TG are preserved in cleared tissues as recently described (Belle et al, 2014;Erturk et al, 2012;Erturk and Bradke, 2013;Erturk et al, 2014;Renier et al, 2014).…”

“…Several important new clearing methods have been developed and used: ScaleA2 (Hama et al, 2011), 3DISCO (Belle et al, 2014;Erturk et al, 2012;Erturk and Bradke, 2013;Erturk et al, 2014), SeeDB (Ke et al, 2013;Ke and Imai, 2014), CLARITY Tomer et al, 2014), PACT/PARS and CUBIC Tainaka et al, 2014).…”

“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 De Felipe and Belmonte, 1999;Felipe et al, 1999). Another advantage of light-sheet fluorescent microscopy is the ability to easily generate high-resolution images of ROIs (Belle et al, 2014;Erturk et al, 2014). Here, we combined light sheet imaging and Imaris software to obtain high-resolution images of labeled corneal afferent neurons in a whole TG.…”

“…For the last three years, several groups have developed new tissue clearing methods for enhanced imaging of entire tissues: ClearT (Kuwajima et al, 2013), SeeDB (Ke et al, 2013;Ke and Imai, 2014), ScaleA (Hama et al, 2011), CLARITY Tomer et al, 2014), PACT/PARS , CU-BIC Tainaka et al, 2014), and 3-dimensional imaging of solvent cleared organ (3DISCO) (Erturk and Bradke, 2013;Erturk et al, 2014). When combined with subsequent lightsheet laser scanning microscopy (Dodt et al, 2007), 3DISCO, offers the possibility of imaging entire transparent organs without the need for sectioning (Dodt et al, 2007;Erturk et al, 2012;Erturk and Bradke, 2013).…”

Analysis of Coronary Vessels in Cleared Embryonic Hearts

“…BABB (a 1:2 mixture of benzyl alcohol and benzyl benzoate) is an inexpensive, commonly used clearing agent, which has the advantage of clearing samples extremely quickly. BABB-based clearing and imaging techniques have been described previously for neurological samples and various organs [10][11][12][13] . Here we describe a robust and straightforward technique for the BABB clearance of immunostained samples followed by confocal microscopy, with specific reference to the examination of blood vessels in murine hearts from E (embryonic day) 11.5 -15.5.…”