A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue

Abstract

For 3-dimensional (3D) imaging of a tissue, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1° clearing of the opaque tissue to render it transparent for microscopy, 2° fluorescence labeling of the tissues and 3° 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most clearing techniques have been applied to the central nervous system and other organs that contain relatively low amounts of connective tissue including extracellular matrix. However, tissues that contain large amounts of extracellular matrix such as dermis in skin or gingiva are difficult to clear. The present survey lists methodologies that are available for clearing of tissues for 3D imaging. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether (DBE) are the most successful methods for clearing connective tissue-rich gingiva and dermis of skin for 3D histochemistry and imaging of fluorescence using light-sheet microscopy.

Mesenchymal stem cells differentially affect the invasion of distinct glioblastoma cell lines

“…To determine differences in proliferation rates of GBM cells in MSC/GBM mixed xenografts, relative changes in fluorescence intensity of GBM cells between 1 day and 3 days after cell implantation were quantified using ImageJ (http://rsbweb.nih.gov/ij/; National Institutes of Health, USA). For confocal imaging, the embryos were fixed at 72 h after cell injection using 4% paraformaldehyde in phosphate-buffered saline for 2 h, and cleared in Sca l e U2, which was modified to enable visualization of carbocyanine-dye-labelled cells [60]. Imaging was performed with a TCS SPE confocal microscope (Leica) as described previously [34].…”

Neuroscience in the third dimension: shedding new light on the brain with tissue clearing

“…In addition, they showed that 3DISCO increased the signal to noise ratio while prolonging the integrity of the fluorescence. Furthermore, this protocol could be applied to a vast array of tissues ranging from lipid-rich structures such as the brain, but also extracellular matrix-rich structures such as gingiva [6]. Overall, 3DISCO offered a standardized protocol for clearing multiple tissues in a simple, efficient and cost-effective manner that was widely applicable.…”

“…Tissue clearing protocols have since diversified into over 10 different clearing agents. The diversity of clearing protocols is beyond the scope of this article, for a comprehensive discussion on this topic you can refer to the following recent reviews [3–6]. What has become apparent in the past five years is that the field has split into two main approaches: i) solvent-based clearing, and ii) aqueous-based clearing techniques.…”

Under the (Light) Sheet after the iDISCO+

“…Additionally, there are many limitations, such as both limited and uneven penetration of the antibodies to the tissue sections, rendering full quantitative evaluation close to impossible. Now imagine there is no obstacle for antibodies to penetrate through the brain tissue and there is also no visibility limit (2), that is, we can see (using microscopy) through the brain (see the overview of clearing techniques [3]). Finally, let's assume that all the labor-intensive procedures in the scenarios are automated and, after few days of processing, the computers spit out quantified data registered per individual brain nuclei.…”