High-resolution 3D imaging of whole organ after clearing: taking a new look at the zebrafish testis

Abstract

Zebrafish testis has become a powerful model for reproductive biology of teleostean fishes and other vertebrates and encompasses multiple applications in applied and basic research. Many studies have focused on 2D images, which is time consuming and implies extrapolation of results. Three-dimensional imaging of whole organs recently became an important challenge to better understand their architecture and allow cell enumeration. Several protocols have thus been developed to enhance sample transparency, a limiting step for imaging large biological samples. However, none of these methods has been applied to the zebrafish testis. We tested five clearing protocols to determine if some of them could be applied with only small modifications to the testis. We compared clearing efficiency at both macroscopic and microscopic levels. CUBIC and PACT were suitable for an efficient transparency, an optimal optical penetration, the GFP fluorescence preservation and avoiding meaningful tissue deformation. Finally, we succeeded in whole testis 3D capture at a cellular resolution with both CUBIC and PACT, which will be valuable in a standard workflow to investigate the 3D architecture of the testis and its cellular content. This paves the way for further development of high content phenotyping studies in several fields including development, genetic or toxicology.

Comparative analysis reveals Ce3D as optimal clearing method for in toto imaging of the mouse intestine

“…Therefore, taken together with their destructive effects on morphology, these protocols were not further considered for testing their compatibility with immunostaining with intestinal neurochemical markers. Of the five aqueous‐based protocols, urea‐based methods such as ScaleS and CUBIC equally induced substantial GFP quenching, probably due to the presence of the high detergent concentration and the denaturating properties of urea . In this study, only UbasM (using much lower detergent concentrations compared to CUBIC), SeeDB2 and Ce3D (both using iohexol solutions) met the requirements in terms of GFP signal retainment, as was also found for other fluorescent reporters .…”

“…Substantial or even complete loss of GFP fluorescence occurred in intestinal samples of CX3CR1 transgenic mice after 3DISCO, iDISCO+, uDISCO, and Visikol clearing, which is in line with previous findings in other tissues . Therefore, taken together with their destructive effects on morphology, these protocols were not further considered for testing their compatibility with immunostaining with intestinal neurochemical markers.…”

“…It has been reported that the potent clearing properties of 3DISCO, iDISCO+, and uDISCO, which might be an advantage for brain tissue, may lead to destructive effects on biological (sub)cellular structures, due to the aggressive properties of ao methanol, THF, and DCM . Similarly, in line with earlier reports on other tissues the CUBIC protocol affected the morphology of the intestinal samples, limiting compatibility of these protocols for posthoc correlative ultrastructural studies.…”

Three‐dimensional reconstruction of prostate cancer architecture with serial immunohistochemical sections: hallmarks of tumour growth, tumour compartmentalisation, and implications for grading and heterogeneity

“…and Van Royen et al ., and that, according to Boag et al ., led to the failure of reconstructions in low‐grade prostate cancer. There are also some other prospective microscopy methods, which have still not been extensively tested for 3D reconstructions of prostate cancer architecture; these should be analysed in further studies …”

“…There are also some other prospective microscopy methods, which have still not been extensively tested for 3D reconstructions of prostate cancer architecture; these should be analysed in further studies. [11][12][13] However, the subtle details of the architecture (e.g. necessary for grading) are very well depicted with reconstructions involving a minimal depth of tissue.…”