New technical approaches for 3D morphological imaging and quantification of measurements

Abstract

3D imaging is becoming more and more popular, as it allows us to identify interactions between structures in organs. Furthermore, it gives the possibility to quantify and size these structures. To allow 3D imaging, the tissue sample has to be transparent. This is usually achieved by using optical tissue clearing protocols. Although using optical tissue clearing often results in perfect 3D images, these protocols have some pitfalls, like long duration of sample preparation (up to several weeks), use of toxic substances, damage to antibody staining, fluorescent proteins or dyes, high refractive indices, and high costs of sample processing.Recently we described [Huang et al., Scientific Reports 9(1): 521 (2019)] a fast, safe, and inexpensive ethyl cinnamate (ECi) based optical tissue clearing protocol. Here, we present extensions of our protocol with respect to the deparaffinization of old paraffin‐embedded samples allowing 3D imaging of the blocks. In addition, we learned to remove ECi from the samples allowing the use of routine immunolabeling protocols. Furthermore, we demonstrate new pictures of lungs after expansion microscopy and adaptation of already existing protocols. The aim of our work is, in summary, to describe the advances in these methodologies, focusing on the morphological imaging of kidneys and lungs.

Introduction to a special issue on kidney development and disease

“…Stereology collaborator Prof. Jens Nyengaard presents in this issue a proof‐of‐concept application of the design‐based nucleator technique for estimating nephron number in human kidneys, using CT scanning and biopsy samples (Nielsen et al, 2020). Collaborator Prof. Norbert Gretz also reports on a new technique, with a description of ethyl cinnamate‐based tissue clearing protocols for the optimization of 3D imaging of paraffin‐embedded tissue (Brenna et al, 2020); great potential to bring old tissue archives back to life. The contemporary application of cationic ferritin‐enhanced magnetic resonance imaging for kidney structural quantification has been extended to assess the renal vasculature in a study by collaborator A/Prof.…”

Optical tissue clearing associated with 3D imaging: application in preclinical and clinical studies

“…Solvent-based clearing agents are more compatible with whole-mount immunostaining and synthetic dyes. In addition, a dehydration step is often required for organ delipidation, allowing a higher sample transparency/translucency (Dobosz 2014 ; Tainaka 2017 ; Cai 2019 ; Hong 2020 ; Huang 2019 ; Brenna 2020 ). Dobosz and coworkers used the BABB technique for visualizing breast tumors, discerning the tumor morphology, vessel architecture, and drug distribution in breast cancer from xenograft mouse models (Dobosz 2014 ).…”

“…Moreover, with the delipidation process also being essential, structures with lipids might appear damaged and disappear; (b) the low clearing efficacy (in particular for BABB) and fluorescence preservation (Kolesová et al 2021 ); (c) some pigments that increase the autofluorescence (such as hemoglobin and melatonin) are not entirely removed by the chemicals. Therefore, when studying mouse models, perfusion with PBS and PFA can optimize these limitations by reducing the autofluorescence and the heme content (Huang et al 2019 ; Brenna et al 2020 ); (d) some chemicals are harmful to the operator; e) the high environmental impact due to the chemicals used; and (f) the long process protocols, which can last from days to weeks. A solution could be found in optimizing the temperature for making more accessible the optical reagent into the tissues (Huang et al 2019 ; Brenna et al 2020 ).…”

“…Therefore, when studying mouse models, perfusion with PBS and PFA can optimize these limitations by reducing the autofluorescence and the heme content (Huang et al 2019 ; Brenna et al 2020 ); (d) some chemicals are harmful to the operator; e) the high environmental impact due to the chemicals used; and (f) the long process protocols, which can last from days to weeks. A solution could be found in optimizing the temperature for making more accessible the optical reagent into the tissues (Huang et al 2019 ; Brenna et al 2020 ). This is the case with ECi clearing, which Klingberg and coworkers ( 2017 ) developed and Huang ( 2019 ) then optimized.…”

Filtering the good from the bad: A focus on kidney development and disease to celebrate John Bertram's long‐standing career in anatomy and renal research

“…The development of these techniques has the potential to make a big impact on the fields of renal biology and clinical medicine, especially as accurate in vivo techniques are developed. Recent advances in these methodologies, feature highly in this Special Issue, with contributions from Norbert Gretz (Brenna et al, 2020), Kevin Bennett (Parvin, Charlton, Baldelomar, Derakhshan, & Bennett, 2020), and Jens Nyengaard (Nielsen et al, 2020) specifically relating to contemporary kidney imaging and quantitation.…”