To better understand cell biology in a more physiologically-relevant context we have progressively moved to the use of 3D cell culture in place of 2D adherent cells on plastic. These spheroids and microtissues (e.g. organoids) can be derived from single cell lines, progenitor cells, or increasingly also co- and tri-cultures to further increase relevance. This immediately challenges the technical limits of confocal and light sheet microscopy due to the level of light scatter, signal loss and related signal:noise ratio that all significantly impacting on the ability to interrogate beyond a few cell layers. This is also the case for tissues and for whole organs from animal models.
To deal with these challenges a wide range of tissue clearing methods have been developed in recent years. However, these have created confusion and none have proved themselves suitably universal necessitating more work to determine best fit of method with cell type, culture system or tissue. Likewise, the choice of nuclear counterstain has been at issue and the point in the protocol at which addition should occur.
Recent papers have sought to improve this situation, and demonstrate that DRAQ5™ has broad utility as a reliable nuclear counterstain in tissue clearing protocols. An example of the use of DRAQ7™ as a counterstain in the CLARITY procedure is also described.
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A broad systematic study from the University of Freiburg (Nürnberg et al.) compared 4 different clearing methods (representative of the common types) against a range of single-cell / tri-cell derived spheroids and co-cultures generated in cavities with different fluorescent readouts for nuclei (DAPI, DRAQ5), proliferation Ki-67, ECFP as constitutive marker and CellTracker dyes. Imaging was performed with confocal microscopy.
In summary, the major conclusions of this work were that tri-cultures proved the most challenging perhaps due to increased cell packing and ECM where no clearing method met the Rose Criterion beyond 90 µm, and where only DAPI was tested as counterstain. ECFP was highly sensitive to all clearing methods.
Results were better in the single-cell and co-culture models, meeting the Rose Criterion (SNR >5) beyond 200 µm when combining glycerol or ScaleS clearing methods with DRAQ5 as nuclear counterstain (which was consistent for all clearing methods). Results were consistently poorer with DAPI and with the other clearing methods - Cytovista, ClearT2 - and referenced against Mowiol and PBS. Additionally Cytovista caused significant shrinkage of the spheroids (necessitating a halving of the z-section depth) while DAPI performed particularly badly in PBS and ClearT2. These results were mostly independent of the cell line used.
In addition to clearing it was also determined that z-compensation was required to achieve good segmentation of nuclei and obtain a quantitative analysis.
Overall, RI-matched 88% glycerol was the simplest and quickest method, performing best overall. Interestingly, the next best was ScaleS where clearing was performed prior to (immuno-) fluorescent stainings unlike the 3 alternatives.
Nürnberg, Elina, et al. "Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward." Frontiers in Molecular Biosciences 7 (2020): 20.
In other work on neutrophil recruitment in Triple-Negative Breast Cancer, SenGupta et al. (Univ. Michigan) generated spheroids by the hanging drop method. 4-day spheroids were DRAQ5-stained (5µM, o/n, 4 degC) and then cleared with FOCM (1-5 min.; ultra-fast optical clearing method, Zhu et al. PNAS 2019) and then imaged by 2-photon microscopy at 1040 nm excitation. FOCM apparently limits expansion/shrinkage and can be used as mounting medium.
SenGupta, Shuvasree, et al. "Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-β and CXCR2 Ligands." Frontiers in immunology 12 (2021): 973.
Mir-Coll et al. (Karolinska Inst.) generated pancreatic islet microtissues by a hanging drop method. For analysis they were first stained with DRAQ5 for 2h and then fixed. Thereafter they were cleared with Visikol HISTO (Visikol) and imaged for Cerulean, EGFP, DsRed2 and DRAQ5 fluorescence by confocal microscopy.
Mir-Coll, Joan, et al. "Human Islet Microtissues as an In Vitro and an In Vivo Model System for Diabetes." International Journal of Molecular Sciences 22.4 (2021): 1813.
In pursuit of standardising comparison of different clearing methods Diosdi et al. (Univ. Szeged) compared spheroids generated with three different carcinoma cell lines and with five clearing protocols (ClearT, ClearT2, CUBIC, ScaleA2, and Sucrose), subsequently stained with DRAQ5 and imaged under identical settings by light sheet microscopy. The resulting images were subjected to experts' evaluation and scored accordingly. ClearT and ClearT2 could not be separated from uncleared controls but also caused significant shrinkage. CUBIC, ScaleA2, and Sucrose gave better results though these varied by cell line. Sucrose caused no overall volumetric change while both CUBIC and ScaleA2 caused some swelling.
DRAQ5 was compatible with all combinations. Interestingly, DRAQ5 was used in PBS for ClearT, ClearT2 and Sucrose and in 4M urea for CUBIC and ScaleA2. This aids its compatibility with multiple protocols and therefore simplifies the decision on choice of chromophores that can be relied upon to perform well, irrespective of the chosen sample preparation methodology.
Diosdi, Akos, et al. "A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids." Computational and Structural Biotechnology Journal 19 (2021): 1233-1243.
A simple protocol for the in situ imaging of kidney across length scales has been proposed by Unnersjö-Jess, et al. (Cologne, Stockholm). The whole nephrectomised organ (mouse/human) was fixed, sectioned to 300 µm slices, followed by an accelerated delipidation (SDS/boric acid, 1h, 70 degC), labeling with antibodies and DRAQ5 as counterstain (10 µM in PBST, 2h, 37 degC) and mounting in fructose/4M urea before imaging from 100X confocal microscopy to conventional immunofluorescence and histology.
Unnersjö-Jess, David, et al. "A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales." Kidney International 99.4 (2021): 1010-1020.
To determine the location of therapeutic human adipose mesenchymal stromal cells (hAMSCs) in a study of the performance of fast-growing cells alongside ganciclovir researchers in Barcelona used a modified CLARITY clearing procedure to analyse brain slices, in a rodent model of treated human glioblastoma infused with GFP-U87 cells and later RFP-hAMSCs. In vivo hydrogel-infused brain slices were cleared by delipidation with SDS/boric acid (4 days, 45 degC) and then mounted between slide and coverslip. DRAQ7 was used as nuclear counterstain, suited due to the DRAQ chromophore's spectral compatible with GFP and RFP. This technique enabled 3D analysis to a depth of 200 µm.
Guerra-Rebollo, M, et al. "Glioblastoma bystander cell therapy: improvements in treatment and insights into the therapy mechanisms." Molecular Therapy-Oncolytics 11 (2018): 39-51.
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