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A Quality Metric for the Systematic Evaluation of Clearing Protocols

Light sheet imaging guidelines to achieve single-cell resolution of cleared 3D multicellular spheroids and organoids

Single-cell Huh-7D12 spheroid imaged using a Leica SP8 DLS microscope system. The spheroid was clarified using the Sucrose protocol. A_Quality_Metric_and_Light_Sheet_Imaging_Guideline_teaser.jpg

3D multicellular spheroids are of interest for studying tumor behavior and evaluating the response of pharmacologically active agents, because they mimic the in vivo tumor environment better than standard 2D cell cultures. However, despite the biological advantages of spheroids, visualizing single cells in the innermost layers of large 3D spheroids with current microscope solutions remains a challenge, mainly due to light scattering causing poor penetration.

The difficulties can be overcome with a combination of fluorescence light sheet microscopy technologies and optical clearing methods. In a recent study, several metrics for evaluating the efficiency of clearing protocols are compared. An imaging guideline for reaching single-cell resolution when analyzing multicellular aggregates with the SP8 DLS microscope system is also described.

One of the DLS main advantages is that it facilitates illumination of the sample from two sides. Spheroids with a diameter of around 250 µm, derived from 3 different cell lines, were analyzed and details inside them visualized using 5 different clearing protocols. The full 3D dataset, which includes 90 fluorescently stained spheroids, has been shared with the community. To characterize the image quality of spheroids, organoids, or other 3D multicellular aggregates, a novel metric is proposed. This metric can serve as a gold standard for the quantitative comparison of optical clearing protocols and help users in choosing the most suitable one for any given experiment.

Figure 1: A: Single-cell Huh-7D12 spheroid imaged using a Leica SP8 DLS microscope system. The spheroid was clarified using the Sucrose protocol; nuclei were stained using DRAQ5. B: Single z-plane from the middle region of the same spheroid.
Figure 2: Brightfield images of uncleared and cleared spheroids according to (from top to bottom): ClearT, ClearT2, CUBIC, ScaleA2, Sucrose protocols, respectively.

Read the full article:

A Diosdi, D Hirling, M Kovacs, T Troth, M Harmati, K Koos, K Buzas, F Piccinini, P Horvath:

A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids

Computational and Structural Biotechnology Journal, 19:1233-1243, February 2021. DOI: 10.1016/j.csbj.2021.01.040

https://www.sciencedirect.com/science/article/pii/S2001037021000441

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