Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids

Schmitz, Alexander; Fischer, Sabine; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H. K.

doi:10.1038/srep43693

Cited by 57 publications

(93 citation statements)

References 56 publications

(80 reference statements)

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“…| 2743 different stacks allows the observation of a great part or even the totality of the sample (Richardson & Lichtman, 2015). In particular, LSFM is the fluorescence-based microscopy technique with the highest penetration depth (>1 cm), and therefore has been broadly used for the imaging of intact spheroids (Achilli et al, 2012;Benninger & Piston, 2013;Combs & Shroff, 2017;Costa et al, 2016;Desmaison et al, 2018;Feuchtinger, Walch, & Dobosz, 2016;Genina et al, 2010;Gualda, Simão, Pinto, Alves, & Brito, 2014;Marques, Oliveira, Chang, Paula-Neto, & Menezes, 2015;Masson et al, 2015;Paiè, Bragheri, Bassi, & Osellame, 2016;Pampaloni, Ansari, & Stelzer, 2013;Reynaud, Kržič, Greger, & Stelzer, 2008;Santi, 2011;Schmitz, Fischer, Mattheyer, Pampaloni, & Stelzer, 2017;Smyrek & Stelzer, 2017;Weber et al, 2015;Wenzel et al, 2014).…”

Section: Conceptmentioning

confidence: 99%

“…The BABB method was initially used to clear mice embryos and whole brains for allowing their imaging by ultramicroscopy. Similarly,Schmitz et al (2017) applied the BABB clearing method and mDSLM to image Draq5 stained T47D spheroids (displaying diameters ranging from 150 μm to more than 500 μm. For that purpose, spheroids were initially dehydrated in ascending ethanol concentrations(50, 70, 85, and 99% (v/v); 5 min each) and then transferred to a BABB (1:1 (v/v)) solution.…”

mentioning

confidence: 99%

“…The obtained results revealed that the shape of the cell nuclei and the cell division is influenced by the spheroid growth conditions, that is, free growth over a nonadhesive surface versus physically confined growth conditions (spheroids embedded in agarose;Desmaison et al, 2018). Similarly,Schmitz et al (2017) applied the BABB clearing method and mDSLM to image Draq5 stained T47D spheroids (displaying diameters ranging from 150 μm to more than 500 μm. After the clearing procedure, the authors could perform the study of several parameters in spheroids, such as spheroids volume, number of cells, and cell nucleus volume by using the automated nuclei segmentation and image computational analysis.…”

mentioning

confidence: 99%

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Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Costa

Silva

Moreira

et al. 2019

Biotech & Bioengineering

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Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a lightscattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI;(b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted. K E Y W O R D Simaging, light scattering, optical clearing, optical microscopy, spheroids.

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Section: Conceptmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Costa

Silva

Moreira

et al. 2019

Biotech & Bioengineering

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“…Nuclei segmentation was performed as previously described in Schmitz et al 2017. Raw image stacks of the nuclei channel were pre-processed in Fiji (ImageJ version 1.51h, Java version 1.8.0_66).…”

Section: Automated Nuclei Segmentationmentioning

confidence: 99%

Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens

Hoette

Koch

Hof

et al. 2019

Preprint

Self Cite

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In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution.The most direct approach to reduce aberrations, improve the contrast, and achieve an optimal resolution is minimizing the impact of changes of the refractive index along an optical path.Many light sheet fluorescence microscopes operate with a large chamber that contains an aqueous immersion medium and an inner specimen holder that contains the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimize the impact of the specimen holder on the optical quality, we use multi-facetted cuvettes fabricated with vacuum-formed ultra-thin fluorocarbon (FEP) foils The ultra-thin FEP-foil cuvettes have a wall thickness of about 12 µm. They are resilient to fluidic exchanges, durable, mechanically stable and yet flexible.We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs, thick tissue sections and dense organoid clusters. The cuvettes outperform many other sample-mounting techniques in terms of full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding, as well as their applicability for multiple-view imaging and automated segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The ultra-thin cuvettes allow the fixation and staining of the specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.

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“…We also assessed performance on the detection of the number of neighbours as this feature can be particularly relevant when studying collective organisation of the cells in various contexts (Blin et al, 2018;Bove et al, 2017;Mesa et al, 2017;Schmitz et al, 2017;Shaya et al, 2017;Toth et al, 2018). Again, only Ilastik and Nessys resulted in an accurate neighbour count with a standard deviation of 2.2 neighbours from GT.…”

Section: Quantification Of Performance Metrics Of Nessys In Comparisomentioning

confidence: 99%

Nessys: a novel method for accurate nuclear segmentation in 3D

Blin

Sadurska

Migueles

et al. 2018

Preprint

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Methods for measuring the properties of individual cells within their native 3D environment will enable a deeper understanding of embryonic development, tissue regeneration, and tumorigenesis. However current methods for segmenting nuclei in 3D tissues are not designed for situations where nuclei are densely packed, non-spherical, heterogeneous in shape, size, or texture, all of which are true of many embryonic and adult tissue types as well as in many cases for cells differentiating in culture.Here we overcome this bottleneck by devising a novel method based on labelling the nuclear envelope (NE) and automatically distinguishing individual nuclei using a tree structured ridge tracing method followed by shape ranking according to a trained classifier. The method is fast and makes it possible to process images that are larger than the computer's memory. We consistently obtain accurate segmentation rates of >90% even for challenging images such as mid-gestation embryos or 3D cultures. We provide a 3D editor and inspector for the manual curation of the segmentation results as well as a program to assess the accuracy of the segmentation.We have also generated a live reporter of the NE that can be used to track live cells in three dimensions over time. We use this to monitor the history of cell interactions and occurrences of neighbour exchange within cultures of pluripotent cells during differentiation.We provide these tools in an open-access user-friendly format. 2 35 40 45 50 2012; Carpenter et al., 2013; Meijering et al., 2016).Here, we report a new approach to overcome these bottlenecks in quantitative image analysis of individual cells in 3D. Rather than relying on staining for nuclear content (for example DAPI or Hoescht staining), we instead detect the nuclear envelope (NE). This makes it easier to identify individual nuclei that are in close contact with each other and does not suffer from segmentation problems associated with textured nuclear staining, unusually shaped nuclei, or cell debris. Furthermore, the NE of individual nuclei are easily discernable by eye in crowded tissues, and so manual correction of any mis-segmented nuclei becomes easier than is the case for DAPI stained nuclei. We provide a user-friendly 3D-4D editing tool to rapidly correct any

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Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids

Cited by 57 publications

References 56 publications

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens

Nessys: a novel method for accurate nuclear segmentation in 3D

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