Optically clearing tissue as an initial step for 3D imaging of core biopsies to diagnose pancreatic cancer

Das, Ronnie; Agrawal, Aishwarya; Upton, Melissa P.; Seibel, Eric J.

doi:10.1117/12.2041114

Cited by 2 publications

(2 citation statements)

References 19 publications

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“…In order to improve this situation, we developed a technique that allows to clear, image and three-dimensionally reconstruct tumour resectates up to the size of a standard histological cassette. In contrast to previously published studies, where needle-core biopsies 21 , 49 , surface scanning or imaging of several mm-thick slices of cleared specimen were performed 6 , our method can be used for clearing of tissue samples in a size-range of several centimetres. A major advantage of our method is the possibility to reconstruct the whole tumour in its spatial vicinity, including the complete tumour margins in 3D, from up to several thousands of optical sections per sample.…”

Section: Discussionmentioning

confidence: 97%

3D histopathology of human tumours by fast clearing and ultramicroscopy

Sabdyusheva-Litschauer

Becker

Saghafi

et al. 2020

Sci Rep

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Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid. (b) Ultrafast active chemical dehydration with 2,2-dimethoxypropane and (c) refractive index matching with dibenzyl ether at up to 56 °C. After clearing, the tumour resectates are imaged. The images are computationally post-processed for contrast enhancement and artefact removal and then 3D reconstructed. Importantly, the sequence a–c is fully reversible, allowing the morphological correlation of one and the same histological structures, once visualized with our novel technique and once visualized by standard H&E- and IHC-staining. After reverting the clearing procedure followed by standard H&E processing, the hallmarks of ductal carcinoma in situ (DCIS) found in the cleared samples could be successfully correlated with the corresponding structures present in H&E and IHC staining. Since the imaging of several thousands of optical sections is a fast process, it is possible to analyse a larger part of the tumour than by mechanical slicing. As this also adds further information about the 3D structure of malignancies, we expect that our technology will become a valuable addition for histological diagnosis in clinical pathology.

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Section: Discussionmentioning

confidence: 97%

3D histopathology of human tumours by fast clearing and ultramicroscopy

Sabdyusheva-Litschauer

Becker

Saghafi

et al. 2020

Sci Rep

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“…38 They fabricated a millimetre-scale custom chamber from glass microscope slides in order to clear a 40-600 lm thick section of tissue with millimetre-scale area. Clearing in the device yielded greatly improved light transmittance and allowed the authors to image pancreatic islet structures in the tissue; however, only one tissue section could be cleared at a time.…”

Section: Introductionmentioning

confidence: 99%

On-chip clearing of arrays of 3-D cell cultures and micro-tissues

et al. 2016

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Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 lm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear T2 , and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear T2 ; however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This onchip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications. Published by AIP Publishing. [http://dx

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Optically clearing tissue as an initial step for 3D imaging of core biopsies to diagnose pancreatic cancer

Cited by 2 publications

References 19 publications

3D histopathology of human tumours by fast clearing and ultramicroscopy

3D histopathology of human tumours by fast clearing and ultramicroscopy

On-chip clearing of arrays of 3-D cell cultures and micro-tissues

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