Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues
Posted on: 20 March 2019
Preprint posted on 13 February 2019
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-019-10534-0
Improvements in optics and materials expand the imaging applications of this fast light-sheet microscope
Selected by Tim Fessenden
Categories: bioengineering, cancer biology, cell biology, developmental biology, pathology
Context
Rich descriptions of normal or diseased tissues using microscopy are circumscribed by the limitations of confocal fluorescence or widefield imaging. These limitations include, among others, sample fixation and embedding techniques, poor light penetrance into a tissue, the maximum axial resolution, and the time needed to scan across a sample. Together these obstacles enact boundaries on the spatial information gleaned from imaging a tissue sample. Moreover, the speed of image acquisition places a constraint on the area of a sample that can reasonably be analyzed. These considerations have collectively taught biologists to think of and analyze small areas of tissues sectioned into thin slices. While the achievements of traditional microscopy of tissue sections need not be defended, simultaneous improvements in tissue preparation, optics, and computation can free biologists and clinicians to image tissue specimens as they are, not as we prepare them to ready them for imaging.
The group of Jonathan T. C. Liu recently published their light sheet microscope optimized for large specimens, namely tissue biopsies1. Light sheet microscopy uses lower light intensity and yields improved sensitivity because only a thin plane of the specimen is illuminated at a time. But achieving these benefits requires focusing two objectives on the sample in close proximity, which severely restricts the size and format of samples. The Liu group overcame this limitation by constructing a light sheet microscope in an open top format, not unlike a document scanner. They demonstrated exceptionally rapid rendering of thick 3 dimensional human biopsy tissues, leveraging the improved imaging quality of light sheet microscopy.
Data
The core of the present work is a systematic exploration of materials and immersion solutions in order to optimize the group’s open-top light sheet microscope for different specimens. This work confronts the sacrifices made in optics to enable the open top, scan bed format. Because the light paths for illumination and emission pass through a plane of glass at oblique angles, the resulting image quality suffers. Further, the refractive index of glass is not matched for many of the solvents used for tissue clearing or expansion microscopy. This limits the type of samples compatible with their original microscope design.
The authors improve this design first with illumination and detection objectives that offer greatly improved axial and lateral resolutions as well as a much greater working distance. As in the preceding design, the authors make use of a solid immersion lens (SIL) on the illumination objective, to match the cylindrically focused illumination beam to the spherically focused emission beam. They also incorporate their recently published method to minimize shadow artifacts when imaging through thick and opaque samples2. To achieve this, the incident light is swept within the plane of the light-sheet, a technique they term multidirectional digital scanned light sheet microscopy (mDSLM).
The bulk of this work is in optimizing two materials critical to improve the versatility of this instrument: the fluid in which the detection objective is immersed, and the transparent holder on which the sample rests above the objectives. Using simulations, the authors test 19 immersion fluids that span sample preparations in aqueous (e.g. CLARITY)3, solvent (e.g. iDISCO)4, or expansion microscopy5 solutions. Against these, they test 13 different glass and 14 different monomer/polymer materials for the sample holding material. For each combination they calculate the maximum thickness of the holding material while retaining acceptable image quality. The resulting matrix identifies the combinations that do not compromise image quality based on the sample type or preparation.
With these results in hand, the authors validate their findings by swapping out the immersion fluid and sample holder material for an array of samples. They demonstrate fast and exceptionally detailed 3D microscopy of human prostate biopsies; cleared mouse brain, lymph node, heart, prostate, and lung samples; and expanded mouse kidney samples prepared at 4x their original dimensions. Leveraging the benefits of the open-top system inherent to its original design, the authors image samples in high throughput fashion, for instance by placing 12 prostate core needle biopsies adjacent to each other on the 10 x 10 cm sample bed.
Implications
In this work the authors describe a conceptually straightforward yet practically challenging solution to a persistent problem in microscopy. That is, how to democratize recently developed microscopy techniques to spread the benefits of improved instruments to more users in more fields? Glaser and colleagues combine light-sheet microscopy with an open-top format, optimized in this work to improve resolution, sensitivity, and imaging depth. In doing so, the authors offer a means to spread the benefits of light-sheet microscopy to minimally processed human biopsies, cleared specimens, and expanded specimens. Many biologists have taken advantage of light-sheet microscopy only by modifying their sample preparation to suit its unique needs. The instrument described here removes this limitation while sacrificing little in terms of image quality.
Many advances in microscopy push at the limits of optics to achieve this or that benchmark. While exciting, the technologies that result from these efforts are often limited to the optimal biological specimens for that instrument, strongly constraining its utility. From the standpoint of adoption and utility, this work offers a contribution to biological imaging that well surpasses that of a narrow technical advance.
References
- Glaser, A. K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 0084 (2017).
- Glaser, A. K. et al. Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging. Sci. Rep. 8, (2018).
- Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332 (2013).
- Renier, N. et al. iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell 159, 896–910 (2014).
- Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy. Science 347, 543–548 (2015).
doi: https://doi.org/10.1242/prelights.9291
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| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
| List by | Nándor Lipták |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
| List by | Jesus Victorino |
Single Cell Biology 2020
A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.
| List by | Alex Eve |
Society for Developmental Biology 79th Annual Meeting
Preprints at SDB 2020
| List by | Irepan Salvador-Martinez, Martin Estermann |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EDBC Alicante 2019
Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.
| List by | Sergio Menchero et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
SDB 78th Annual Meeting 2019
A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.
| List by | Alex Eve |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Young Embryologist Network Conference 2019
Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London
| List by | Alex Eve |
Pattern formation during development
The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.
| List by | Alexa Sadier |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
Zebrafish immunology
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.
| List by | Shikha Nayar |
Also in the pathology category:
October in preprints – DevBio & Stem cell biology
Each month, preLighters with expertise across developmental and stem cell biology nominate a few recent developmental and stem cell biology (and related) preprints they’re excited about and explain in a single paragraph why. Short, snappy picks from working scientists — a quick way to spot fresh ideas, bold methods and papers worth reading in full. These preprints can all be found in the October preprint list published on the Node.
| List by | Deevitha Balasubramanian et al. |
October in preprints – Cell biology edition
Different preLighters, with expertise across cell biology, have worked together to create this preprint reading list for researchers with an interest in cell biology. This month, most picks fall under (1) Cell organelles and organisation, followed by (2) Mechanosignaling and mechanotransduction, (3) Cell cycle and division and (4) Cell migration
| List by | Matthew Davies et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
COVID-19 / SARS-CoV-2 preprints
List of important preprints dealing with the ongoing coronavirus outbreak. See http://covidpreprints.com for additional resources and timeline, and https://connect.biorxiv.org/relate/content/181 for full list of bioRxiv and medRxiv preprints on this topic
| List by | Dey Lab, Zhang-He Goh |
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Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
| List by | Pablo Ranea Robles |