A tunable refractive index matching medium for live imaging cells, tissues and model organisms

Boothe, Tobias; Hilbert, Lennart; Heide, Michael; Berninger, Lea; Huttner, Wieland B.; Zaburdaev, Vasily; Vastenhouw, Nadine L.; Myers, Eugene W.; Drechsel, David; Rink, Jochen C.

doi:10.7554/elife.27240

Cited by 152 publications

(136 citation statements)

References 24 publications

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“…We detected primary miR-430 transcripts at the prominent transcription sites, suggesting a role for miR-430 transcription in their formation (SI Figure 11). To follow potential DNA remodeling at these sites by microscopy with sufficient optical resolution, we cultured embryonic cells in a refractive index-matched medium that we recently developed (Boothe et al, 2017). We monitored promoter-recruited RNA polymerase II as well as elongating RNA polymerase II by live cellcompatible antibody fragments (Hayashi-Takanaka et al, 2011;Stasevich, Hayashi-Takanaka, et al, 2014).…”

Section: Microenvironments Form By Rna Accumulation and Expel Not Tramentioning

confidence: 99%

“…15, 2017; High stage. These cells were mounted in refractive index matched medium exactly as previously described (Boothe et al, 2017). During the time required to mount the cells and start microscopy, cells had undergone one to two divisions.…”

Section: Preparation Of Live Cells For Fluorescence Microscopymentioning

confidence: 99%

“…miR-430 primary transcripts were labeled by RNA fluorescence in situ hybridization (RNA FISH) using a primary transcript probe kindly provided by Antonius van Boxtel (van Boxtel et al, 2015). FISH probes were in vitro transcribed from linearized pGEMt_miR-430_ISH plasmid (NdeI restriction enzyme, New England BioLabs) using T7 polymerase (in vitro transcription mix: 2 µl transcription buffer (Roche), 2 µl DIG RNA labeling mix (Roche), 2 µl DTT stock (0.1 mM stock concentration), 1 µl RNAse inhibitor (Roche), 8 µl linearized DNA, 4 µl nuclease-free H2O, 1 µl T7 polymerase (produced inhouse at Max Planck Institute of Molecular Cell Biology and Genetics), left to incubate at 37°C for 2 hours).…”

Section: Fish Labeling Of Primary Mir-430 Transcriptsmentioning

confidence: 99%

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Transcription organizes euchromatin similar to an active microemulsion

Hilbert

Sato

Kimurâ

et al. 2017

Preprint

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Graphical abstract HighlightsThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/234112 doi: bioRxiv preprint first posted online Dec. 15, 2017; Page 2 of 46 SummaryThe three-dimensional organization of the genome is essential for development and health. Although the organization of euchromatin (transcriptionally permissive chromatin) dynamically adjusts to changes in transcription, the underlying mechanisms remain elusive. Here, we studied how transcription organizes euchromatin, using experiments in zebrafish embryonic cells and theory. We show that transcription establishes an interspersed pattern of chromatin domains and RNA-enriched microenvironments. Specifically, accumulation of RNA in the vicinity of transcription sites creates microenvironments that locally remodel chromatin by displacing not transcribed chromatin. Ongoing transcriptional activity stabilizes the interspersed pattern of chromatin domains and RNA-enriched microenvironments by establishing contacts between chromatin and RNA. We explain our observations with an active microemulsion model based on two macromolecular mechanisms: RNA/RNA-binding protein complexes generally segregate from chromatin, while transcribed chromatin is retained among RNA/RNA-binding protein accumulations. We propose that microenvironments might be central to genome architecture and serve as gene regulatory hubs.

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Section: Microenvironments Form By Rna Accumulation and Expel Not Tramentioning

confidence: 99%

Section: Preparation Of Live Cells For Fluorescence Microscopymentioning

confidence: 99%

Section: Fish Labeling Of Primary Mir-430 Transcriptsmentioning

confidence: 99%

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Transcription organizes euchromatin similar to an active microemulsion

Hilbert

Sato

Kimurâ

et al. 2017

Preprint

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“…It is also likely that other clearing solutions could be substituted for BABB (Richardson & Lichtmann, 2015), and that clearing of live larvae may be possible using the refractive‐index tunable and nontoxic clearing method recently by Boothe et al . (Boothe et al ., 2017). Unlike mammalian tissue (Hama et al ., 2011; Ke et al ., 2013), we did not observe significant tissue shrinkage using the BABB clearing method.…”

Section: Discussionmentioning

confidence: 99%

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

McConnell

Amos

2018

Journal of Microscopy

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SummaryIn a previous paper, we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently‐stained 12.5‐day old mouse embryos. Here, we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show subcellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal discs are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.

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“…Recently, Iodixanol has been investigated as a nontoxic medium supplement and RI-matching agent that improves image quality in live-imaging experiments involving primary cell cultures, planarians, zebrafish and human cerebral organoids (Boothe et al, 2017).…”

Section: Outlook and Future Directionsmentioning

confidence: 99%

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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A tunable refractive index matching medium for live imaging cells, tissues and model organisms

Cited by 152 publications

References 24 publications

Transcription organizes euchromatin similar to an active microemulsion

Transcription organizes euchromatin similar to an active microemulsion

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

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

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