Ultrarapid cryo-arrest of living cells on a microscope enables multiscale imaging of out-of-equilibrium molecular patterns

Huebinger, Jan; Grecco, Hernán E.; Masip, Martín E.; Christmann, Jens; Fuhr, G.; Bastiaens, Philippe I. H.

doi:10.1126/sciadv.abk0882

Cited by 10 publications

(13 citation statements)

References 63 publications

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“…The sample was cryofixed by rapid freezing using liquid propane at 88 K (in an amount of 40-60 ml) to avoid destruction of cell structures by large ice crystal formation [26],[27],[28]. Immediately after applying the cryogen to the sample, circulation of liquid nitrogen in the cooling block was started to stabilize the temperature of the cooling block for Raman measurement.…”

Section: Resultsmentioning

confidence: 99%

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

Mizushima,

Kumamoto,

Tamura

et al. 2023

Preprint

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Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise-ratio (SNR) in measurements of biological specimens is severely limited due to the small cross-section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and significantly enhanced spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Finally, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.

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Section: Resultsmentioning

confidence: 99%

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

Mizushima,

Kumamoto,

Tamura

et al. 2023

Preprint

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“…To investigate whether this change in EGFR-RPTPγ interaction dynamics is reflected in the nanoscale organization of EGFR (Ibach et al , 2015; Masip et al , 2016) in relation to RPTPγ, we assessed the colocalization of Alexa647-SNAP-EGFR and RPTPγ-mCitrine after receptor-saturating EGF stimulation by super resolution radial fluctuation microscopy after ultra-rapid cryo-arrest on a fluorescence microscope (Huebinger et al , 2021). In serum-starved, quiescent cells, a co-organization of EGFR nanoclusters within larger RPTPγ patches along the PM could be observed.…”

Section: Resultsmentioning

confidence: 99%

“…Ultra-rapid cryo-arrest of a stable MCF7 cell line expressing SNAP-EGFR and RPTPγ-mCitrine was done as recently described (Huebinger et al , 2021). For this, the cells were grown on fibronectin-coated (5 μg/cm 2 , F0895, Sigma-Aldrich) circular microscopy cover slides (No.1; Ø 4 mm) that were mounted to chambers from biocompatible silicone (4-well micro-Inserts; ibidi GmbH, Gräfelfing, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Before the experiment, Snap-EGFR was labeled with 0.5 μM Snap-Surface Alexa647 (New England Biolabs GmbH, Frankfurt, Germany) for at least 60’. The cover slides were mounted to the ultra-rapid cryo-arrest device (Huebinger et al , 2021), which was placed on top of an epifluorescence microscope. The cells were cryo-arrested during observation on the microscope.…”

Section: Methodsmentioning

confidence: 99%

“…Super-Resolution radial fluctuation (SRRF) under ultrarapid cryo-arrest Ultra-rapid cryo-arrest of a stable MCF7 cell line expressing SNAP-EGFR and RPTPg-mCitrine was done as recently described (Huebinger et al, 2021). For this, the cells were grown on fibronectin-coated…”

Section: Fluorescence Anisotropy Microscopymentioning

confidence: 99%

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RPTPγ is a redox-regulated suppressor of promigratory EGFR signaling

Joshi

Stanoev

Soetje

et al. 2022

Preprint

Self Cite

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Spatially-organized interaction dynamics between proto-oncogenic epidermal growth factor receptor (EGFR) and protein tyrosine phosphatases (PTPs) determine EGFR′s phosphorylation response to growth factors and thereby cellular behavior within developing tissues. We show here, that and how the coupling between EGFR and RPTPγ activity leads to migratory signaling responses to very low, physiological growth factor stimuli while suppressing aberrant, spontaneous signaling. Single cell imaging of EGFR phosphorylation and PTP oxidation revealed that RPTPγ fully suppresses spontaneous EGFR phosphorylation, while EGF-induced NADPH-oxidase activity enables promigratory signaling responses at the plasma membrane by H2O2-mediated oxidative inhibition of RPTPγ′s phosphatase activity. The EGF-dependent toggle switch dynamics between interacting EGFR monomers and RPTPγ thereby enables autocatalytically amplified phosphorylation responses to very low, physiological, EGF levels even at sparse receptor expression. This signaling mechanism is distinct from the proliferative signaling stemming from liganded endosomal EGFR complexes at high growth factor concentrations. Accordingly, RPTPγ knock-out results in spontaneous promigratory EGFR signaling but loss of proliferative signaling. We thereby provide evidence of RPTPγ′s suppressor function of oncogenic, promigratory EGFR-signaling from the plasma membrane.

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Time Resolved Cryo‐Correlative Light and Electron Microscopy

Szabo,

Burg

2024

Adv Funct Materials

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Complex materials exhibit fascinating features especially in situations far from equilibrium. Thus, methods for investigating structural dynamics with sub‐second time resolution are becoming a question of interest at varying spatial scales. With novel microscopy techniques steadily improving, the temporal and spatial limits of multiple imaging methods are investigated with an emphasis on the important role of correlative imaging and cryo‐fixation. A deep‐dive is taken into cryo‐correlative light and electron microscopy (CLEM) as a starting point for multimodal investigations of ultrastructural dynamics at high spatiotemporal resolution. The focus is on highlighting the different microscopy methods that capture the following key aspects: 1) samples are as close to native state as possible 2) dynamic process information is captured, 3) high structural resolution is enabled. Additionally, the size of samples that can be imaged under these conditions is looked at and approaches not only focusing on single molecules, but larger structures are highlighted.

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Ultrarapid cryo-arrest of living cells on a microscope enables multiscale imaging of out-of-equilibrium molecular patterns

Abstract: Fluorescence imaging under cryo-arrest allows resolution of dynamic signaling system features that are otherwise obscured.

Cited by 10 publications

References 63 publications

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

RPTPγ is a redox-regulated suppressor of promigratory EGFR signaling

Time Resolved Cryo‐Correlative Light and Electron Microscopy

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