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High-resolution label-free 3D mapping of extracellular pH of single living cells.

ABSTRACT: Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H⁺ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity?>?0.01 units, 2?ms response time, and 50?nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC6898398 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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High-resolution label-free 3D mapping of extracellular pH of single living cells.

Zhang Yanjun Y Takahashi Yasufumi Y Hong Sung Pil SP Liu Fengjie F Bednarska Joanna J Goff Philip S PS Novak Pavel P Shevchuk Andrew A Gopal Sahana S Barozzi Iros I Magnani Luca L Sakai Hideki H Suguru Yoshimoto Y Fujii Takuto T Erofeev Alexander A Gorelkin Peter P Majouga Alexander A Weiss Dominik J DJ Edwards Christopher C Ivanov Aleksandar P AP Klenerman David D Sviderskaya Elena V EV Edel Joshua B JB Korchev Yuri Y

Nature communications 20191206 1

Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H<sup>+</sup> in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensi ...[more]

PMID: 31811139

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Project description:BackgroundChronic kidney disease (CKD) is a global burden affecting both children and adults. Novel imaging modalities hold great promise to visualize and quantify structural, functional, and molecular organ damage. The aim of the study was to visualize and quantify murine renal vasculature using label-free raster scanning optoacoustic mesoscopy (RSOM) in explanted organs from mice with renal injury.Material and methodsFor the experiments, freshly bisected kidneys of alpha 8 integrin knock-out (KO) and wildtype mice (WT) were used. A total of n=7 female (n=4 KO, n=3 WT) and n=6 male animals (n=2 KO, n=4 WT) aged 6 weeks were examined with RSOM optoacoustic imaging systems (RSOM Explorer P50 at SWL 532nm and/or ms-P50 imaging system at 532 nm, 555 nm, 579 nm, and 606 nm). Images were reconstructed using a dedicated software, analyzed for size and vascular area and compared to standard histologic sections.ResultsRSOM enabled mapping of murine kidney size and vascular area, revealing differences between kidney sizes of male (m) and female (f) mice (merged frequencies (MF) f vs. m: 52.42±6.24 mm2 vs. 69.18±15.96 mm2, p=0.0156) and absolute vascular area (MF f vs. m: 35.67±4.22 mm2 vs. 49.07±13.48 mm2, p=0.0036). Without respect to sex, the absolute kidney area was found to be smaller in knock-out (KO) than in wildtype (WT) mice (WT vs. KO: MF: p=0.0255) and showed a similar trend for the relative vessel area (WT vs. KO: MF p=0.0031). Also the absolute vessel areas of KO compared to WT were found significantly different (MF p=0.0089). A significant decrease in absolute vessel area was found in KO compared to WT male mice (MF WT vs. KO: 54.37±9.35 mm2 vs. 34.93±13.82 mm2, p=0.0232). In addition, multispectral RSOM allowed visualization of oxygenated and deoxygenated parenchymal regions by spectral unmixing.ConclusionThis study demonstrates the capability of RSOM for label-free visualization of differences in vascular morphology in ex vivo murine renal tissue at high resolution. Due to its scalability optoacoustic imaging provides an emerging modality with potential for further preclinical and clinical imaging applications.

Project description:BACKGROUND:Cardiovascular magnetic resonance (CMR) T1? mapping can be used to detect ischemic or non-ischemic cardiomyopathy without the need of exogenous contrast agents. Current 2D myocardial T1? mapping requires multiple breath-holds and provides limited coverage. Respiratory gating by diaphragmatic navigation has recently been exploited to enable free-breathing 3D T1? mapping, which, however, has low acquisition efficiency and may result in unpredictable and long scan times. This study aims to develop a fast respiratory motion-compensated 3D whole-heart myocardial T1? mapping technique with high spatial resolution and predictable scan time. METHODS:The proposed electrocardiogram (ECG)-triggered T1? mapping sequence is performed under free-breathing using an undersampled variable-density 3D Cartesian sampling with spiral-like order. Preparation pulses with different T1? spin-lock times are employed to acquire multiple T1?-weighted images. A saturation prepulse is played at the start of each heartbeat to reset the magnetization before T1? preparation. Image navigators are employed to enable beat-to-beat 2D translational respiratory motion correction of the heart for each T1?-weighted dataset, after which, 3D translational registration is performed to align all T1?-weighted volumes. Undersampled reconstruction is performed using a multi-contrast 3D patch-based low-rank algorithm. The accuracy of the proposed technique was tested in phantoms and in vivo in 11 healthy subjects in comparison with 2D T1? mapping. The feasibility of the proposed technique was further investigated in 3 patients with suspected cardiovascular disease. Breath-hold late-gadolinium enhanced (LGE) images were acquired in patients as reference for scar detection. RESULTS:Phantoms results revealed that the proposed technique provided accurate T1? values over a wide range of simulated heart rates in comparison to a 2D T1? mapping reference. Homogeneous 3D T1? maps were obtained for healthy subjects, with septal T1? of 58.0 ± 4.1?ms which was comparable to 2D breath-hold measurements (57.6 ± 4.7?ms, P?=?0.83). Myocardial scar was detected in 1 of the 3 patients, and increased T1? values (87.4 ± 5.7?ms) were observed in the infarcted region. CONCLUSIONS:An accelerated free-breathing 3D whole-heart T1? mapping technique was developed with high respiratory scan efficiency and near-isotropic spatial resolution (1.7 × 1.7 × 2?mm3) in a clinically feasible scan time of ~?6 mins. Preliminary patient results suggest that the proposed technique may find applications in non-contrast myocardial tissue characterization.

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High-resolution label-free 3D mapping of extracellular pH of single living cells.

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High-resolution label-free 3D mapping of extracellular pH of single living cells.

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