Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions

Knoepp, Fenja; Wahl, Joël; Andersson, Anders G.; Borg, J.; Weißmann, Norbert; Ramser, Kerstin

doi:10.3390/s18103238

Cited by 3 publications

(7 citation statements)

References 22 publications

(31 reference statements)

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“…The Raman signal was filtered out from the laser light using a filter cube containing a dichroic mirror (Semrock, Rochester, NY, USA) and a 532-nm edge filter (Semrock). An in-house-designed gas-tight microfluidic system was used to register Raman spectra on the cultured PASMC at different oxidation states, i.e., normoxic and hypoxic conditions (48). The sample was illuminated with a laser output power of 3.1 mW with an integration time of 60 s. Acquired Raman data were processed in two steps.…”

Section: Lentivirus Production and Mouse Pasmc Transductionmentioning

confidence: 99%

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing

et al. 2020

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Mitochondria play an important role in sensing both acute and chronic hypoxia in the pulmonary vasculature, but their primary oxygen-sensing mechanism and contribution to stabilization of the hypoxia-inducible factor (HIF) remains elusive. Alteration of the mitochondrial electron flux and increased superoxide release from complex III has been proposed as an essential trigger for hypoxic pulmonary vasoconstriction (HPV). We used mice expressing a tunicate alternative oxidase, AOX, which maintains electron flux when respiratory complexes III and/or IV are inhibited. Respiratory restoration by AOX prevented acute HPV and hypoxic responses of pulmonary arterial smooth muscle cells (PASMC), acute hypoxia-induced redox changes of NADH and cytochrome c, and superoxide production. In contrast, AOX did not affect the development of chronic hypoxia-induced pulmonary hypertension and HIF-1α stabilization. These results indicate that distal inhibition of the mitochondrial electron transport chain in PASMC is an essential initial step for acute but not chronic oxygen sensing.

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Section: Lentivirus Production and Mouse Pasmc Transductionmentioning

confidence: 99%

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing

et al. 2020

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“…Raman spectroscopy can detect a range of biomarkers (e.g., DNA, lipids, and proteins) in a single spectrum without staining or destruction of the sample, [23,24] and is currently one of the most precise options to measure the cellular and mitochondrial redox state in living cells. In addition, Raman spectroscopy can be combined with patchclamp electrophysiology, [25,26] which enables high-resolution recordings of membrane potential and ionic currents-even on single-channel level-in excised membrane patches, individual cells, or tissue sections. [27] Due to its unrivaled signal-to-noise ratio, patch-clamp electrophysiology is regarded as the gold standard for the measurement of cellular electrophysiological responses to hypoxia.…”

Section: Introductionmentioning

confidence: 99%

“…More recently, we developed a sealed microfluidic system that allowed simultaneous Raman spectroscopy and the general possibility to perform patch-clamp electrophysiology via a mobile patch-clamp pipette. [26] We have now further improved our approach, constructing an innovative microfluidic system that combines Raman spectroscopy and patch-clamp electrophysiology-the current gold standards for the investigation of the cellular redox state and electrophysiological properties-with simultaneous live-cell imaging to determine the contraction of single living PASMCs that are acutely exposed to precisely defined levels of pO 2 . PASMCs are specialized oxygen sensing cells that contract in response to acute hypoxia which helps the pulmonary vasculature to divert blood from poorly oxygenated to well-oxygenated lung areas and thereby optimize arterial pO 2 .…”

Section: Introductionmentioning

confidence: 99%

“…[3,4,8,9] However, studying the electrophysiological response to acute hypoxia requires precise regulation of pO 2 and rapid switching between normoxia and hypoxia, while still allowing access for the patch pipette. Application of acute hypoxia in open patch-clamp chambers by continuous perfusion with solutions purged with hypoxic gas mixtures [8,26,28] often leads to reoxygenation due to dilution with ambient O 2 , rendering tight control over pO 2 impossible and delaying the application of acute hypoxia to the cells. [26] The need for precise regulation of pO 2 resulted in the development of a microfluidic system with a fixed patch-clamp pipette and optical tweezers to steer single suspended cells towards the pipette.…”

Section: Introductionmentioning

confidence: 99%

“…Application of acute hypoxia in open patch-clamp chambers by continuous perfusion with solutions purged with hypoxic gas mixtures [8,26,28] often leads to reoxygenation due to dilution with ambient O 2 , rendering tight control over pO 2 impossible and delaying the application of acute hypoxia to the cells. [26] The need for precise regulation of pO 2 resulted in the development of a microfluidic system with a fixed patch-clamp pipette and optical tweezers to steer single suspended cells towards the pipette. [29] However, no full patch-clamp protocol could be established and the measurement of adherent cells, such as pulmonary arterial smooth muscle cells (PASMCs), was inoperable.…”

Section: Introductionmentioning

confidence: 99%

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A Microfluidic System for Simultaneous Raman Spectroscopy, Patch‐Clamp Electrophysiology, and Live‐Cell Imaging to Study Key Cellular Events of Single Living Cells in Response to Acute Hypoxia

et al. 2021

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The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood and studies of acute oxygen sensing, in particular, have produced inconsistent results. Current methods cannot simultaneously measure the key cellular events in acute hypoxia (i.e., changes in redox state, electrophysiological properties, and mechanical responses) at controlled partial pressures of oxygen (pO2). The lack of such a comprehensive method essentially contributes to the discrepancies in the field. A sealed microfluidic system that combines i) Raman spectroscopy, ii) patch‐clamp electrophysiology, and iii) live‐cell imaging under precisely controlled pO2 have therefore been developed. Merging these modalities allows label‐free and simultaneous observation of oxygen‐dependent alterations in multiple cellular redox couples, membrane potential, and cellular contraction. This technique is adaptable to any cell type and allows in‐depth insight into acute oxygen sensing processes underlying various physiologic and pathologic conditions.

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Microbial single-cell applications under anoxic conditions

Keating,

Fiege,

Diender

et al. 2024

Appl Environ Microbiol

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The field of microbiology traditionally focuses on studying microorganisms at the population level. Nevertheless, the application of single-cell level methods, including microfluidics and imaging techniques, has revealed heterogeneity within populations, making these methods essential to understand cellular activities and interactions at a higher resolution. Moreover, single-cell sorting has opened new avenues for isolating cells of interest from microbial populations or complex microbial communities. These isolated cells can be further interrogated in downstream single-cell “omics” analyses, providing physiological and functional information. However, applying these methods to study anaerobic microorganisms under in situ conditions remains challenging due to their sensitivity to oxygen. Here, we review the existing methodologies for the analysis of viable anaerobic microorganisms at the single-cell level, including live-imaging, cell sorting, and microfluidics (lab-on-chip) applications, and we address the challenges involved in their anoxic operation. Additionally, we discuss the development of non-destructive imaging techniques tailored for anaerobes, such as oxygen-independent fluorescent probes and alternative approaches.

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Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions

Cited by 3 publications

References 22 publications

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing

A Microfluidic System for Simultaneous Raman Spectroscopy, Patch‐Clamp Electrophysiology, and Live‐Cell Imaging to Study Key Cellular Events of Single Living Cells in Response to Acute Hypoxia

Microbial single-cell applications under anoxic conditions

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