Photofunctional Surfaces for Quantitative Fluorescence Microscopy: Monitoring the Effects of Photogenerated Reactive Oxygen Species at Single Cell Level with Spatiotemporal Resolution

2015 ◽  
Vol 7 (10) ◽  
pp. 5944-5949 ◽  
Author(s):  
Linda Stegemann ◽  
Klaus C. Schuermann ◽  
Cristian A. Strassert ◽  
Hernán E. Grecco
Keyword(s):  
Reactive Oxygen Species ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Reactive Oxygen ◽  
Single Cell Level ◽  
Oxygen Species ◽  
Cell Level ◽  
Spatiotemporal Resolution ◽  
Quantitative Fluorescence

scholarly journals Cathelicidin Peptides Restrict Bacterial Growth via Membrane Perturbation and Induction of Reactive Oxygen Species

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Dean A. Rowe-Magnus ◽  
Adenine Y. Kao ◽  
Antonio Cembellin Prieto ◽  
Meng Pu ◽  
Cheng Kao
Keyword(s):  
Reactive Oxygen Species ◽  
Antimicrobial Peptides ◽  
Single Cell ◽  
Reactive Oxygen ◽  
Cellular Damage ◽  
Primary Effect ◽  
Gram Negative Bacteria ◽  
Oxygen Species ◽  
Gram Positive ◽  
Gram Negative

ABSTRACT All metazoans produce antimicrobial peptides (AMPs) that have both broad antimicrobial and immunomodulatory activity. Cathelicidins are AMPs that preferentially kill Gram-negative bacteria in vitro, purportedly by assembling into higher-order structures that perforate the membrane. We utilized high-resolution, single-cell fluorescence microscopy to examine their mechanism of action in real time. Engineered cathelicidins rapidly bound to Gram-negative and Gram-positive cells and penetrated the cytoplasmic membrane. Rapid failure of the peptidoglycan superstructure in regions of active turnover caused leakage of cytoplasmic contents and the formation of membrane-bound blebs. A mutation anticipated to destabilize interactions between cathelicidin subunits had no effect on bactericidal activity, suggesting that cathelicidins have activities beyond perforating the membrane. Nanomolar concentrations of cathelicidins, although not bactericidal, reduced the growth rate of Gram-negative and Gram-positive bacteria. The cells exhibited expression changes in multiple essential processes, including protein synthesis, peptidoglycan biosynthesis, respiration, and the detoxification of reactive oxygen species (ROS). Time-lapse imaging revealed that ROS accumulation preceded bleb formation, and treatments that reduced cellular ROS levels overcame these bactericidal effects. We propose that that the primary effect of cathelicidins is to induce the production of ROS that damage bacterial molecules, leading to slowed growth or cell death. Given their low circulating levels in vivo, AMPs may serve to slow bacterial population expansion so that cellular immunity systems can respond to and battle the infection. IMPORTANCE Antimicrobial peptides (AMPs) are an important part of the mammalian innate immune system in the battle against microbial infection. How AMPs function to control bacteria is not clear, as nearly all activity studies use nonphysiological levels of AMPs. We monitored peptide action in live bacterial cells over short time frames with single-cell resolution and found that the primary effect of cathelicidin peptides is to increase the production of oxidative molecules that cause cellular damage in Gram-positive and Gram-negative bacteria.

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

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yanjun Zhang ◽  
Yasufumi Takahashi ◽  
Sung Pil Hong ◽  
Fengjie Liu ◽  
Joanna Bednarska ◽  
...  
Keyword(s):  
Single Cell ◽  
Cancer Cells ◽  
Extracellular Ph ◽  
Single Cell Level ◽  
Ph Sensing ◽  
Label Free ◽  
Cell Level ◽  
Spatiotemporal Resolution ◽  
Ion Conductance ◽  
Scanning Ion Conductance Microscopy

AbstractDynamic 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.

scholarly journals Detection of PLGA-based nanoparticles at a single-cell level by synchrotron radiation FTIR spectromicroscopy and correlation with X-ray fluorescence microscopy

2014 ◽  
pp. 2791 ◽  
Author(s):  
Giovanni Maria Severini ◽  
Lorella Pascolo ◽  
Barbara Bortot ◽  
Nuria Benseny-Cases ◽  
Alessandra Gianoncelli ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
X Ray

Measuring bacterial adaptation dynamics at the single-cell level using a microfluidic chemostat and time-lapse fluorescence microscopy

The Analyst ◽  
2014 ◽  
Vol 139 (20) ◽  
pp. 5254-5262 ◽  
Author(s):  
Zhicheng Long ◽  
Anne Olliver ◽  
Elisa Brambilla ◽  
Bianca Sclavi ◽  
Marco Cosentino Lagomarsino ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Cell ◽  
Time Lapse ◽  
Single Cell Level ◽  
Cell Level ◽  
Gfp Expression ◽  
Bacterial Adaptation ◽  
E Coli ◽  
Cell Dimensions

We grewE. coliin a microfluidic chemostat and monitored the dynamics of cell dimensions and reporter GFP expression in individual cells during nutritional upshift or downshift.

Singlet oxygen and ROS in a new light: low-dose subcellular photodynamic treatment enhances proliferation at the single cell level

2014 ◽  
Vol 13 (9) ◽  
pp. 1235-1240 ◽  
Author(s):  
Alfonso Blázquez-Castro ◽  
Thomas Breitenbach ◽  
Peter R. Ogilby
Keyword(s):  
Reactive Oxygen Species ◽  
Laser Beam ◽  
Hela Cells ◽  
Low Dose ◽  
Single Cell Level ◽  
Oxygen Species ◽  
Cell Level ◽  
Photodynamic Treatment ◽  
Two Photon ◽  
Photon Excitation

Two-photon excitation of a sensitizer with a focused laser beam was used to create a spatially-localized subcellular population of reactive oxygen species, ROS, stimulating proliferation in single HeLa cells.

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