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Fully Automated Computational Approach for Precisely Measuring Organelle Acidification with Optical pH Sensors.

ABSTRACT: pH balance and regulation within organelles are fundamental to cell homeostasis and proliferation. The ability to track pH in cells becomes significantly important to understand these processes in detail. Fluorescent sensors based on micro- and nanoparticles have been applied to measure intracellular pH; however, an accurate methodology to precisely monitor acidification kinetics of organelles in living cells has not been established, limiting the scope of this class of sensors. Here, silica-based fluorescent microparticles were utilized to probe the pH of intracellular organelles in MDA-MB-231 and MCF-7 breast cancer cells. In addition to the robust, ratiometric, trackable, and bioinert pH sensors, we developed a novel dimensionality reduction algorithm to automatically track and screen massive internalization events of pH sensors. We found that the mean acidification time is comparable among the two cell lines (ΔT_MCF-7 = 16.3 min; ΔT_MDA-MB-231 = 19.5 min); however, MCF-7 cells showed a much broader heterogeneity in comparison to MDA-MB-231 cells. The use of pH sensors and ratiometric imaging of living cells in combination with a novel computational approach allow analysis of thousands of events in a computationally inexpensive and faster way than the standard routes. The reported methodology can potentially be used to monitor pH as well as several other parameters associated with endocytosis.

SUBMITTER: Chandra A

PROVIDER: S-EPMC9052195 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Fully Automated Computational Approach for Precisely Measuring Organelle Acidification with Optical pH Sensors.

Chandra Anil A Chandra Anil A Prasad Saumya S Alemanno Francesco F De Luca Maria M Rizzo Riccardo R Romano Roberta R Gigli Giuseppe G Bucci Cecilia C Barra Adriano A Del Mercato Loretta L LL

ACS applied materials & interfaces 20220411 16

pH balance and regulation within organelles are fundamental to cell homeostasis and proliferation. The ability to track pH in cells becomes significantly important to understand these processes in detail. Fluorescent sensors based on micro- and nanoparticles have been applied to measure intracellular pH; however, an accurate methodology to precisely monitor acidification kinetics of organelles in living cells has not been established, limiting the scope of this class of sensors. Here, silica-bas ...[more]

PMID: 35404562

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Project description:Photosynthetic algae and cyanobacteria have been proposed for producing biofuels through a direct photoconversion process. To accelerate the efforts of discovering and screening microbes for biofuel production, sensitive and high throughput methods to measure photosynthetic activity need to be developed. Here we report the development of new ratiometric optical oxygen and pH dual sensors with three emission colors for measuring photosynthetic activities directly. The dual sensor system can measure oxygen (O(2)) generation and pH increase resulted from carbon dioxide (CO(2)) consumption simultaneously. The sensor was prepared by a copolymerization of three monomeric probes, an intra-reference probe (IRP) which does not respond to pH or O(2), a probe for pH sensing (pHS), and an O(2) probe for O(2) sensing (OS) with 2-hydroxyethyl methacrylate (HEMA) and acrylamide (AM). After polymerization, the three probes were chemically immobilized in an ion and O(2) permeable poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrix. The resulted sensing films (membranes) exhibited three emission colors with well separated emission spectra, covering blue, green, and red emission windows, under 380 nm light excitation. Responses of the sensors to pH and dissolved O(2) were investigated in buffers and cyanobacterial cell cultures (Synechocystis sp. PCC 6803). In spite of the strong autofluorescence from cyanobacteria, the sensors were able to determine the pH values and dissolved O(2) concentrations accurately and reproducibly. The measured results using the optical sensors were well in accordance with measurements using electrodes with minimal experimental variations. The sensors were further applied for evaluation of photosynthetic activities of Synechocystis sp. PCC 6803 at the exponential and stationary phases. The results were consistent with biological observation that the photosynthetic activity in the exponential phase was higher than that in the stationary phase.

Dataset Information

Fully Automated Computational Approach for Precisely Measuring Organelle Acidification with Optical pH Sensors.

Publications

Fully Automated Computational Approach for Precisely Measuring Organelle Acidification with Optical pH Sensors.

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