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A Novel Quantum Dot-Based pH Probe for Long-Term Fluorescence Lifetime Imaging Microscopy Experiments in Living Cells.

ABSTRACT: The use of two nanoparticles for quantitative pH measurements in live cells by means of fluorescence lifetime imaging microscopy (FLIM) is investigated here. These nanoparticles are based on CdSe/ZnS quantum dots (QDs), functionalized with N-acetylcysteine (CdSe/ZnS-A) and with a small peptide containing D-penicillamine and histidine (CdSe/ZnS-PH). CdSe/ZnS-A has tendency to aggregate and nonlinear pH sensitivity in a complex medium containing salts and macromolecules. On the contrary, CdSe/ZnS-PH shows chemical stability, low toxicity, efficient uptake in C3H10T1/2 cells, and good performance as an FLIM probe. CdSe/ZnS-PH also has key advantages over a recently reported probe based on a CdSe/ZnS QD functionalized with D-penicillamine (longer lifetimes and higher pH-sensitivity). A pH(±2σ) of 6.97 ± 0.14 was determined for C3H10T1/2 cells by FLIM employing this nanoprobe. In addition, the fluorescence lifetime signal remains nearly constant for C3H10T1/2 cells treated with CdSe/ZnS-PH for 24 h. These results show the promising applications of this nanoprobe to monitor the intracellular pH and cell state employing the FLIM technique.

SUBMITTER: Herrera-Ochoa D

PROVIDER: S-EPMC8778634 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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A Novel Quantum Dot-Based pH Probe for Long-Term Fluorescence Lifetime Imaging Microscopy Experiments in Living Cells.

Herrera-Ochoa Diego D Pacheco-Liñán Pedro J PJ Bravo Iván I Garzón-Ruiz Andrés A

ACS applied materials & interfaces 20220110 2

The use of two nanoparticles for quantitative pH measurements in live cells by means of fluorescence lifetime imaging microscopy (FLIM) is investigated here. These nanoparticles are based on CdSe/ZnS quantum dots (QDs), functionalized with N-acetylcysteine (CdSe/ZnS-A) and with a small peptide containing D-penicillamine and histidine (CdSe/ZnS-PH). CdSe/ZnS-A has tendency to aggregate and nonlinear pH sensitivity in a complex medium containing salts and macromolecules ...[more]

PMID: 35001616

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Project description:Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.

Dataset Information

A Novel Quantum Dot-Based pH Probe for Long-Term Fluorescence Lifetime Imaging Microscopy Experiments in Living Cells.

Publications

A Novel Quantum Dot-Based pH Probe for Long-Term Fluorescence Lifetime Imaging Microscopy Experiments in Living Cells.

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