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Background: Intracellular acidosis in the ischemic penumbra can contribute to further cell death, effectively enlarging the infarct core. Restoring the acid-base balance may enhance tissue survivability after cerebral ischemia. Objective: This study investigated whether translocating protons out of penumbral neurons could mitigate tissue acidification and induce neuroprotection in a rodent model of acute cerebral ischemia. Methods: We modulated the penumbral neurons via a light-driven pump to translocate protons out (i.e., archaerhodopsin/ArchT group) or into (i.e., channelrhodopsin-2/ChR2 group) neurons after focal cerebral ischemia in rats. Intracellular pH values were imaged via neutral red (NR) fluorescence and cerebral blood flow (CBF) was monitored through laser speckle contrast imaging (LSCI). Global CBF responses to electrical stimulation of the hindlimbs were obtained 24 h and 48 h after ischemia to assess neurological function. Behavioral and histological outcomes were evaluated 48 h after ischemia. A control group without gene modification was included. Results: The reduction of relative pH (RpH), the amplitude of negative peak of hypoemic response (RNP) and the hemispheric lateralization index (LI) in ArchT group were significantly less than those of the ChR2 or control group. Moreover, RpH was strongly correlated with RNP (r = 0.60) and LI (r24h = 0.80, r48h = 0.59). In addition, behavioral and histological results supported a neuroprotective effect of countering neuronal acidosis in penumbra through optogenetic stimulation. Conclusion(s): These results indicate that countering intracellular acidosis by optogenetically translocating protons out of penumbral neurons during the acute ischemic stage could induce protection after ischemic brain injury.

Intracellular pH is a key physiological factor for controlling the activities and functions of cells and lysosome is a vital subcellular organelle. Thus, developing a novel lysosome-targeting fluorescence probe for selective and sensitive detection of lysosomal pH in living cells is very important. In this work, we synthesized a series of fluorescence probes based on imidazole-fused benzothiadiazole. The optical properties of these probes were easily adjusted by modifying the substituents with different electron-withdrawing/donating ability in imidazole moiety. All of them showed acid-response and decreased fluorescence intensity during pH values changing from 4.0 to 8.5. The introduction of morpholine group allowed them to specifically respond to the changes of lysosomal pH. Among them, probe MIBTAA possessed a suitable pK a value (5.3) and showed good linear response to pH (R 2  = 0.9918) with red emission when pH changed from 4.4 to 5.6. The probe was successfully applied for monitoring pH variation in living cells induced by proton-pump inhibitor Baf-A1 and chloroquine, indicating its great potential for pH imaging in biological applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Intracellular acidosis in the ischemic penumbra can contribute to further cell death, effectively enlarging the infarct core. Restoring the acid-base balance may enhance tissue survivability after cerebral ischemia., Objective: This study investigated whether translocating protons out of penumbral neurons could mitigate tissue acidification and induce neuroprotection in a rodent model of acute cerebral ischemia., Methods: We modulated the penumbral neurons via a light-driven pump to translocate protons out (i.e., archaerhodopsin/ArchT group) or into (i.e., channelrhodopsin-2/ChR2 group) neurons after focal cerebral ischemia in rats. Intracellular pH values were imaged via neutral red (NR) fluorescence and cerebral blood flow (CBF) was monitored through laser speckle contrast imaging (LSCI). Global CBF responses to electrical stimulation of the hindlimbs were obtained 24 h and 48 h after ischemia to assess neurological function. Behavioral and histological outcomes were evaluated 48 h after ischemia. A control group without gene modification was included., Results: The reduction of relative pH (R pH ), the amplitude of negative peak of hypoemic response (R NP ) and the hemispheric lateralization index (LI) in ArchT group were significantly less than those of the ChR2 or control group. Moreover, R pH was strongly correlated with R NP (r = 0.60) and LI (r 24h  = 0.80, r 48h  = 0.59). In addition, behavioral and histological results supported a neuroprotective effect of countering neuronal acidosis in penumbra through optogenetic stimulation., Conclusion(s): These results indicate that countering intracellular acidosis by optogenetically translocating protons out of penumbral neurons during the acute ischemic stage could induce protection after ischemic brain injury., Competing Interests: Declaration of competing interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intracellular pH, especially cytoplasmic pH (~7.2) plays a crucial role in cell functions and metabolism. A ratiometric fluorescent probe namely, 6-(2-(benzothiazol-2-yl)vinyl)naphthalen-2-ol (BTNO) was facilely synthesized by the condensation of 6-hydroxy-2-naphthaldehyde and 2-methylbenzothiazole. BTNO exhibited a remarkable ratiometric emission (F 456 /F 526 ) enhancement in response to a pH change with a linear range of pH = 9.50-7.00 and a pKa value of 7.91 ± 0.03, which is desirable for measuring and monitoring the cytoplasmic pH fluctuations. In addition, because of the high fluorescence quantum yield of BTNO (Φ = 0.88 in DMSO and 0.61 in water relative to quinine sulfate solution in 0.1 M H 2 SO 4 ), the interferences of the probe on the physiological functions could be greatly reduced. This could also provide enhanced measurement sensitivity. The successful demonstration of BTNO in detecting and monitoring the intracellular pH changes in live HeLa cells via a ratiometric approach confirmed that BTNO held a practical potential in biomedical research., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Genetically-Encoded Voltage Indicators (GEVIs) are capable of converting changes in membrane potential into an optical signal. Here, we focus on recent insights into the mechanism of ArcLight-type probes and the consequences of utilizing a pH-dependent Fluorescent Protein (FP). A negative charge on the exterior of the β-can of the FP combined with a pH-sensitive FP enables voltage-dependent conformational changes to affect the fluorescence of the probe. This hypothesis implies that interaction/dimerization of the FP creates a microenvironment for the probe that is altered via conformational changes. This mechanism explains why a pH sensitive FP with a negative charge on the outside of the β-can is needed, but also suggests that pH could affect the optical signal as well. To better understand the effects of pH on the voltage-dependent signal of ArcLight, the intracellular pH (pHi) was tested at pH 6.8, 7.2, or 7.8. The resting fluorescence of ArcLight gets brighter as the pHi increases, yet only pH 7.8 significantly affected the ΔF/F. ArcLight could also simultaneously report voltage and pH changes during the acidification of a neuron firing multiple action potentials revealing different buffering capacities of the soma versus the processes of the cell., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Cerebral acidosis is a consequence of secondary injury mechanisms following traumatic brain injury (TBI), including excitotoxicity and ischemia, with potentially significant clinical implications. However, there remains an unmet clinical need for technology for non-invasive, high resolution pH imaging of human TBI for studying metabolic changes following injury. The current study examined 17 patients with TBI and 20 healthy controls using amine chemical exchange saturation transfer echoplanar imaging (CEST EPI), a novel pH-weighted molecular MR imaging technique, on a clinical 3T MR scanner. Results showed significantly elevated pH-weighted image contrast (MTR asym at 3 ppm) in areas of T2 hyperintensity or edema (P < 0.0001), and a strong negative correlation with Glasgow Coma Scale (GCS) at the time of the MRI exam (R 2  = 0.4777, P = 0.0021), Glasgow Outcome Scale - Extended (GOSE) at 6 months from injury (R 2  = 0.5334, P = 0.0107), and a non-linear correlation with the time from injury to MRI exam (R 2  = 0.6317, P = 0.0004). This evidence suggests clinical feasibility and potential value of pH-weighted amine CEST EPI as a high-resolution imaging tool for identifying tissue most at risk for long-term damage due to cerebral acidosis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Current imaging techniques, such as position emission tomography, magnetic resonance imaging and optical imaging, have been developed and applied to imaging acidity in live tissues for years, but they still have some limitations. In the present study, a reliable and interference-free pH imaging method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was established for the first time. A novel reactive matrix, named glycosyl-3-aminoquinoline (Gly-3AQ), was designed and synthesized here not only to overcome the background interference in the low mass range of MALDI but also provide a new function of acid-responsiveness for the matrix. This matrix was responsive to pH change due to its acid-catalyzed hydrolysis and the hydrolysis product 3-aminoquinoline (3-AQ) has an increase of peak intensity with pH decrease. The Gly-3AQ-based MALDI pH detection method is demonstrated to have good acid-responsiveness in the range of pH 2.0-7.0, and when pH changes 0.5, relative abundance of 3-AQ changes about 1.5 folds. The method showed a very prominent advantage, that is, free-interference when analyzing real samples. Validation for the method revealed pH calibration curve had good linearity (R > 0.9953) with relative standard deviation less than 13.8%. This method was applied to analyze and visualize acidity variation of citrus during development and the changed acidity underwent relative quantitation and statistical analysis. The newly developed reactive matrix has potential application to in-situ MSI of acidity in the biomedical field., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Intracellular pH underlies most cellular processes. There is emerging evidence of a pH-signaling role in plant cells and microorganisms. Dysregulation of pH is associated with human diseases, such as cancer and Alzheimer's disease., Scope of Review: In this review, we attempt to provide a summary of the progress that has been made in the field during the past two decades. First, we present an overview of the current state of the design and applications of fluorescent protein (FP)-based pH indicators. Then, we turn our attention to the development and applications of hybrid pH sensors that combine the capabilities of non-GFP fluorophores with the advantages of genetically encoded tags. Finally, we discuss recent advances in multicolor pH imaging and the applications of genetically encoded pH sensors in multiparameter imaging., Major Conclusions: Genetically encoded pH sensors have proven to be indispensable noninvasive tools for selective targeting to different cellular locations. Although a variety of genetically encoded pH sensors have been designed and applied at the single cell level, there is still much room for improvements and future developments of novel powerful tools for pH imaging. Among the most pressing challenges in this area is the design of brighter redshifted sensors for tissue research and whole animal experiments., General Significance: The design of precise pH measuring instruments is one of the important goals in cell biochemistry and may give rise to the development of new powerful diagnostic tools for various diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)