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31P MRSI allows for the non‐invasive mapping of pH and magnesium ion content (Mg) in vivo, by translating the chemical shifts of inorganic phosphate and adenosine‐5′‐triphosphate (ATP) to pH and Mg via suitable calibration equations, such as the modified Henderson–Hasselbalch equation. However, the required constants in these calibration equations are typically only determined for physiological conditions, posing a particular challenge for their application to diseased tissue, where the biochemical conditions might change manyfold. In this article, we propose a multi‐parametric look‐up algorithm aiming at the condition‐independent determination of pH and Mg by employing multiple quantifiable 31P spectral properties simultaneously. To generate entries for an initial look‐up table, measurements from 114 model solutions prepared with varying chemical properties were made at 9.4 T. The number of look‐up table entries was increased by inter‐ and extrapolation using a multi‐dimensional function developed based on the Hill equation. The assignment of biochemical parameters, that is, pH and Mg, is realized using probability distributions incorporating specific measurement uncertainties on the quantified spectral parameters, allowing for an estimation of most plausible output values. As proof of concept, we applied a version of the look‐up algorithm employing only the chemical shifts of γ‐ and β‐ATP for the determination of pH and Mg to in vivo 3D 31P MRSI data acquired at 7 T from (i) the lower leg muscles of healthy volunteers and (ii) the brains of patients with glioblastoma. The resulting volumetric maps showed plausible values for pH and Mg, partly revealing differences from maps generated using the conventional calibration equations. [ABSTRACT FROM AUTHOR]

Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although few methods can accurately assess their effect in vivo. In this study, we investigated whether MRI-CEST (Magnetic Resonance Imaging—Chemical Exchange Saturation Transfer) tumor pH imaging can be a useful tool to evaluate in vivo the therapeutic efficacy of several Proton Pump Inhibitors (PPIs) in breast cancer. Cell viability and extracellular pH assays were carried out in breast cancer cells cultured at physiological pH (7.4) or acid-adapted (pH of 6.5 and 6.8) following the exposure to inhibitors of V-ATPase (Lansoprazole, Esomeprazole) or NHE1 (Amiloride, Cariporide) at several concentrations. Next, triple-negative breast cancer 4T1 tumor-bearing mice were treated with Lansoprazole or Amiloride and MRI-CEST tumor pH imaging was utilized to assess the in vivo efficacy. Only Lansoprazole induced, in addition to breast cancer cell toxicity, a significant inhibition of proton extrusion. A significant reduction in tumor volume, prolonged survival, and increase in extracellular tumor pH after 1 and 2 weeks were observed after Lansoprazole treatment, whereas no significant changes were detected upon Amiloride treatment. Our results suggested that MRI-CEST tumor pH imaging can monitor the therapeutic efficacy of PPIs in breast cancer murine models. [ABSTRACT FROM AUTHOR]

Amide proton transfer (APT) imaging, a variant of chemical exchange saturation transfer MRI, has shown promise in detecting ischemic tissue acidosis following impaired aerobic metabolism in animal models and in human stroke patients due to the sensitivity of the amide proton exchange rate to changes in pH within the physiological range. Recent studies have demonstrated the possibility of using APT‐MRI to detect acidosis of the ischemic penumbra, enabling the assessment of stroke severity and risk of progression, monitoring of treatment progress, and prognostication of clinical outcome. This paper reviews current APT imaging methods actively used in ischemic stroke research and explores the clinical aspects of ischemic stroke and future applications for these methods. [ABSTRACT FROM AUTHOR]

Amine‐weighted chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is particularly valuable as an amine‐ and pH‐sensitive imaging technique in brain tumors, targeting the intrinsically high concentration of amino acids with exchangeable amine protons and reduced extracellular pH in brain tumors. Amine‐weighted CEST MRI contrast is dependent on the glioma genotype, likely related to differences in degree of malignancy and metabolic behavior. Amine‐weighted CEST MRI may provide complementary value to anatomic imaging in conventional and exploratory therapies in brain tumors, including chemoradiation, antiangiogenic therapies, and immunotherapies. Continual improvement and clinical testing of amine‐weighted CEST MRI has the potential to greatly impact patients with brain tumors by understanding vulnerabilities in the tumor microenvironment that may be therapeutically exploited. [ABSTRACT FROM AUTHOR]

Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although few methods can accurately assess their effect in vivo. In this study, we investigated whether MRI-CEST (Magnetic Resonance Imaging—Chemical Exchange Saturation Transfer) tumor pH imaging can be a useful tool to evaluate in vivo the therapeutic efficacy of several Proton Pump Inhibitors (PPIs) in breast cancer. Cell viability and extracellular pH assays were carried out in breast cancer cells cultured at physiological pH (7.4) or acid-adapted (pH of 6.5 and 6.8) following the exposure to inhibitors of V-ATPase (Lansoprazole, Esomeprazole) or NHE1 (Amiloride, Cariporide) at several concentrations. Next, triple-negative breast cancer 4T1 tumor-bearing mice were treated with Lansoprazole or Amiloride and MRI-CEST tumor pH imaging was utilized to assess the in vivo efficacy. Only Lansoprazole induced, in addition to breast cancer cell toxicity, a significant inhibition of proton extrusion. A significant reduction in tumor volume, prolonged survival, and increase in extracellular tumor pH after 1 and 2 weeks were observed after Lansoprazole treatment, whereas no significant changes were detected upon Amiloride treatment. Our results suggested that MRI-CEST tumor pH imaging can monitor the therapeutic efficacy of PPIs in breast cancer murine models.

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 (MTRasym at 3 ppm) in areas of T2 hyperintensity or edema (P

Purpose: Chemical exchange saturation transfer (CEST) imaging measurement depends not only on the labile proton concentration and pH‐dependent exchange rate but also on experimental conditions, including the relaxation delay and radiofrequency (RF) saturation time. Our study aimed to extend a quasi–steady‐state (QUASS) solution to a modified multi‐slice CEST MRI sequence and test if it provides enhanced pH imaging after acute stroke. Methods: Our study derived the QUASS solution for a modified multislice CEST MRI sequence with an unevenly segmented RF saturation between image readout and signal averaging. Numerical simulation was performed to test if the generalized QUASS solution corrects the impact of insufficiently long relaxation delay, primary and secondary saturation times, and multi‐slice readout. In addition, multiparametric MRI scans were obtained after middle cerebral artery occlusion, including relaxation and CEST Z‐spectrum, to evaluate the performance of QUASS CEST MRI in a rodent acute stroke model. We also performed Lorentzian fitting to isolate multi‐pool CEST contributions. Results: The QUASS analysis enhanced pH‐weighted magnetization transfer asymmetry contrast over the routine apparent CEST measurements in both contralateral normal (−3.46% ± 0.62% (apparent) vs. −3.67% ± 0.66% (QUASS), P < 0.05) and ischemic tissue (−5.53% ± 0.68% (apparent) vs. −5.94% ± 0.73% (QUASS), P < 0.05). Lorentzian fitting also showed significant differences between routine and QUASS analysis of ischemia‐induced changes in magnetization transfer, amide, amine, guanidyl CEST, and nuclear Overhauser enhancement (−1.6 parts per million) effects. Conclusion: Our study demonstrated that generalized QUASS analysis enhanced pH MRI contrast and improved quantification of the underlying CEST contrast mechanism, promising for further in vivo applications. [ABSTRACT FROM AUTHOR]

Stainless steel plays an important role in industry due to its anti-corrosion characteristic. It is known, however, that local corrosion can damage stainless steel under certain conditions. In this study, we developed a novel measurement system to observe crevice corrosion, which is a local corrosion that occurs inside a narrow gap. In addition to pH imaging inside the crevice, another imaging technique using an infrared light was combined to simultaneously visualize surface roughening of the test piece. According to experimental results, the lowering of local pH propagated inside the crevice, and after that, the surface roughening started and expanded due to propagation of corrosion. The real-time measurement of the pH distribution and the surface roughness can be a powerful tool to investigate the crevice corrosion. [ABSTRACT FROM AUTHOR]

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.

31 P MRSI allows for the non-invasive mapping of pH and magnesium ion content (Mg) in vivo, by translating the chemical shifts of inorganic phosphate and adenosine-5'-triphosphate (ATP) to pH and Mg via suitable calibration equations, such as the modified Henderson-Hasselbalch equation. However, the required constants in these calibration equations are typically only determined for physiological conditions, posing a particular challenge for their application to diseased tissue, where the biochemical conditions might change manyfold. In this article, we propose a multi-parametric look-up algorithm aiming at the condition-independent determination of pH and Mg by employing multiple quantifiable 31 P spectral properties simultaneously. To generate entries for an initial look-up table, measurements from 114 model solutions prepared with varying chemical properties were made at 9.4 T. The number of look-up table entries was increased by inter- and extrapolation using a multi-dimensional function developed based on the Hill equation. The assignment of biochemical parameters, that is, pH and Mg, is realized using probability distributions incorporating specific measurement uncertainties on the quantified spectral parameters, allowing for an estimation of most plausible output values. As proof of concept, we applied a version of the look-up algorithm employing only the chemical shifts of γ- and β-ATP for the determination of pH and Mg to in vivo 3D 31 P MRSI data acquired at 7 T from (i) the lower leg muscles of healthy volunteers and (ii) the brains of patients with glioblastoma. The resulting volumetric maps showed plausible values for pH and Mg, partly revealing differences from maps generated using the conventional calibration equations., (© 2024 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Acute stroke imaging plays a vital and time-sensitive role in therapeutic decision-making. Current clinical workflows widely use computed tomography (CT) and magnetic resonance (MR) techniques including CT and MR perfusion to estimate the volume of ischemic penumbra at risk for infarction without acute intervention. The use of imaging techniques aimed toward evaluating the metabolic derangements underlying a developing infarct may provide additional information for differentiating the penumbra from benign oligemia and infarct core. The authors review several modalities of metabolic imaging including PET, hydrogen and oxygen spectroscopy, sodium MRI, and pH-weighted MRI., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Low pH is associated with intervertebral disc (IVD)-generated low back pain (LBP). The purpose of this work was to develop an in vivo pH level-dependent magnetic resonance imaging (MRI) method for detecting discogenic LBP, without using exogenous contrast agents. Methods: The ratio of R1ρ dispersion and chemical exchange saturation transfer (CEST) (RROC) was used for pH-level dependent imaging of the IVD while eliminating the effect of labile proton concentration. The technique was validated by numerical simulations and studies on phantoms and ex vivo porcine spines. Four male (ages 42.8 ± 18.3) and two female patients (ages 55.5 ± 2.1) with LBP and scheduled for discography were examined with the method on a 3.0 Tesla MR scanner. RROC measurements were compared with discography outcomes using paired t-test. Results: Simulation and phantom results indicated RROC is a concentration independent and pH level-dependent technique. Porcine spine study results found higher RROC value was related to lower pH level. Painful discs based on discography had significant higher RROC values than those with negative diagnosis (P < 0.05). Conclusion: RROC imaging is a promising pH level dependent MRI technique that has the potential to be a noninvasive imaging tool to detect painful IVDs in vivo. © 2014 Wiley Periodicals, Inc.

Purpose: Chemical exchange saturation transfer MRI provides new approaches for investigating tumor microenvironment, including tumor acidosis that plays a key role in tumor progression and resistance to therapy. Following iopamidol injection, the detection of the contrast agent inside the tumor tissue allows measurements of tumor extracellular pH. However, accurate tumor pH quantifications are hampered by the low contrast efficiency of the CEST technique and by the low SNR of the acquired CEST images, hence in a reduced detectability of the injected agent. This work aims to investigate a novel denoising method for improving both tumor pH quantification and accuracy of CEST‐MRI pH imaging. Methods: An hybrid denoising approach was investigated for CEST‐MRI pH imaging based on the combination of the nonlocal mean filter and the anisotropic diffusion tensor method. The denoising approach was tested in simulated and in vitro data and compared with previously reported methods for CEST imaging and with established denoising approaches. Finally, it was validated with in vivo data to improve the accuracy of tumor pH maps. Results: The proposed method outperforms current denoising methods in CEST contrast quantification and detection of the administered contrast agent at several increasing noise levels with simulated data. In addition, it achieved a better pH quantification in in vitro data and demonstrated a marked improvement in contrast detection and a substantial improvement in tumor pH accuracy in in vivo data. Conclusion: The proposed approach effectively reduces the noise in CEST images and increases the sensitivity detection in CEST‐MRI pH imaging. [ABSTRACT FROM AUTHOR]

Chemical exchange saturation transfer (CEST) is a novel molecular magnetic resonance imaging (MRI) method that makes full use of the chemical exchange between water protons and exchangeable protons from solute molecules. Through chemical exchanges, the MRI signal of bulk water decreases when saturating at specific frequency of solute protons. Important biological information related to the chemical exchange processes could be extracted from the amplitude of water signal decrease. For example, the CEST signals have been used for in vivo pH imaging since the proton exchange rate is often pH-dependent. CEST signals originated from endogenous proteins/peptides and exogenous small molecule/metal chelate probes have been used for in vivo pH imaging. Using the ratiometric methods or the amine and amide concentration-independent detection (AACID) methods, in vivo pH maps have been acquired from kidneys, ischemic brain and tumors. In this paper, we thoroughly reviewed the progresses during the past twenty years in the field of in vivo pH imaging with CEST contrast. Principles, methods and applications were discussed, as well as development trends and future directions.

Stainless steel plays an important role in industry due to its anti-corrosion characteristic. It is known, however, that local corrosion can damage stainless steel under certain conditions. In this study, we developed a novel measurement system to observe crevice corrosion, which is a local corrosion that occurs inside a narrow gap. In addition to pH imaging inside the crevice, another imaging technique using an infrared light was combined to simultaneously visualize surface roughening of the test piece. According to experimental results, the lowering of local pH propagated inside the crevice, and after that, the surface roughening started and expanded due to propagation of corrosion. The real-time measurement of the pH distribution and the surface roughness can be a powerful tool to investigate the crevice corrosion.

Purpose: In vivo 31P MRSI enables noninvasive mapping of absolute pH values via the pH‐dependent chemical shifts of inorganic phosphates (Pi). A particular challenge is the quantification of extracellular Pi with low SNR in vivo. The purpose of this study was to demonstrate feasibility of assessing both intra‐ and extracellular pH across the whole human brain via volumetric 31P MRSI at 7T. Methods: 3D 31P MRSI data sets of the brain were acquired from three healthy volunteers and three glioma patients. Low‐rank denoising was applied to enhance the SNR of 31P MRSI data sets that enables detection of extracellular Pi at high spatial resolutions. A robust two‐compartment quantification model for intra‐ and extracellular Pi signals was implemented. Results: In particular low‐rank denoising enabled volumetric mapping of intra‐ and extracellular pH in the human brain with voxel sizes of 5.7 mL. The average intra‐ and extracellular pH measured in white matter of healthy volunteers were 7.00 ± 0.00 and 7.33 ± 0.03, respectively. In tumor tissue of glioma patients, both the average intra‐ and extracellular pH increased to 7.12 ± 0.01 and 7.44 ± 0.01, respectively, compared to normal appearing tissue. Conclusion: Mapping of pH values via 31P MRSI at 7T using the proposed two‐compartment quantification model improves reliability of pH values obtained in vivo, and has the potential to provide novel insights into the pH heterogeneity of various tissues. [ABSTRACT FROM AUTHOR]

Due to the complex nature of probe/substrate interactions in scanning electrochemical microscopy (SECM), SECM has been primarily limited to well‐defined probe geometries and slow scan speeds that reduce imaging throughput. Herein, we show that in situ colorimetric visualization of concentration gradients using pH indicator dyes during SECM measurements can be a powerful tool for understanding the coupled influences of probe geometry and scan speed on the dynamics of localized plumes of electroactive species that mediate probe/substrate interactions. Colorimetric images of plumes generated at a band electrode reveal that probe geometry strongly influences linescan signal distortion and hysteresis at scan speeds surpassing the conventional SECM "speed limit". Combining mechanistic information from in situ colorimetric imaging with transport models, this article reports design principles that have the potential to enable quantitative SECM with novel probe geometries and at imaging rates that are at least an order of magnitude faster than currently employed. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. 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. • Countering of intracellular acidosis in penumbra was protective after ischemia. • The hypoemic response was compromised by translocating protons out of neurons. • Modulation of penumbral neurons after ischemic injury reduced the hemispheric lateralization index. [ABSTRACT FROM AUTHOR]

The dogma that the synaptic cleft acidifies during neurotransmission is based on the corelease of neurotransmitters and protons from synaptic vesicles, and is supported by direct data from sensory ribbon-type synapses. However, it is unclear whether acidification occurs at non-ribbon-type synapses. Here we used genetically encoded fluorescent pH indicators to examine cleft pH at conventional neuronal synapses. At the neuromuscular junction of female Drosophila larvae, we observed alkaline spikes of over 1 log unit during fictive locomotion in vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only ~--0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca2+ movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca2+/H + antiporting activity of the plasma membrane Ca2+-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage gated Ca2+ channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extrasynaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non-ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses. [ABSTRACT FROM AUTHOR]

• A pH-sensitive fluorescent protein enables a voltage dependent optical signal. • A negative charge on the fluorescent protein improves the optical signals. • Dimerization via the fluorescent protein domain is required for large signals. • Voltage induced conformational changes alter the position of the negative charge. • ArcLight is capable of optically resolving voltage and pH simultaneously. 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. [ABSTRACT FROM AUTHOR]

• Electrophilic fluorination of naphthofluorescein (NF) resulted in mono-, di- and trifluorinated derivatives. • Optical and chemical characterization of products shows bathochromic and acidic shifts in pK a with increasing fluorination. • NF, 4-MFNF and 4,10-DFNF can distinguish pH in a biologically-relevant range using Selective Bandwidth Quenching. • Measurements of pH using Cerenkov Radiation Energy Transfer (CRET) are possible with NF; not with 4-MFNF and 4,10-DFNF. • Quantum yield of fluorescence shown to be decreased due to fluorination of NF. In this paper, we report the synthesis and characterization of fluorinated derivatives of naphthofluorescein (NF), a fluorescent pH-sensitive probe that can be used for functional Cerenkov imaging. The compounds were prepared using electrophilic fluorination with dilute fluorine gas under acidic conditions. The fluorination of the NF molecule occurred in the ortho positions to the hydroxyl moiety, producing mono-, di-, and tri-substituted derivatives. The properties of the fluorinated naphthofluoresceins were similar to the parent compound, retaining pH sensitivity and fluorescence capability, but showed a more acidic pK a with increasing fluorination degree and a bathochromic shift in both absorbance and fluorescence. NF and its two major fluorinated derivatives were shown to attenuate Cerenkov radiation in the basic form; the greatest attenuation was observed at wavelengths coinciding with the absorption maxima. NF also showed potential as a Cerenkov Radiation Energy Transfer (CRET) probe. Fluorinated naphthofluoresceins provide a new family of molecular imaging probes for the detection of pH in tissue and organs by using a combination of PET and Cerenkov imaging. [ABSTRACT FROM AUTHOR]

Cancer cells are characterized by a metabolic shift in cellular energy production, orchestrated by the transcription factor HIF-1α, from mitochondrial oxidative phosphorylation to increased glycolysis, regardless of oxygen availability (Warburg effect). The constitutive upregulation of glycolysis leads to an overproduction of acidic metabolic products, resulting in enhanced acidification of the extracellular pH (pHe ~ 6.5), which is a salient feature of the tumor microenvironment. Despite the importance of pH and tumor acidosis, there is currently no established clinical tool available to image the spatial distribution of tumor pHe. The purpose of this review is to describe various imaging modalities for measuring intracellular and extracellular tumor pH. For each technique, we will discuss main advantages and limitations, pH accuracy and sensitivity of the applied pH-responsive probes and potential translatability to the clinic. Particular attention is devoted to methods that can provide pH measurements at high spatial resolution useful to address the task of tumor heterogeneity and to studies that explored tumor pH imaging for assessing treatment response to anticancer therapies. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Image 1 Highlights • Novel acid-responsive reactive matrix, Gly-3AQ, has been synthesized for the first time. • Gly-3AQ has a broad range of acid-responsiveness (pH 2.0–7.0) and good selectivity when analyzing real samples. • Using Gy-3AQ-based MALDI imaging, acidity variation of citrus juice sac during development was clearly visualized. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Unlabelled Image Highlights • Fluorescent protein (FP)-based pH indicators, advantages and shortcomings. • Redshifted FP-based pH indicators overcome cellular autofluorescence. • FRET-based pH indicators as ratiometric pH-sensing probes. • Hybrid pH sensors combine capabilities of non-GFP fluorophores with advantages of genetically encoded tags. [ABSTRACT FROM AUTHOR]

Ischemic stroke is a leading cause of death and disability worldwide. Yet, the effective therapy of focal cerebral ischemia has been an unresolved challenge. We propose here that ischemic tissue acidosis, a sensitive metabolic indicator of injury progression in cerebral ischemia, can be harnessed for the targeted delivery of neuroprotective agents. Ischemic tissue acidosis, which represents the accumulation of lactic acid in malperfused brain tissue is significantly exacerbated by the recurrence of spreading depolarizations. Deepening acidosis itself activates specific ion channels to cause neurotoxic cellular Ca2+ accumulation and cytotoxic edema. These processes are thought to contribute to the loss of the ischemic penumbra. The unique metabolic status of the ischemic penumbra has been exploited to identify the penumbra zone with imaging tools. Importantly, acidosis in the ischemic penumbra may also be used to guide therapeutic intervention. Agents with neuroprotective promise are suggested here to be delivered selectively to the ischemic penumbra with pH-responsive smart nanosystems. The administered nanoparticels release their cargo in acidic tissue environment, which reliably delineates sites at risk of injury. Therefore, tissue pH-targeted drug delivery is expected to enrich sites of ongoing injury with the therapeutical agent, without the risk of unfavorable off-target effects.

Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme’s intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity.

Many diseases can overrule natural pH regulatory mechanisms and alter the extracellular pH (pHe). A non-invasive method that resolves pHe in vivo with high spatial and temporal resolution could therefore improve diagnosis and monitoring of diseases, contributing to the concept of precision medicine. During the last decades, several techniques have been proposed to image pHe non-invasively. The majority of these methods rely on magnetic resonance because of its good spatial resolution, high penetration depth, non-ionizing radiation and excellent complimentary soft tissue contrast. Dissolution dynamic nuclear polarization (DNP) is an emerging concept to enhance nuclear magnetic resonance (NMR) signals by more than four orders of magnitude, making it possible to observe in vivo metabolic processes in real-time. Here, we summarize and review recent developments in pHe imaging techniques based on hyperpolarization methods and give an overview of recently discovered hyperpolarized pH sensor molecules that have been applied in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Acid-base homeostasis is an essential process for maintaining proper pH levels in the body, with alterations in this process potentially signaling pathology. While monitoring urine pH, blood gases and other assays can provide information on the overall systemic pH, it is desirable to get more localized knowledge to assist diagnosis. Various non-invasive methods have been developed with Chemical Exchange Saturation Transfer (CEST) MRI now emerging as a particularly intriguing technology for detecting pH. In this review, we provide an overview of the paramagnetic and diamagnetic CEST probes available for producing pH maps, describe in depth the various methods to collect CEST MRI data and post-process the resulting images to create pH maps and describe how this has been applied for detecting renal injury, mapping the pH of tumors and monitoring the health of transplanted therapeutic cells. Finally, we will provide an outlook of the possibilities to translate this technology into patients. [ABSTRACT FROM AUTHOR]

DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau 1/2 <1 s for 7.2 → 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.

A series of intra-molecular hydrogen bonded imidazoles and related heterocyclic compounds were screened for their N-H chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) contrast properties. Of the compounds, imidazole-4,5-dicarboxamides (I45DCs) were found to provide the strongest contrast, with the contrast produced at a large chemical shift from water (7.8 ppm) and strongly dependent on pH. We have tested several probes based on this scaffold, and demonstrated that these probes could be applied for in vivo detection of kidney pH after intravenous administration. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The gastric acidity scale has been imaged by integration of the pH-conversion model (pH-CM) into 3D-gastro electrical impedance tomography (3D- g -EIT) under the influence of spatial mean hydrogen-specific conductivity σ H + . The pH-CM uses Debye-Hückel-Onsager (DHO) interionic equation, molar conductivity ratio, ω H + , viscosity η state (k) and relative permittivity ε state (k) in order to convert the imaged conductivity distribution σ to the pH distribution pH *. With the proposed integration, phantoms of empty (S 1), liquid storage (S 2 - S 4), and solid meal storage (S 5) states are measured to reflect the dilution state. In the case of empty (S 1), the artificial gastric juice is made based on 99.50% of H 2 O and 0.50% solution consisting of HCl (1.4263 g/L), KCl (0.8647 g/L), and NaCl (2.8559 g/L). In the case of the liquid storage (S 2 - S 4) state, 200 mL of H 2 O is added three times as a dilution of artificial gastric juice in each S 2 , S 3 , and S 4. In the solid meal storage (S 5) state, an acid solid meal is blended into the artificial gastric content. The modified constants, α , and β are calculated based on the variation of viscosity η state (k) and permittivity ε state (k) of each state. The conductivity of gastric-shape models in five states is studied in the simulation. In the experiments, the simulated models were measured as gastric phantoms. Integrating pH-CM into 3D- g -EIT successfully images the pH * in the simulations and experiments. As a result, the estimated acidity scale 〈 pH 〉 is inversely proportional to σ H + with mean pH error evaluation of simulations e ˆ pH (sim) and experiments e ˆ pH (sim) are 15.8% and 8.5%, respectively. • A new pH estimation 〈 pH 〉 based on a conversion model from hydrogen-specific conductivity 〈 σ H + 〉 to concentration H + • The imaging of artificial gastric pH distribution pH∗ by integrating 3D- g -EIT and DHO equation. • The evaluation of acidity scale 〈 pH 〉 based on gastric-shaped phantoms during empty, liquid, and solid storage states. [ABSTRACT FROM AUTHOR]

The assessment of intracellular dynamics is crucial for understanding the function and formation process of bacterial organelle, just as it is for the inquisition of their eukaryotic counterparts. The methods for imaging magnetosome organelles in a magnetotactic bacterial cell using live-cell fluorescence imaging by highly inclined and laminated optical sheet (HILO) microscopy are presented in this chapter. Furthermore, we introduce methods for pH imaging in magnetosome lumen as an application of fluorescence magnetosome imaging., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose Low pH is associated with intervertebral disc (IVD)-generated low back pain (LBP). The purpose of this work was to develop an in vivo pH level-dependent magnetic resonance imaging (MRI) method for detecting discogenic LBP, without using exogenous contrast agents. Methods The ratio of R1ρ dispersion and chemical exchange saturation transfer (CEST) (RROC) was used for pH-level dependent imaging of the IVD while eliminating the effect of labile proton concentration. The technique was validated by numerical simulations and studies on phantoms and ex vivo porcine spines. Four male (ages 42.8 ± 18.3) and two female patients (ages 55.5 ± 2.1) with LBP and scheduled for discography were examined with the method on a 3.0 Tesla MR scanner. RROC measurements were compared with discography outcomes using paired t-test. Results Simulation and phantom results indicated RROC is a concentration independent and pH level-dependent technique. Porcine spine study results found higher RROC value was related to lower pH level. Painful discs based on discography had significant higher RROC values than those with negative diagnosis ( P < 0.05). Conclusion RROC imaging is a promising pH level dependent MRI technique that has the potential to be a noninvasive imaging tool to detect painful IVDs in vivo. Magn Reson Med 73:1196-1205, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Purpose: Chemical exchange saturation transfer (CEST) imaging measurement depends not only on the labile proton concentration and pH-dependent exchange rate but also on experimental conditions, including the relaxation delay and radiofrequency (RF) saturation time. Our study aimed to extend a quasi-steady-state (QUASS) solution to a modified multi-slice CEST MRI sequence and test if it provides enhanced pH imaging after acute stroke., Methods: Our study derived the QUASS solution for a modified multislice CEST MRI sequence with an unevenly segmented RF saturation between image readout and signal averaging. Numerical simulation was performed to test if the generalized QUASS solution corrects the impact of insufficiently long relaxation delay, primary and secondary saturation times, and multi-slice readout. In addition, multiparametric MRI scans were obtained after middle cerebral artery occlusion, including relaxation and CEST Z-spectrum, to evaluate the performance of QUASS CEST MRI in a rodent acute stroke model. We also performed Lorentzian fitting to isolate multi-pool CEST contributions., Results: The QUASS analysis enhanced pH-weighted magnetization transfer asymmetry contrast over the routine apparent CEST measurements in both contralateral normal (-3.46% ± 0.62% (apparent) vs. -3.67% ± 0.66% (QUASS), P &lt; 0.05) and ischemic tissue (-5.53% ± 0.68% (apparent) vs. -5.94% ± 0.73% (QUASS), P &lt; 0.05). Lorentzian fitting also showed significant differences between routine and QUASS analysis of ischemia-induced changes in magnetization transfer, amide, amine, guanidyl CEST, and nuclear Overhauser enhancement (-1.6 parts per million) effects., Conclusion: Our study demonstrated that generalized QUASS analysis enhanced pH MRI contrast and improved quantification of the underlying CEST contrast mechanism, promising for further in vivo applications., (© 2022 International Society for Magnetic Resonance in Medicine.)

Chemical exchange saturation transfer (CEST) enables indirect detection of small metabolites in tissue by MR imaging. To optimize and interpret creatine-CEST imaging we characterized the dependence of the exchange-rate constant ksw of creatine guanidinium protons in aqueous creatine solutions as a function of pH and temperature T in vitro. Model solutions in the low pH range (pH = 5-6.4) were measured by means of water-exchange (WEX)-filtered 1H NMR spectroscopy on a 3 T whole-body MR tomograph. An extension of the Arrhenius equation with effective base-catalyzed Arrhenius parameters yielded a general expression for ksw(pH, T). The defining parameters were identified as the effective base-catalyzed rate constant kb,eff(298.15 K) = (3.009 ± 0.16) × 109 Hz l/mol and the effective activation energy EA,b,eff = (32.27 ± 7.43) kJ/mol at a buffer concentration of cbuffer = (1/15) M. As expected, a strong dependence of ksw on temperature was observed. The extrapolation of the exchange-rate constant to in vivo conditions (pH = 7.1, T = 37 °C) led to the value of the exchange-rate constant ksw = 1499 Hz. With the explicit function ksw(pH, T) available, absolute-pH CEST imaging could be realized and experimentally verified in vitro. By means of our calibration method it is possible to adjust the guanidinium proton exchange-rate constant ksw to any desired value by preparing creatine model solutions with a specific pH and temperature. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Imaging all the people: Using ionizable diblock copolymers a series of nanoprobes encoded with different 19F reporters for specific pH transitions is prepared for use in MRI. The pH response of the nanoprobes is extremely sharp (ΔpHON/OFF≈0.25 pH), and results from the disassembly of polymer micelles (see scheme). A collection of three nanoprobes provides the proof of concept and allows for a qualitative measurement of environmental pH values. [ABSTRACT FROM AUTHOR]

Noninvasive localized measurement of extracellular pH in cancer tissues can have a significant impact on the management of cancer. Despite its significance, there are limited approaches for rapid and noninvasive measurement of local pH in a clinical environment. In this study, we demonstrate the potential of noninvasive topical delivery of Alexa-647 labeled pHLIP (pH responsive peptide conjugated with Alexa Fluor® 647) to image changes in extracellular pH associated with head and neck squamous cell carcinoma using widefield and high resolution imaging. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of Alexa-647 labeled pHLIP in intact fresh human tissue specimens using widefield and high-resolution fluorescence imaging. Using topical delivery, Alexa-647 labeled pHLIP can be rapidly delivered throughout the epithelium of intact tissues with a depth exceeding 700 µm. Following labeling with Alexa-647 labeled pHLIP, the mean fluorescent contrast increased four to eight fold higher in clinically abnormal tissues as compared to paired clinically normal biopsies. Furthermore, the imaging approach showed significant differences in fluorescence contrast between the cancer and the normal biopsies across diverse patients and different anatomical sites (unpaired comparison). The fluorescence contrast differences between clinically abnormal and normal tissues were in agreement with the pathologic evaluation. Topical application of fluorescently labeled pHLIP can detect and differentiate normal from cancerous tissues using both widefield and high resolution imaging. This technology will provide an effective tool to assess tumor margins during surgery and improve detection and prognosis of head and neck cancer. [ABSTRACT FROM AUTHOR]

It has been proposed that the spatial mismatch between deficits on perfusion-weighted imaging (PWI) and diffusion-weighted imaging (DWI) in MRI can be used to decide regarding thrombolytic treatment in acute stroke. However, uncertainty remains about the meaning and reversibility of the perfusion deficit and even part of the diffusion deficit. Thus, there remains a need for continued development of imaging technology that can better define a potentially salvageable ischemic area at risk of infarction. Amide proton transfer imaging is a novel MRI method that can map tissue pH changes, thus providing the potential to separate the PWI/DWI mismatch into an acidosis-based penumbra and a zone of benign oligemia. In this totally noninvasive method, the pH dependence of the chemical exchange between amide protons in endogenous proteins and peptides and water protons is exploited. Early results in animal models of ischemia show promise to derive an acidosis penumbra. Possible translation to the clinic and hurdles standing in the way of achieving this are discussed. [ABSTRACT FROM AUTHOR]

Abstract: The medulla contains central chemosensitive cells important for the maintenance of blood gas and pH homeostasis. To identify the intrinsic chemosensitive cells, we measured responses of intracellular Ca2+ ([Ca2+]i) and H+ ([H+]i), and membrane potential of rat primary-cultured medullary cells to 6-s exposure to acidosis. The cells showed transient [Ca2+]i increases to extracellular pH 6.8, which was inhibited by the specific ASIC1a blocker (psalmotoxin-1), but did not respond to pH 7.1 in the HEPES-buffered solution. Isocapnic acidosis induced no changes in [Ca2+]i, whereas hypercapnic acidosis induced a remarkable Ca2+ response and an increase in membrane potential in the HCO3 −-buffered solution (pH 7.1). In glia-rich cultures, intracellular acidification preceded the hypercapnic acidosis-induced Ca2+ response, and acetazolamide, a carbonic anhydrase inhibitor suppressed these responses. Transient receptor potential (TRP) channel broad-spectrum blockers Ni2+ and ruthenium red, and a TRPV1- and TRPM8-specific blocker N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-carbox-amide attenuated the hypercapnic acidosis-induced Ca2+ response. Subpopulations of cells that exhibited the hypercapnic acidosis-induced Ca2+ response also responded to the application of capsaicin (TRPV1 agonist) and menthol (TRPM8 agonist). These results suggest that the TRP channel family partially mediates the fast hypercapnic acidosis-induced Ca2+ response via changes in [H+]i and is a candidate of central chemosensing proteins. [ABSTRACT FROM AUTHOR]

We have studied CI- transport mechanisms in freshwater rainbow trout gill mitochondrion-rich (MR) cells using intmceUular pH (pHi) imaging. Scanning electron microscopy demonstrated maintenance of cellular polarity in isolated MR cells. MR cell subtypes were identified by Na+ introduction to the bath, and CI- transport mechanisms were subsequently examined. CI--free exposure resulted in an aikalinization of pHi in both MR cell subtypes, which was dependent on HCO3- in the bath and inhibited by 1 mM DIDS. Recovery of pHi from an acidified state in Na+-free conditions was also DIDS sensitive. These results are the first functional evidence for CI-/HCO3- exchangers in fish gill MR cells. A direct switch from NaCI to CI--free conditions caused a pHi acidification in a subset of MR cells, which was enhanced in die absence of HCO3-. The acidification was replaced by an alkalinization when CI- removal was performed in the presence of NPPB (500 μM) or EIPA (500 μM). Finally, we found that the Na+-induced alkalinization of pHi found in a previous study is inhibited by EIPA. This inhibitor profile's results suggest the presence of a Cl--dependent Na+/H+ exchange mechanism. [ABSTRACT FROM AUTHOR]