High-resolution extracellular pH imaging of liver cancer with multiparametric MR using Deep Image Prior.

Noninvasive extracellular pH (pHe) mapping with Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) using MR spectroscopic imaging (MRSI) has been demonstrated on 3T clinical MR scanners at 8 × 8 × 10 mm3 spatial resolution and applied to study various liver cancer treatments. Although pHe imaging at higher resolution can be achieved by extending the acquisition time, a postprocessing method to increase the resolution is preferable, to minimize the duration spent by the subject in the MR scanner. In this work, we propose to improve the spatial resolution of pHe mapping with BIRDS by incorporating anatomical information in the form of multiparametric MRI and using an unsupervised deep-learning technique, Deep Image Prior (DIP). Specifically, we used high-resolution T 1 , T 2 , and diffusion-weighted imaging (DWI) MR images of rabbits with VX2 liver tumors as inputs to a U-Net architecture to provide anatomical information. U-Net parameters were optimized to minimize the difference between the output super-resolution image and the experimentally acquired low-resolution pHe image using the mean-absolute error. In this way, the super-resolution pHe image would be consistent with both anatomical MR images and the low-resolution pHe measurement from the scanner. The method was developed based on data from 49 rabbits implanted with VX2 liver tumors. For evaluation, we also acquired high-resolution pHe images from two rabbits, which were used as ground truth. The results indicate a good match between the spatial characteristics of the super-resolution images and the high-resolution ground truth, supported by the low pixelwise absolute error.

A versatile look-up algorithm for mapping pH values and magnesium ion content using 31P MRSI.

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 ß-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.

Amide proton transfer imaging in stroke.

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.

Amine-weighted chemical exchange saturation transfer magnetic resonance imaging in brain tumors.

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.

Quasi-steady-state amide proton transfer (QUASS APT) MRI enhances pH-weighted imaging of acute stroke.

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.

Simultaneous In Situ Imaging of pH and Surface Roughening during the Progress of Crevice Corrosion of Stainless Steel.

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.

Neuronal Glutamatergic Synaptic Clefts Alkalinize Rather Than Acidify during Neurotransmission.

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.SIGNIFICANCE STATEMENT Neurotransmission is highly sensitive to the pH of the extracellular milieu. This is readily evident in the neurological symptoms that accompany systemic acid/base imbalances. Imaging data from sensory ribbon-type synapses show that neurotransmission itself can acidify the synaptic cleft, likely due to the corelease of protons and glutamate. It is not clear whether the same phenomenon occurs at conventional neuronal synapses due to the difficulties in collecting such data. If it does occur, it would provide for an additional layer of activity-dependent modulation of neurotransmission. Our findings of alkalinization, rather than acidification, within the cleft of two different neuronal synapses encourages a reassessment of the scope of activity-dependent pH influences on neurotransmission and short-term synaptic plasticity.

Evaluation of a similarity anisotropic diffusion denoising approach for improving in vivo CEST-MRI tumor pH imaging.

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.

Imaging tumor acidosis: a survey of the available techniques for mapping in vivo tumor pH.

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.

Volumetric mapping of intra- and extracellular pH in the human brain using 31 P MRSI at 7T.

PURPOSE: In vivo 31 P 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 31 P MRSI at 7T. METHODS: 3D 31 P 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 31 P 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 31 P 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.

Using bidirectional chemical exchange for improved hyperpolarized [13 C]bicarbonate pH imaging.

PURPOSE: Rapid chemical exchange can affect SNR and pH measurement accuracy for hyperpolarized pH imaging with [13 C]bicarbonate. The purpose of this work was to investigate chemical exchange effects on hyperpolarized imaging sequences to identify optimal sequence parameters for high SNR and pH accuracy. METHODS: Simulations were performed under varying rates of bicarbonate-CO2 chemical exchange to analyze exchange effects on pH quantification accuracy and SNR under different sampling schemes. Four pulse sequences, including 1 new technique, a multiple-excitation 2D EPI (multi-EPI) sequence, were compared in phantoms using hyperpolarized [13 C]bicarbonate, varying parameters such as tip angles, repetition time, order of metabolite excitation, and refocusing pulse design. In vivo hyperpolarized bicarbonate-CO2 exchange measurements were made in transgenic murine prostate tumors to select in vivo imaging parameters. RESULTS: Modeling of bicarbonate-CO2 exchange identified a multiple-excitation scheme for increasing CO2 SNR by up to a factor of 2.7. When implemented in phantom imaging experiments, these sampling schemes were confirmed to yield high pH accuracy and SNR gains. Based on measured bicarbonate-CO2 exchange in vivo, a 47% CO2 SNR gain is predicted. CONCLUSION: The novel multi-EPI pulse sequence can boost CO2 imaging signal in hyperpolarized 13 C bicarbonate imaging while introducing minimal pH bias, helping to surmount a major hurdle in hyperpolarized pH imaging.

Development of fluorinated naphthofluoresceins for Cerenkov imaging.

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 pKa 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.

Deuteration of Hyperpolarized 13 C-Labeled Zymonic Acid Enables Sensitivity-Enhanced Dynamic MRI of pH.

Aberrant pH is characteristic of many pathologies such as ischemia, inflammation or cancer. Therefore, a non-invasive and spatially resolved pH determination is valuable for disease diagnosis, characterization of response to treatment and the design of pH-sensitive drug-delivery systems. We recently introduced hyperpolarized [1,5-13 C2 ]zymonic acid (ZA) as a novel MRI probe of extracellular pH utilizing dissolution dynamic polarization (DNP) for a more than 10000-fold signal enhancement of the MRI signal. Here we present a strategy to enhance the sensitivity of this approach by deuteration of ZA yielding [1,5-13 C2 , 3,6,6,6-D4 ]zymonic acid (ZAd ), which prolongs the liquid state spin lattice relaxation time (T1 ) by up to 39 % in vitro. Measurements with ZA and ZAd on subcutaneous MAT B III adenocarcinoma in rats show that deuteration increases the signal-to-noise ratio (SNR) by up to 46 % in vivo. Furthermore, we demonstrate a proof of concept for real-time imaging of dynamic pH changes in vitro using ZAd , potentially allowing for the characterization of rapid acidification/basification processes in vivo.

Microchamber arrays with an integrated long luminescence lifetime pH sensor.

A pH probe with a microsecond luminescence lifetime was obtained via covalent coupling of 6-carboxynaphthofluorescein (CNF) moieties to ruthenium-tris-(1,10-phenanthroline)(2+). The probe was covalently attached to amino-modified poly-(2-hydroxyethyl)methacrylate (pHEMA) and showed a pH-dependent FRET with luminescence lifetimes of 681 to 1260 ns and a working range from ca. pH 6.5 to 9.0 with a pKa of 7.79 ± 0.14. The pH sensor matrix was integrated via spin coating as ca. 1- to 2-µm-thick layer into "CytoCapture" cell culture dishes of 6 mm in diameter. These contained a microcavity array of square-shaped regions of 40 µm length and width and 15 µm depth that was homogeneously coated with the pH sensor matrix. The sensor layer showed fast response times in both directions. A microscopic setup was developed that enabled imaging of the pH inside the microchamber arrays over many hours. As a proof of principle, we monitored the pH of Escherichia coli cell cultures grown in the microchamber arrays. The integrated sensor matrix allowed pH monitoring spatially resolved in every microchamber, and the differences in cell growth between individual chambers could be resolved and quantified.

Detection of low back pain using pH level-dependent imaging of the intervertebral disc using the ratio of R1ρ dispersion and -OH chemical exchange saturation transfer (RROC).

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.

Characterization of creatine guanidinium proton exchange by water-exchange (WEX) spectroscopy for absolute-pH CEST imaging in vitro.

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 k(sw) 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 k(sw) (pH, T). The defining parameters were identified as the effective base-catalyzed rate constant k(b,eff) (298.15 K) = (3.009 ± 0.16) × 109 Hz l/mol and the effective activation energy E(A,b,eff) = (32.27 ± 7.43) kJ/mol at a buffer concentration of c(buffer) = (1/15) M. As expected, a strong dependence of k(sw) 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 k(sw) = 1499 Hz. With the explicit function k(sw) (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 k(sw) to any desired value by preparing creatine model solutions with a specific pH and temperature.

Metabolic Imaging of Acute Ischemic Stroke (PET, 1Hydrogen Spectroscopy, 17Oxygen Imaging, 23Sodium MRI, pH Imaging).

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.

Ratiometric i-Motif-Based Sensor for Precise Long-Term Monitoring of pH Micro Alterations in the Nucleoplasm and Interchromatin Granules.

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 (tau1/2 <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.

Clinically Translatable Hyperpolarized 13C Bicarbonate pH Imaging Method for Use in Prostate Cancer.

Solid tumors such as prostate cancer (PCa) commonly develop an acidic microenvironment with pH 6.5-7.2, owing to heterogeneous perfusion, high metabolic activity, and rapid cell proliferation. In preclinical prostate cancer models, disease progression is associated with a decrease in tumor extracellular pH, suggesting that pH imaging may reflect an imaging biomarker to detect aggressive and high-risk disease. Therefore, we developed a hyperpolarized carbon-13 MRI method to image the tumor extracellular pH (pHe) and prepared it for clinical translation for detection and risk stratification of PCa. This method relies on the rapid breakdown of hyperpolarized (HP) 1,2-glycerol carbonate (carbonyl-13C) via base-catalyzed hydrolysis to produce HP 13CO32-, which is neutralized and converted to HP H13CO3-. After injection, HP H13CO3- equilibrates with HP 13CO2 in vivo and enables the imaging of pHe. Using insights gleaned from mechanistic studies performed in the hyperpolarized state, we solved issues of polarization loss during preparation in a clinical polarizer system. We successfully customized a reaction apparatus suitable for clinical application, developed clinical standard operating procedures, and validated the radiofrequency pulse sequence and imaging data acquisition with a wide range of animal models. The results demonstrated that we can routinely produce a highly polarized and safe HP H13CO3- contrast agent suitable for human injection. Preclinical imaging studies validated the reliability and accuracy of measuring acidification in healthy kidney and prostate tumor tissue. These methods were used to support an Investigational New Drug application to the U.S. Food and Drug Administration. This methodology is now ready to be implemented in human trials, with the ultimate goal of improving the management of PCa.

Live-Cell Fluorescence Imaging of Magnetosome Organelle for Magnetotaxis Motility.

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.