Genetically encoded red fluorescent copper(I) sensors for cellular copper(I) imaging

Copper ranks among the most important metal ions in living organism, owing to its key catalytic effect in a range of biochemical processes. Dysregulation of in vivo copper(I) metabolism is extremely toxic and would cause serious diseases in human, such as Wilson’s and Menkes. Thus, it would be highly valuable to have a proper approach to monitor the dynamics of copper(I) in vivo, as it is directly related to the onset of human copper(I)-related diseases. Under these circumstance, developing fluorescent protein based copper(I) sensors is highly demanded. However, these established sensors are mostly based on green or yellow FPs. Fluorescent copper(I) sensors with a spectra in the red range are more desirable due to lower phototoxicity, less auto-fluorescent noise and better penetration of red light. In the present work, we grafted a special red FP into three different location of a copper(I) binding protein, and generate a series of red fluorescent copper(I) sensors. Despite their limited in vivo sensitivity toward copper(I), these sensors are viable for cellular copper(I) imaging. Furthermore, these red fluorescent copper(I) sensors are a good starting point to develop superior copper(I) biosensors capable of imaging copper(I) fluctuations within a truly biologically relevant concentration, and further effort to realize this endeavor is under way.     

Co-Encapsulating the Fusogenic Peptide INF7 and Molecular Imaging Probes in Liposomes Increases Intracellular Signal and Probe Retention

Liposomes are promising vehicles to deliver diagnostic and therapeutic agents to cells in vivo. After uptake into cells by endocytosis, liposomes are degraded in the endolysosomal system. Consequently, the encapsulated cargo molecules frequently remain sequestered in endosomal compartments; this limits their usefulness in many applications (e.g. gene delivery). To overcome this, various fusogenic peptides have been developed to facilitate delivery of liposomally-encapsulated molecules into the cytosol. One such peptide is the pH-sensitive influenza-derived peptide INF7. Liposomal delivery of imaging agents is an attractive approach for enabling cell imaging and cell tracking in vivo, but can be hampered by inadequate intracellular accumulation and retention of probes caused by exocytosis (and possible degradation) of endosome-entrapped probes. Such signal loss could be minimized by facilitating escape of probe molecules from endolysosomal compartments into the cytosol. We investigated the ability of co-encapsulated INF7 to release liposomally-delivered rhodamine fluorophores into the cytosol after endosomal acidification/maturation. We co-encapsulated INF7 and fluorescent rhodamine derivatives having vastly different transport properties to show that after endocytosis by CV1 cells, the INF7 peptide is activated by acidic endosomal pH and facilitates efficient release of the fluorescent tracers into the cytosol. Furthermore, we show that INF7-facilitated escape from endosomes markedly enhanced retention of tracers that cannot be actively extruded from the cytosol. Minimizing loss of intracellular probes improves cellular imaging by increasing the signal-to-noise ratio of images and lengthening the time window that imaging can be performed. In particular, this will enhance in vivo electron paramagnetic resonance imaging, an emergent magnetic resonance imaging modality requires exogenous paramagnetic imaging agents and is highly promising for cellular and molecular imaging.     

Genetically Encoded Sensors to Elucidate Spatial Distribution of Cellular Zinc

Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn²⁺ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn²⁺, these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn²⁺ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn²⁺ is taken up into mitochondria following glutamate/Zn²⁺ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn²⁺ signals under different cellular conditions.Although mammalian cells are known to concentrate transition metals, it is now well established that under resting conditions, “free” (e.g. unbound) metals are maintained at extremely low levels. Estimates of the total Zn²⁺ concentration in mammalian cells typically range from 100 to 500 μm (1); yet free Zn²⁺ concentrations are tightly buffered by proteins such as metallothionein to maintain cytosolic Zn²⁺ concentrations in the picomolar to nanomolar range (2–5). However, there is emerging evidence that this static picture is dramatically altered by different cellular conditions, such as redox perturbations caused by oxidative stress (6, 7) and cellular signals such as nitric oxide (8). Consequently, there is a pool of labile Zn²⁺ that, if mobilized by cellular signals, would result in the generation of transient Zn²⁺ signals. Recent studies suggest that these Zn²⁺ signals influence critical biological processes, such as mitochondrial function (7, 9, 10). Elucidation of the sources and dynamics of these Zn²⁺ signals would greatly advance our understanding of the interplay between metal regulation and cellular function.There has been a huge effort in the past few years to develop sensitive and selective fluorescent probes to monitor Zn²⁺ in biological systems. The majority of this work has focused on the generation of small molecule fluorescent indicators (reviewed by Que et al. (11)). Yet there are also examples of sensors based partially on Zn²⁺-binding proteins, such as carbonic anhydrase (12) and metallothionein (13), and peptide scaffolds (14). Although many of these sensors have begun to provide insight into Zn²⁺ concentrations within cells, one limitation is that it is challenging to explicitly target them to subdomains within the cell. Localized probes are necessary to generate a complete picture of cellular Zn²⁺ homeostasis in mammalian cells. For this reason, sensors that are genetically encoded (i.e. generated by translation of a nucleic acid sequence) are attractive platforms for engineering metal-specific sensors. Encoded sensors provide additional benefits such as retention of the sensor over days to weeks permitting long term imaging and the ability to systematically vary the sensor concentration to evaluate the extent to which the sensor perturbs resting Zn²⁺ concentrations.Here we present genetically encoded sensors designed with a “Zn²⁺-sensing domain” sandwiched between two fluorescent proteins. The fluorescent proteins are chosen so that they are capable of undergoing fluorescence resonance energy transfer (FRET).² Because the mechanism of FRET involves dipole-dipole coupling, it is exquisitely dependent on the distance and orientation of the fluorophores with respect to one another. Therefore, if the binding of Zn²⁺ induces a conformational change in the sensor, it will alter the energy transfer between the two fluorescent proteins. The advantage of using FRET as the optical readout is that the donor emission will decrease and the acceptor emission will increase upon Zn²⁺ binding. Hence, by taking the ratio of the acceptor to the donor emission, we can create a ratiometric sensor. These sensors are targeted to the cytosol, mitochondria, and plasma membrane by attachment of signal sequences and fusion to other proteins. These sensors reveal differences in the spatial distribution of Zn²⁺ and highlight the power and utility of localized probes.     

Using lanthanide-based resonance energy transfer for in vitro and in vivo studies of biological processes

This review describes key directions in the development of different probes based on complex compounds of lanthanides for in vitro and in vivo researches. The role of microsecond fluorescence of lanthanides for overcoming problems of background fluorescence is considered. The basic classes of synthetic and genetically encoded complex compounds of lanthanides are summarized. Main principles of designing lanthanide-based molecular sensors, including FRET sensors based on lanthanides and colored fluorescent proteins are described. Their applications in bioanalytical research and cellular bioimaging are described. The main principles of cellular bioimaging using lanthanides are formulated, questions of their delivery into cells are considered, and the problem of their potential toxicity for living organisms is discussed. A technique using multiphoton excitation of lanthanides is described.     

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein–based or yellow fluorescent protein–based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes “ROBINc” and “ROBINy.” ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.     

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development^1-5, physiology^6-11, disease^12-16, and host-microbe interactions^17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae²⁶. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results.We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.     

Synthesis and evaluation of radioactive/fluorescent peptide probes for imaging of legumain activity

Legumain or asparaginyl endopeptidase is an enzyme overexpressed in some cancers and involved in cancer migration, invasion, and metastasis. We have developed radioiodine- ([¹²⁵I]I-LCP) or fluorescein-labeled peptides (FL-LCP) with a cell-permeable d-Arg nonamer fused to an anionic d-Glu nonamer via a legumain-cleavable linker, to function as peptide probes that measure and monitor legumain activity. Non-cleavable probes of FL-NCP and [¹²⁵I]I-NCP were similarly prepared and evaluated as negative control probes by altering their non-cleavable sequence. Model peptides with the legumain-cleavable or non-cleavable sequence (LCP and NCP, respectively) reacted with recombinant human legumain, and only LCP was digested by this enzyme. [¹²⁵I]I-LCP uptake in legumain-positive HCT116 cells was significantly higher than that of [¹²⁵I]I-NCP (11.2 ± 0.44% vs 1.75 ± 0.06% dose/mg). The accumulation of FL-LCP in the HCT116 cells was rather low (4.75 ± 0.29% dose/mg protein), but not significantly different from the levels of FL-NCP. It is possible that low concentrations of [¹²⁵I]I-LCP (40 pM) can be effectively internalized after legumain cleavage. On the other hand, the cellular uptake of much higher concentrations of the FL-LCP derivative (1 mM) may be restricted by high concentrations of polyanions. The in vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [¹²⁵I]I-LCP was 1.34% injected dose per gram (% ID/g) at 30 min. The tumor/blood and tumor/muscle ratios at 30 min were 0.63 and 1.77, respectively, indicating that the [¹²⁵I]I-LCP accumulation in tumors was inadequate for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties, [¹²⁵I]I-LCP has been demonstrated to be an effective scaffold for the development of nuclear medicine imaging probes to monitor legumain activity in living subjects.     

Evidence for in vivo compartmentation of phenylalanyl-tRNA ligase in amphibian oocytes

The in vivo activity of phenylalanyl-tRNA ligase of Xenopus laevis oocytes was assayed by measuring the esterification of microinjected yeast tRNA^Phe with [¹⁴C]phenylalanine added to the extracellular medium. The three enzyme substrates, ATP, phenylalanine, and tRNA^Phe, are present in the in vivo assay at saturating concentrations as seen by the fact that microinjection into the cell of additional amounts of these compounds does not increase the quantity of [¹⁴C]Phe-tRNA^Phe formed. The in vivo activity of Phe-tRNA ligase in oocytes at several stages of development is less than 10% of the in vitro activity measured in homogenates of the same cells. The in vivo assay of Phe-tRNA ligase in oocytes that have been microinjected with this enzyme partially purified from X. laevis ovary shows that the enzyme is not inhibited by the cellular conditions. The conclusion drawn from these experiments is that a large fraction of the Phe-tRNA ligase present in oocytes is in a cellular compartment which is not available to the injected tRNA.     

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into >250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8⁺ and CD4⁺ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8⁺ T cell-mediated killing of FTA target cells and CD4⁺ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since >250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g. the cumulative magnitude of the response) and a qualitative (e.g. functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.     

More gating charges are needed to open a Shaker K+ channel than are needed to open an rBIIA Na+ channel

This study presents what is, to our knowledge, a novel technique by means of which the ratio of the single gating charges of voltage-gated rat brain IIA (rBIIA) sodium and Shaker potassium ion channels was estimated. In the experiment, multiple tandems of enhanced green fluorescent protein were constructed and inserted into the C-terminals of Na⁺ and K⁺ ion channels. cRNA of Na⁺ and K⁺ ion channels was injected and expressed in Xenopus laevis oocytes. The two electrode voltage-clamp technique allowed us to determine the total gating charge of sodium and potassium ion channels, while a relative measure of the amount of expressed channels could be established on the basis of the quantification of the fluorescence intensity of membrane-bound channels marked by enhanced green fluorescent proteins. As a result, gating charge and fluorescence intensity were found to be positively correlated. A relative comparison of the single gating charges of voltage-gated sodium and potassium ion channels could thus be established: the ratio of the single gating charges of the Shaker potassium channel and the rBIIA sodium channel was found to be 2.5 ± 0.4. Assuming the single channel gating charge of the Shaker K⁺ channel to be ∼13 elementary charges (well supported by other studies), this leads to approximately six elementary charges for the rBIIA sodium channel, which includes a fraction of gating charge that is missed during inactivation.     

Fluorescent probes for the structure and function of metallothionein

Fluorescence methods have been instrumental in demonstrating that the structure of human metallothionein in vivo depends on the availability of metal ions and the redox environment. Differential chemical modifications of its cysteine thiols with fluorescent probes allowed determination of three states: metallothionein (zinc-bound thiolate), thionein (free thiols), and thionin (disulfides). Interrogation of its zinc-binding properties with fluorescent chelating agents revealed that the affinities for the seven zinc ions vary over four orders of magnitude. Attachment of fluorescent labels generated metallothionein FRET (fluorescence resonance energy transfer) sensors for investigating its structure and function in living cells.     

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on Förster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.     

Live monitoring of plant redox and energy physiology with genetically encoded biosensors

Genetically encoded biosensors pave the way for understanding plant redox dynamics and energy metabolism on cellular and subcellular levels.

ADVANCES

• Methodological advances in fluorescent protein-based in vivo biosensing have been instrumental for several paradigm shifts in our understanding of cell physiology, metabolism and signaling.
• An increasing number of genetically encoded biosensors has been used to dissect the dynamics of several distinct redox couples and energy physiology in plants.
• In vivo monitoring using biosensors has pioneered the simultaneous read-out of different physiological parameters in different subcellular locations by parallelized plate reader-based, multiwell fluorimetry, or expression strategies for multiple sensors in parallel.
• Sensing dynamic changes in hydrogen peroxide levels is possible with sensors of the HyPer family, or roGFP fusion variants with a thiol peroxidase.
• Peredox and SoNar family sensors enable direct visualization of NADH/NAD⁺, while iNAP family sensors respond to NADPH concentration in plants.
• Sensor variants with different sensitivity ranges enable use of the most appropriate variant for the specific in vivo environment or experimental scope.

     

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides^{1, 2}. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy³. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid⁴ and N-acetylgalactosamine^{5, 6}, in developing zebrafish and in other living organisms.Download video file.^{(41M, mov)}     

Facile synthesis of anthracene-appended amino acids as highly selective and sensitive fluorescent Fe3+ ion sensors

We designed new fluorescent chemical sensors for Fe³⁺ ion detection, by conjugating amino acids as receptors into an anthracene fluorophore. The conjugates were synthesized in solid phase by Fmoc-chemistry. Fluorescence sensors containing Asp (1) and Glu (2) both had exclusive selectivity for Fe³⁺ in 100% aqueous solution and in a mixed organic–aqueous solvent system. Other metal ions did not interfere with the detection ability of the sensors for Fe³⁺. The sensors detect Fe³⁺ ions via a chelation-enhanced fluorescent quenching effect. The binding affinity, reversible monitoring, and pH sensitivity of the sensors were investigated. In addition, detection of fluoride ion among halide ions was done by a chemosensing ensemble method with 1–Fe³⁺ and 2–Fe³⁺ complexes.     

An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.     

Involvement of transferrin in the uptake of 67Ga in inflammatory and normal tissues

The results from gel chromatography and electrophoresis showed that ⁶⁷Ga is exclusively bound with transferrin both in vitro and in vivo, but high concentrations of sodium citrate strongly inhibited the binding of ⁶⁷Ga to transferrin. The influence of sodium citrate on the uptake of ⁶⁷Ga into inflammatory and normal soft tissues was also investigated. Sodium citrate decreased the uptake of ⁶⁷Ga into the liver and spleen, but had no influence on the uptake of ⁶⁷Ga into inflammatory tissue. These results suggest that the uptake of ⁶⁷Ga into normal soft tissues occurs in a transferrin-bound form but into inflammatory tissue in an unbound form.     

pH敏感的荧光探针及在活细胞中的应用

细胞内的pH是细胞内多种酶活性和生理活动的重要调节因素,准确、动态的监测细胞内pH变化对研究细胞内的活动至关重要。一些荧光小分子可以感应pH的变化,同时具有较高的灵敏度和特异性,对细胞损伤较小且标记操作简单,已逐渐发展成为一种监测细胞内pH变化的有效方法。本文主要介绍目前常用pH敏感的荧光探针及其在活细胞研究中的进展。     

Investigating Acid Production by Streptococcus mutans with a Surface-Displayed pH-Sensitive Green Fluorescent Protein

Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.