Two-photon Fluorescence Microscopy Research Articles

Predict Early Recurrence of Resectable Hepatocellular Carcinoma Using Multi-Dimensional Artificial Intelligence Analysis of Liver Fibrosis.

Simple SummaryHepatocellular carcinoma (HCC) is the third most commonly diagnosed cancer in the world, and surgical resection is the commonly used curative management of early-stage disease. However, the recurrence rate is high after resection, and liver fibrosis has been thought to increase the risk of recurrence. Conventional histological staging of fibrosis is highly subjective to observer variations. To overcome this limitation, we used a fully quantitative fibrosis assessment tool, qFibrosis (utilizing second harmonic generation and two-photon excitation fluorescence microscopy), with multi-dimensional artificial intelligence analysis to establish a fully-quantitative, accurate fibrotic score called a “combined index”, which can predict early recurrence of HCC after curative intent resection. Therefore, we can pay more attention on the patients with high risk of early recurrence.Background: Liver fibrosis is thought to be associated with early recurrence of hepatocellular carcinoma (HCC) after resection. To recognize HCC patients with higher risk of early recurrence, we used a second harmonic generation and two-photon excitation fluorescence (SHG/TPEF) microscopy to create a fully quantitative fibrosis score which is able to predict early recurrence. Methods: The study included 81 HCC patients receiving curative intent hepatectomy. Detailed fibrotic features of resected hepatic tissues were obtained by SHG/TPEF microscopy, and we used multi-dimensional artificial intelligence analysis to create a recurrence prediction model “combined index” according to the morphological collagen features of each patient’s non-tumor hepatic tissues. Results: Our results showed that the “combined index” can better predict early recurrence (area under the curve = 0.917, sensitivity = 81.8%, specificity = 90.5%), compared to alpha fetoprotein level (area under the curve = 0.595, sensitivity = 68.2%, specificity = 47.6%). Using a Cox proportional hazards analysis, a higher “combined index” is also a poor prognostic factor of disease-free survival and overall survival. Conclusions: By integrating multi-dimensional artificial intelligence and SHG/TPEF microscopy, we may locate patients with a higher risk of recurrence, follow these patients more carefully, and conduct further management if needed.

Segmentation-Less, Automated, Vascular Vectorization.

Recent advances in two-photon fluorescence microscopy (2PM) have allowed large scale imaging and analysis of blood vessel networks in living mice. However, extracting network graphs and vector representations for the dense capillary bed remains a bottleneck in many applications. Vascular vectorization is algorithmically difficult because blood vessels have many shapes and sizes, the samples are often unevenly illuminated, and large image volumes are required to achieve good statistical power. State-of-the-art, three-dimensional, vascular vectorization approaches often require a segmented (binary) image, relying on manual or supervised-machine annotation. Therefore, voxel-by-voxel image segmentation is biased by the human annotator or trainer. Furthermore, segmented images oftentimes require remedial morphological filtering before skeletonization or vectorization. To address these limitations, we present a vectorization method to extract vascular objects directly from unsegmented images without the need for machine learning or training. The Segmentation-Less, Automated, Vascular Vectorization (SLAVV) source code in MATLAB is openly available on GitHub. This novel method uses simple models of vascular anatomy, efficient linear filtering, and vector extraction algorithms to remove the image segmentation requirement, replacing it with manual or automated vector classification. Semi-automated SLAVV is demonstrated on three in vivo 2PM image volumes of microvascular networks (capillaries, arterioles and venules) in the mouse cortex. Vectorization performance is proven robust to the choice of plasma- or endothelial-labeled contrast, and processing costs are shown to scale with input image volume. Fully-automated SLAVV performance is evaluated on simulated 2PM images of varying quality all based on the large (1.4×0.9×0.6 mm3 and 1.6×108 voxel) input image. Vascular statistics of interest (e.g. volume fraction, surface area density) calculated from automatically vectorized images show greater robustness to image quality than those calculated from intensity-thresholded images.

Non-Diffracting Light Wave: Fundamentals and Biomedical Applications

The light propagation in the medium normally experiences diffraction, dispersion, and scattering. Studying the light propagation is a century-old problem as the photons may attenuate and wander. We start from the fundamental concepts of the non-diffracting beams, and examples of the non-diffracting beams include but are not limited to the Bessel beam, Airy beam, and Mathieu beam. Then, we discuss the biomedical applications of the non-diffracting beams, focusing on linear and nonlinear imaging, e.g., light-sheet fluorescence microscopy and two-photon fluorescence microscopy. The non-diffracting photons may provide scattering resilient imaging and fast speed in the volumetric two-photon fluorescence microscopy. The non-diffracting Bessel beam and the Airy beam have been successfully used in volumetric imaging applications with faster speed since a single 2D scan provides information in the whole volume that adopted 3D scan in traditional scanning microscopy. This is a significant advancement in imaging applications with sparse sample structures, especially in neuron imaging. Moreover, the fine axial resolution is enabled by the self-accelerating Airy beams combined with deep learning algorithms. These additional features to the existing microscopy directly realize a great advantage over the field, especially for recording the ultrafast neuronal activities, including the calcium voltage signal recording. Nonetheless, with the illumination of dual Bessel beams at non-identical orders, the transverse resolution can also be improved by the concept of image subtraction, which would provide clearer images in neuronal imaging.

Fluorescence Probe for Imaging N-Methyl-d-aspartate Receptors and Monitoring GSH Selectively Using Two-Photon Microscopy.

N-Methyl-d-aspartate (NMDA) is an excitotoxic amino acid used to identify a specific subset of glutamate receptors. The activity of NMDA receptors is closely related to the redox level of the biological system. Glutathione (GSH) as an antioxidant plays a key role with regard to modulation of the redox environment. In this work we designed and developed a GSH-specific fluorescent probe with the capability of targeting NMDA receptors, which was composed of a two-photon naphthalimide fluorophore, a GSH-reactive group sulfonamide, and an ifenprodil targeting group for the NMDA receptor. This probe exhibited high selectivity toward GSH in comparison to other similar amino acids. Two-photon fluorescence microscopy allowed this probe to successfully monitor GSH in neuronal cells and hippocampal tissues with an excitation at 750 nm. It could serve as a potential practical imaging tool to explore the function of GSH and related biological processes in the brain.

Two Photon Fluorescence Microscopy of the Unstained Human Cochlea Reveals Organ of Corti Cytoarchitecture.

Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide, and it typically originates from the cochlea. Methods to visualize intracochlear cells in living people are currently lacking, limiting not only diagnostics but also therapies for SNHL. Two-photon fluorescence microscopy (TPFM) is a high-resolution optical imaging technique. Here we demonstrate that TPFM enables visualization of sensory cells and auditory nerve fibers in an unstained, non-decalcified adult human cochlea.

Single-laser-based simultaneous four-wavelength excitation source for femtosecond two-photon fluorescence microscopy.

Multicolor labeling of biological samples with large volume is required for omic-level of study such as the construction of nervous system connectome. Among the various imaging method, two photon microscope has multiple advantages over traditional single photon microscope for higher resolution and could image large 3D volumes of tissue samples with superior imaging depth. However, the growing number of fluorophores for labeling underlines the urgent need for an ultrafast laser source with the capability of providing simultaneous plural excitation wavelengths for multiple fluorophores. Here, we propose and demonstrate a single-laser-based four-wavelength excitation source for two-photon fluorescence microscopy. Using a sub-100 fs 1,070-nm Yb:fiber laser to pump an ultrashort nonlinear photonic crystal fiber in the low negative dispersion region, we introduced efficient self-phase modulation and acquired a blue-shifted spectrum dual-peaked at 812 and 960 nm with 28.5% wavelength conversion efficiency. By compressing the blue-shift near-IR spectrum to 33 fs to ensure the temporal overlap of the 812 and 960 nm peaks, the so-called sum frequency effect created the third virtual excitation wavelength effectively at 886 nm. Combined with the 1,070 nm laser source as the fourth excitation wavelength, the all-fiber-format four-wavelength excitation source enabled simultaneous four-color two-photon imaging in Brainbow AAV-labeled (TagBFP, mTFP, EYFP, and mCherry) brain samples. With an increased number of excitation wavelengths and improved excitation efficiency than typical commercial femtosecond lasers, our compact four-wavelength excitation approach can provide a versatile, efficient, and easily accessible solution for multiple-color two-photon fluorescence imaging in the field of neuroscience, biomolecular probing, and clinical applications with at least four spectrally-distinct fluorophores.

Heterogeneity and Development of Fine Astrocyte Morphology Captured by Diffraction-Limited Microscopy.

The fine processes of single astrocytes can contact many thousands of synapses whose function they can modulate through bi-directional signaling. The spatial arrangement of astrocytic processes and neuronal structures is relevant for such interactions and for the support of neuronal signaling by astrocytes. At the same time, the geometry of perisynaptic astrocyte processes is variable and dynamically regulated. Studying these fine astrocyte processes represents a technical challenge, because many of them cannot be fully resolved by diffraction-limited microscopy. Therefore, we have established two indirect parameters of astrocyte morphology, which, while not fully resolving local geometry by design, provide statistical measures of astrocyte morphology: the fraction of tissue volume that astrocytes occupy and the density of resolvable astrocytic processes. Both are straightforward to obtain using widely available microscopy techniques. We here present the approach and demonstrate its robustness across various experimental conditions using mainly two-photon excitation fluorescence microscopy in acute slices and in vivo as well as modeling. Using these indirect measures allowed us to analyze the morphology of relatively large populations of astrocytes. Doing so we captured the heterogeneity of astrocytes within and between the layers of the hippocampal CA1 region and the developmental profile of astrocyte morphology. This demonstrates that volume fraction (VF) and segment density are useful parameters for describing the structure of astrocytes. They are also suitable for online monitoring of astrocyte morphology with widely available microscopy techniques.

Large-scale, cell-resolution volumetric mapping allows layer-specific investigation of human brain cytoarchitecture.

Although neuronal density analysis on human brain slices is available from stereological studies, data on the spatial distribution of neurons in 3D are still missing. Since the neuronal organization is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than relying on sparse sampling methods. To achieve this goal, we implement a new tissue transformation protocol to clear and label human brain tissues and we exploit the high-resolution optical sectioning of two-photon fluorescence microscopy to perform 3D mesoscopic reconstruction. We perform neuronal mapping of 100mm3 human brain samples and evaluate the volume and density distribution of neurons from various areas of the cortex originating from different subjects (young, adult, and elderly, both healthy and pathological). The quantitative evaluation of the density in combination with the mean volume of the thousands of neurons identified within the specimens, allow us to determine the layer-specific organization of the cerebral architecture.

Visualization of Platelet Integrins via Two-Photon Microscopy Using Anti-transmembrane Domain Peptides Containing a Blue Fluorescent Amino Acid.

The fluorescent reporters commonly used to visualize proteins can perturb both protein structure and function. Recently, we found that 4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable for one-photon imaging applications. Here, we demonstrate its utility in two-photon fluorescence microscopy by using it to image integrins on cell surfaces. Specifically, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their N-termini to image integrins on cell surfaces. CHAMP (computed helical anti-membrane protein) peptides spontaneously insert into membrane bilayers to target integrin transmembrane domains and cause integrin activation. We found that 4CNI labeling did not perturb the ability of CHAMP peptides to insert into membranes, bind to integrins, or cause integrin activation. We then used two-photon fluorescence microscopy to image 4CNI-containing integrins on the surface of platelets. Compared to a 4CNI-labeled scrambled peptide that uniformly decorated cell surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue foci. To confirm that these foci represented CN peptide-containing integrins, we co-stained platelets with integrin-specific fluorescent monoclonal antibodies and found that CN peptide and antibody fluorescence coincided. Because 4CNI can readily be biosynthetically incorporated into proteins with little if any effect on protein structure and function, it provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. In addition, these results clearly demonstrate that the two-photon excitation cross section of 4CN-Trp is sufficiently large to make it a useful two-photon fluorescence reporter for biological applications.

Three-dimensional microvascular network reconstruction from in vivo images with adaptation of the regional inhomogeneity in the signal-to-noise ratio.

Quantification of angiographic images with two-photon laser scanning fluorescence microscopy (2PLSM) relies on proper segmentation of the vascular images. However, the images contain inhomogeneities in the signal-to-noise ratio (SNR) arising from regional effects of light scattering and absorption. The present study developed a semiautomated quantification method for volume images of 2PLSM angiography by adjusting the binarization threshold according to local SNR along the vessel centerlines. A phantom model made with fluorescent microbeads was used to incorporate a region-dependent binarization threshold. The recommended SNR for imaging was found to be 4.2-10.6 that provide the true size of imaged objects if the binarization threshold was fixed at 50% of SNR. However, angiographic images in the mouse cortex showed variable SNR up to 45 over the depths. To minimize the errors caused by variable SNR and a spatial extent of the imaged objects in an axial direction, the microvascular networks were three-dimensionally reconstructed based on the cross-sectional diameters measured along the vessel centerline from the XY-plane images with adapted binarization threshold. The arterial volume was relatively constant over depths of 0-500µm, and the capillary volume (1.7% relative to the scanned volume) showed the larger volumes than the artery (0.8%) and vein (0.6%). The present methods allow consistent segmentation of microvasculature by adapting the local inhomogeneity in the SNR, which will be useful for quantitative comparison of the microvascular networks, such as under disease conditions where SNR in the 2PLSM images varies over space and time.

Low noise, self-phase-modulation-enabled femtosecond fiber sources tunable in 740-1236 nm for wide two-photon fluorescence microscopy applications

We have demonstrated widely tunable Yb:fiber-based laser sources, aiming to replace Ti:sapphire lasers for the nJ-level ultrafast applications, especially for the uses of nonlinear light microscopy. We investigated the influence of different input parameters to obtain an expansive spectral broadening, enabled by self-phase modulation and further reshaped by self-steepening, in the normal dispersion regime before the fiber damage. We also discussed the compressibility and intensity fluctuations of the demonstrated pulses, to reach the transform-limited duration with a very low intensity noise. Most importantly, we have demonstrated clear two-photon fluorescence images from UV-absorbing fluorophores to deep red dye stains.

Two-photon fluorescent probe for cellular peroxynitrite: Fluorescence detection, imaging, and identification of peroxynitrite-specific products

A new naphthalene-based boronate probe, NAB-BE, for the fluorescence-based detection of inflammatory oxidants, including peroxynitrite, hypochlorous acid, and hydrogen peroxide, is reported. The chemical reactivity and fluorescence properties of the probe and the products are described. The major, phenolic oxidation product, NAB-OH, is formed in case of all three oxidants tested. This product shows green fluorescence, with a maximum at 512 nm, and can be excited either at 340 nm or in the near infrared region (745 nm) for two-photon fluorescence imaging. Peroxynitrite is the fastest of the oxidants tested and, in addition to the phenolic product, leads to the formation of a nitrated product, NAB-NO2, which can serve as a fingerprint for peroxynitrite. The probe was applied to detect peroxynitrite in activated macrophages using fluorimetry and two-photon fluorescence microscopy, and both NAB-OH and NAB-NO2 products were detected in cell extracts by liquid chromatography-mass spectrometry. The combined use of fluorometric high-throughput analyses, fluorescence imaging, and liquid chromatography-mass spectrometry-based product identification and quantitation is proposed for most comprehensive and rigorous characterization of oxidants in biological systems.

Autofluorescence enhancement for label-free imaging of myelinated fibers in mammalian brains

Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics. Different technologies try to address this goal; however, they are limited either by the ineffective labeling of the fibers or in the achievable resolution. The possibility of discriminating between different adjacent myelinated axons gives the opportunity of providing more information about the fiber composition and architecture within a specific area. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a tissue preparation method to perform label-free fluorescence imaging of myelinated fibers that is user friendly and easy to handle. We exploit the high axial and radial resolution of two-photon fluorescence microscopy (TPFM) optical sectioning to decipher the mixture of various fiber orientations within the sample of interest. We demonstrate its broad applicability by performing mesoscopic reconstruction at a sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in control and Reeler mouse's hippocampal sections. Our study provides a novel method for 3D label-free imaging of nerve fibers in fixed samples at high resolution, below micrometer level, that overcomes the limitation related to the myelinated axons exogenous labeling, improving the possibility of analyzing brain connectivity.

Synchronously pumped Raman laser for simultaneous degenerate and nondegenerate two-photon microscopy.

Two-photon fluorescence microscopy is a nonlinear imaging modality frequently used in deep-tissue imaging applications. A tunable-wavelength multicolor short-pulse source is usually required to excite fluorophores with a wide range of excitation wavelengths. This need is most typically met by solid-state lasers, which are bulky, expensive, and complicated systems. Here, we demonstrate a compact, robust fiber system that generates naturally synchronized femtosecond pulses at 1050 nm and 1200 nm by using a combination of gain-managed and Raman amplification. We image the brain of a mouse and view the blood vessels, neurons, and other cell-like structures using simultaneous degenerate and nondegenerate excitation.

Improving the Way We See: Adaptive Optics Based Optical Microscopy for Deep-Tissue Imaging

With the recent developments in optical imaging tools and techniques, scientists are now able to image deeper regions of the tissue with greater resolution and accuracy. However, light scattering while imaging deeper regions of a biological tissue remains a fundamental issue. Presence of lipids, proteins and nucleic acids in the tissue makes it inhomogeneous for a given wavelength of light. Two-photon fluorescence (TPF) microscopy supplemented with improved invasive optical tools allows functional imaging in awake behaving mammals in an unprecedented manner. Similarly, improved optical methods conjugated with previously existing scanning laser ophthalmoscopy (SLO) has paved diffraction-limited retinal imaging. With the evolving technology, scientists are now able to resolve biological structures and function at the sub-cellular level. Wavefront correcting methods like adaptive optics (AO) has been implemented in correcting tissue or optical-based distortions, shaping the excitation beam in 3D-holography to target multiple neurons. And more recently, AO-based SLO is implemented for eye imaging both in research and clinical settings. In this review, we discuss some of the recent improvements in TPF microscopy with the application of AO for wavefront corrections and its recent application in brain imaging as well as ophthalmoscopy.

Study of Optical Modulation based on Binary Masks with Finite Pixels

Binary holography has been applied with fast switching digital masks, e.g., digital micromirror device (DMD), to modulate light in recent years for a wide range of optical and scientific applications, such as trapping of cold atoms and 3D random-access two-photon fluorescence microscopy. However, the binarized modulation errors associated with the effect of discrete sampling, finite pixels, and intrinsic noises of algorithms have yet to be systematically investigated. In this paper, we present the development of an analytical model for a general binary mask with finite pixels. The model has established a deterministic link between the quality of the reconstructed wavefront to important parameters of the binary mask, including pixel size, pixel geometry, fill factor, and aperture. Based on the model, three case studies are presented, including (1) beam shaping, (2) 3D random-access scanning, and (3) multi-focus generation based on binary masks. The results show the model can precisely predict the wavefront distortion, power efficiency, position accuracy, and intensity uniformity for multi-focus generation as a function of binary mask parameters. As digital systems are highly repeatable, the predicted errors, e.g., position errors, can be compensated by adding appropriate phase to the designed holograms. The model and the simulation results may provide useful guidance to select appropriate binary devices and optimization algorithms for specific optical engineering applications, e.g., nanofabrication or nonlinear microscopy.

Simultaneous Two-Photon Fluorescence Microscopy of NADH and FAD Using Pixel-to-Pixel Wavelength-Switching

Two-photon fluorescence (TPF) microscopy of intrinsic fluorophores provides physiological and pathological information from biological tissues. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are two endogenous fluorescent coenzymes existing on the intracellular scale. Autofluorescence images of NADH and FAD have been applied to noninvasively record changes during metabolism, according to their distributions and concentrations. However, the widely used sequential (non-simultaneous) excitation scheme results in artifacts caused by sample motion or laser power fluctuation. The single-wavelength illumination scheme suffers from low excitation efficiency and spectral bleed-through. In this paper, we demonstrate a new imaging system simultaneously capturing autofluorescence images from NADH and FAD, with high excitation efficiency and negligible spectral bleed-through. Two temporally multiplexed and spatially overlapped excitation beams were achieved with fast-switching light paths based on an electro-optic modulator. The switching beams were centered at 750 and 860 nm, enabling independent excitations of NADH and FAD. Autofluorescence images of NADH and FAD were acquired at the wavelength ranges of 415–455 nm and 500–550 nm, respectively. The electro-optic modulator was synchronized with the pixel clock from the microscope, achieving pixel-to-pixel wavelength-switching. The capability of the system was demonstrated by performing TPF imaging of freshly excised mouse colon tissues. The microenvironment of the colon wall was depicted by the distributions of colonocytes, goblet cells, and crypts of Lieberkühn, and the relative concentrations of NADH and FAD were estimated. The experimental results show that the system can effectively perform simultaneous imaging of NADH and FAD, and is considered a promising tool for investigations into metabolism-associated processes and diseases.

Treatment with Cyclic AMP Activators Reduces Glioblastoma Growth and Invasion as Assessed by Two-Photon Microscopy.

(1) Background: Despite progress in surgery and radio-chemotherapy of glioblastoma (GB), the prognosis remains very poor. GB cells exhibit a preference for hypoxia to maintain their tumor-forming capacity. Enhancing oxidative phosphorylation—known as the anti-Warburg effect—with cyclic AMP activators has been demonstrated to drive GB cells from proliferation to differentiation thereby reducing tumor growth in a cell culture approach. Here we re-evaluate this treatment in a more clinically relevant model. (2) Methods: The effect of treatment with dibutyryl cyclic AMP (dbcAMP, 1 mM) and the cAMP activator forskolin (50µM) was assessed in a GB cell line (U87GFP+, 104 cells) co-cultured with mouse organotypic brain slices providing architecture and biochemical properties of normal brain tissue. Cell viability was determined by propidium-iodide, and gross metabolic effects were excluded in the extracellular medium. Tumor growth was quantified in terms of area, volume, and invasion at the start of culture, 48 h, 7 days, and 14 days after treatment. (3) Results: The tumor area was significantly reduced following dbcAMP or forskolin treatment (F2,249 = 5.968, p = 0.0029). 3D volumetric quantification utilizing two-photon fluorescence microscopy revealed that the treated tumors maintained a spheric shape while the untreated controls exhibited the GB typical invasive growth pattern. (4) Conclusions: Our data demonstrate that treatment with a cAMP analog/activator reduces GB growth and invasion.

High-speed label-free two-photon fluorescence microscopy of metabolic transients during neuronal activity.

The brain is an especially active metabolic system, requiring a large supply of energy following neuronal activation. However, direct observation of cellular metabolic dynamics associated with neuronal activation is challenging with currently available imaging tools. In this study, an optical imaging approach combining imaging of calcium transients and the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is utilized to track the metabolic dynamics in hippocampal neuron cultures. Results show distinct cellular components for the NAD(P)H response following neuronal activity, where notable differences in the NAD(P)H dynamics between neurons and astrocytes can be directly observed. Additionally, tracking of these responses across a large field of view is demonstrated for metabolic profiling of neuronal activation. Observation of neuronal dynamics using these methods allows for closer examination of the complex metabolic machinery of the brain, and may lead to a better understanding of the cellular metabolism of neuronal activation.

Determination of Hydrogen Sulfide in Endoplasmic Reticulum by Two-Photon Fluorescence

An endoplasmic reticulum (ER) targetable two-photon fluorescent probe termed ER-N3 is presented for the determination of hydrogen sulfide (H2S) in living cells and mice. The results demonstrate that ER-N3 was highly selective and sensitive to H2S. Confocal fluorescence imaging in living cells indicated that ER-N3 was accumulated in the endoplasmic reticulum and visualized exogenous and endogenous H2S production. Moreover, a burst of H2S upregulated by the nitric oxide donor sodium nitroprusside was monitored. Meanwhile, the increase of H2S ended following the addition of DL-propargylglycine, an inhibitor for cystathionine β-synthase and cystathionine γ-lyase that are the main sources of H2S. Importantly, the exogenous H2S was visualized in abdominal tissue of mice by utilizing two-photon fluorescence microscopy. The ER-N3 fluorescent probe may be an excellent tool to image H2S fluctuations in the endoplasmic reticulum and characterize the relationship between H2S and disease in vivo.