Increased nicotinamide adenine dinucleotide pool promotes colon cancer progression by suppressing reactive oxygen species level.

Nicotinamide adenine dinucleotide (NAD) exists in an oxidized form (NAD+ ) and a reduced form (NADH). NAD+ plays crucial roles in cancer metabolism, including in cellular signaling, energy production and redox regulation. However, it remains unclear whether NAD(H) pool size (NAD+ and NADH) could be used as biomarker for colon cancer progression. Here, we showed that the NAD(H) pool size and NAD+ /NADH ratio both increased during colorectal cancer (CRC) progression due to activation of the NAD+ salvage pathway mediated by nicotinamide phosphoribosyltransferase (NAMPT). The NAMPT expression was upregulated in adenoma and adenocarcinoma tissues from CRC patients. The NADH fluorescence intensity measured by two-photon excitation fluorescence (TPEF) microscopy was consistently increased in CRC cell lines, azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC tissues and tumor tissues from CRC patients. The increases in the NAD(H) pool inhibited the accumulation of excessive reactive oxygen species (ROS) levels and FK866, a specific inhibitor of NAMPT, treatment decreased the CRC nodule size by increasing ROS levels in AOM/DSS mice. Collectively, our results suggest that NAMPT-mediated upregulation of the NAD(H) pool protects cancer cells against detrimental oxidative stress and that detecting NADH fluorescence by TPEF microscopy could be a potential method for monitoring CRC progression.

A Ratiometric Two-Photon Fluorescent Probe for Tracking Lysosomal ATP: Direct In†Cellulo Observation of Lysosomal Membrane Fusion Processes.

Vesicles exchange their contents through membrane fusion processes, kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We report a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging, the lysosomal membrane fusion process in cells has been directly observed and the concentration of its content, lysosomal ATP, has been measured. Results show that the kiss-and-run process between lysosomes proceeds through repeated transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.

Two-Photon Absorbing Dyes with Minimal Autofluorescence in Tissue Imaging: Application to in Vivo Imaging of Amyloid-ß Plaques with a Negligible Background Signal.

Fluorescence imaging of tissues offer an essential means for studying biological systems. Autofluorescence becomes a serious issue in tissue imaging under excitation at UV-vis wavelengths where biological molecules compete with the fluorophore. To address this critical issue, a novel class of fluorophores that can be excited at ∼900 nm under two-photon excitation conditions and emits in the red wavelength region (≥600 nm) has been disclosed. The new π-extended dipolar dye system shows several advantageous features including minimal autofluorescence in tissue imaging and pronounced solvent-sensitive emission behavior, compared with a widely used two-photon absorbing dye, acedan. As an important application of the new dye system, one of the dyes was developed into a fluorescent probe for amyloid-ß plaques, a key biomarker of Alzheimer's disease. The probe enabled in vivo imaging of amyloid-ß plaques in a disease-model mouse, with negligible background signal. The new dye system has great potential for the development of other types of two-photon fluorescent probes and tags for imaging of tissues with minimal autofluorescence.

Toward a selective, sensitive, fast-responsive, and biocompatible two-photon probe for hydrogen sulfide in live cells.

Hydrogen sulfide has emerged as an exciting endogenous gasotransmitter in addition to nitric oxide and carbon dioxide. Noninvasive detection methods for hydrogen sulfide thus become indispensable tools for studying its diverse roles in biological systems. Accordingly, fluorescent probes for hydrogen sulfide have received great attention in recent years. A practically useful fluorescent probe for bioimaging of hydrogen sulfide should be selective, sensitive, fast-responsive, biocompatible, observable in the biological optical window, and capable of deep-tissue imaging. These sensing properties, however, are extremely difficult to achieve at the same time. Disclosed here is the two-photon fluorescent probe that meets all of these criteria. The probe belongs to a Michael acceptor system, which raised a serious selectivity issue over the competing biothiols such as cysteine and glutathione. We have addressed the selectivity issue by optimizing the electronic and steric interactions between biothiols and the probe, in addition to achieving very high sensitivity, fast-response, and biocompatibility. Also, the sensing mechanism suggested in the literature was revised. The probe thus enables us to image the endogenously produced hydrogen sulfide with negligible interference from other biothiols in live cells. The excellent sensing properties of the probe combined with its capability of bioimaging thus make it a practically useful tool for further studying biological roles of hydrogen sulfide.

Dark-field polarization-sensitive optical coherence tomography.

Polarization-sensitive optical coherence tomography (PS-OCT) is a functional OCT providing both structural and birefringent information of the sample, and it has been applied to the studies of various organs having polarization properties. Fiber-based PS-OCT is sensitive to specular reflection from the sample surface, because signal saturation due to the strong specular reflection can make the polarization measurement difficult. We developed a dark-field PS-OCT which can avoid the specular reflection problem. Dark-field PS-OCT was implemented by adapting a hybrid method of Bessel-beam illumination and Gaussian-beam detection, and a PS-OCT method based on passive delay unit (PDU). The new system was characterized in comparison with the conventional Gaussian-beam based method in both polarization components and various samples including the human skin. Dark-field PS-OCT performed as good as the conventional PS-OCT without the specular reflection artifact. Dark-field PS-OCT may be useful in practical situations where the specular reflection is unavoidable.

Gradient index lens based combined two-photon microscopy and optical coherence tomography.

We report a miniaturized probe-based combined two-photon microscopy (TPM) and optical coherence tomography (OCT) system. This system is to study the colorectal cancer in mouse models by visualizing both cellular and structural information of the colon in 3D with TPM and OCT respectively. The probe consisted of gradient index (GRIN) lenses and a 90° reflecting prism at its distal end for side-viewing, and it was added onto an objective lens-based TPM and OCT system. The probe was 2.2 mm in diameter and 60 mm in length. TPM imaging was performed by raster scanning of the excitation focus at the imaging speed of 15.4 frames/s. OCT imaging was performed by combining the linear sample translation and probe rotation along its axis. This miniaturized probe based dual-modal system was characterized with tissue phantoms containing fluorescent microspheres, and applied to image mouse colonic tissues ex vivo as a demonstration. As OCT and TPM provided structural and cellular information of the tissues respectively, this probe based multi-modal imaging system can be helpful for in vivo studies of preclinical animal models such as mouse colonic tumorigenesis.

Unique photothermal response and sustained photothermal effect of pH-responsive gold-nanoparticle aggregates.

Hot gold: The photothermal response upon pulsed laser irradiation is studied for pH-responsive gold-nanoparticle aggregates and compared to that of gold nanorods. The aggregates show a slight red shift in the absorption spectrum and retain the photothermal effect, whereas the nanorods lose the photothermal effect and exhibit a stark blue shift in the absorption.

Combined two-photon microscopy and optical coherence tomography using individually optimized sources.

The combination of two-photon microscopy (TPM) and optical coherence tomography (OCT) is useful in conducting in-vivo tissue studies, because they provide complementary information regarding tissues. In the present study, we developed a new combined system using separate light sources and scanners for individually optimal imaging conditions. TPM used a Ti-Sapphire laser and provided molecular and cellular information in microscopic tissue regions. Meanwhile, OCT used a wavelength-swept source centered at 1300 nm and provided structural information in larger tissue regions than TPM. The system was designed to do simultaneous imaging by combining light from both sources. TPM and OCT had the field of view values of 300 µm and 800 µm on one side respectively with a 20x objective. TPM had resolutions of 0.47 µm and 2.5 µm in the lateral and axial directions respectively, and an imaging speed of 40 frames/s. OCT had resolutions of 5 µm and 8 µm in lateral and axial directions respectively, a sensitivity of 97dB, and an imaging speed of 0.8 volumes per second. This combined system was tested with simple microsphere specimens, and was then applied to image small intestine and ear tissues of mouse models ex-vivo. Molecular, cellular, and structural information of the tissues were visualized using the proposed combined system.

Three-photon tissue imaging using moxifloxacin.

Moxifloxacin is an antibiotic used in clinics and has recently been used as a clinically compatible cell-labeling agent for two-photon (2P) imaging. Although 2P imaging with moxifloxacin labeling visualized cells inside tissues using enhanced fluorescence, the imaging depth was quite limited because of the relatively short excitation wavelength (<800 nm) used. In this study, the feasibility of three-photon (3P) excitation of moxifloxacin using a longer excitation wavelength and moxifloxacin-based 3P imaging were tested to increase the imaging depth. Moxifloxacin fluorescence via 3P excitation was detected at a >1000 nm excitation wavelength. After obtaining the excitation and emission spectra of moxifloxacin, moxifloxacin-based 3P imaging was applied to ex vivo mouse bladder and ex vivo mouse small intestine tissues and compared with moxifloxacin-based 2P imaging by switching the excitation wavelength of a Ti:sapphire oscillator between near 1030 and 780 nm. Both moxifloxacin-based 2P and 3P imaging visualized cellular structures in the tissues via moxifloxacin labeling, but the image contrast was better with 3P imaging than with 2P imaging at the same imaging depths. The imaging speed and imaging depth of moxifloxacin-based 3P imaging using a Ti:sapphire oscillator were limited by insufficient excitation power. Therefore, we constructed a new system for moxifloxacin-based 3P imaging using a high-energy Yb fiber laser at 1030 nm and used it for in vivo deep tissue imaging of a mouse small intestine. Moxifloxacin-based 3P imaging could be useful for clinical applications with enhanced imaging depth.

Two-photon microscopy of Paneth cells in the small intestine of live mice.

Paneth cells are one of the principal epithelial cell types in the small intestine, located at the base of intestinal crypts. Paneth cells play key roles in intestinal host-microbe homeostasis via granule secretion, and their dysfunction is implicated in pathogenesis of several diseases including Crohn's disease. Despite their physiological importance, study of Paneth cells has been hampered by the limited accessibility and lack of labeling methods. In this study, we developed a simple in vivo imaging method of Paneth cells in the intact mouse small intestine by using moxifloxacin and two-photon microscopy (TPM). Moxifloxacin, an FDA-approved antibiotic, was used for labeling cells and its fluorescence was strongly observed in Paneth cell granules by TPM. Moxifloxacin labeling of Paneth cell granules was confirmed by molecular counterstaining. Comparison of Paneth cells in wild type, genetically obese (ob/ob), and germ-free (GF) mice showed different granule distribution. Furthermore, Paneth cell degranulation was observed in vivo. Our study suggests that TPM with moxifloxacin labeling can serve as a useful tool for studying Paneth cell biology and related diseases.

In vivo longitudinal visualization of bone marrow engraftment process in mouse calvaria using two-photon microscopy.

Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level. However, the current method used to access the mouse calvaria allows for only a few imaging times in the same mouse because of scar formation and inflammation induced by multiple surgeries. Longitudinal imaging of the BM may help better understand its microenvironment. In this study, a mouse calvarial window model was developed for longitudinal imaging that involves attaching a cover glass window onto the mouse calvaria and sealing the surrounding exposed area with cyanoacrylate glue and dental cement. The model was used for the longitudinal two-photon microscopy (TPM) imaging of the BM engraftment process. The same BM cavity sites were imaged multiple times over 4 weeks after BM transplantation (BMT). Temporal changes in the BM microenvironment, such as the reconstitution of transplanted BM cells and the recovery of vasculature, were observed and analysed qualitatively and quantitatively. Longitudinal intravital microscopy using the mouse calvarial window model was successfully demonstrated and may be useful for further BM studies.

Brain tumor delineation enhanced by moxifloxacin-based two-photon/CARS combined microscopy.

Delineating brain tumor margin is critical for maximizing tumor removal while sparing adjacent normal tissue for better clinical outcome. We describe the use of moxifloxacin-based two-photon (TP)/coherent anti-Stokes Raman scattering (CARS) combined microscopy for differentiating normal mouse brain tissue from metastatic brain tumor tissue based on histoarchitectural and biochemical differences. Moxifloxacin, an FDA-approved compound, was used to label cells in the brain, and moxifloxacin-based two-photon microscopy (TPM) revealed tumor lesions with significantly high cellular density and invading edges in a metastatic brain tumor model. Besides, label-free CARS microscopy showed diminishing of lipid signal due to the destruction of myelin at the tumor site compared to a normal brain tissue site resulting in a complementary contrast for tumor detection. This study demonstrates that moxifloxacin-based TP/CARS combined microscopy might be advantageous for tumor margin identification in the brain that has been a long-standing challenge in the operating room.

Two-Photon In Vivo Imaging with Porous Silicon Nanoparticles.

A major obstacle in luminescence imaging is the limited penetration of visible light into tissues and interference associated with light scattering and autofluorescence. Near-infrared (NIR) emitters that can also be excited with NIR radiation via two-photon processes can mitigate these factors somewhat because they operate at wavelengths of 650-1000 nm where tissues are more transparent, light scattering is less efficient, and endogenous fluorophores are less likely to absorb. This study presents photolytically stable, NIR photoluminescent, porous silicon nanoparticles with a relatively high two-photon-absorption cross-section and a large emission quantum yield. Their ability to be targeted to tumor tissues in vivo using the iRGD targeting peptide is demonstrated, and the distribution of the nanoparticles with high spatial resolution is visualized.

In vivo characterization of early-stage radiation skin injury in a mouse model by two-photon microscopy.

Ionizing radiation (IR) injury is tissue damage caused by high energy electromagnetic waves such as X-ray and gamma ray. Diagnosis and treatment of IR injury are difficult due to its characteristics of clinically latent post-irradiation periods and the following successive and unpredictable inflammatory bursts. Skin is one of the many sensitive organs to IR and bears local injury upon exposure. Early-stage diagnosis of IR skin injury is essential in order to maximize treatment efficiency and to prevent the aggravation of IR injury. In this study, early-stage changes of the IR injured skin at the cellular level were characterized in an in vivo mouse model by two-photon microscopy (TPM). Various IR doses were applied to the mouse hind limbs and the injured skin regions were imaged daily for 6 days after IR irradiation. Changes in the morphology and distribution of the epidermal cells and damage of the sebaceous glands were observed before clinical symptoms. These results showed that TPM is sensitive to early-stage changes of IR skin injury and may be useful for its diagnosis.

A Dipolar Anthracene Dye: Synthesis, Optical Properties and Two-photon Tissue Imaging.

Two-photon microscopy is a powerful tool for studying biological systems. In search of novel two-photon absorbing dyes for bioimaging, we synthesized a new anthracene-based dipolar dye (anthradan) and evaluated its two-photon absorbing and imaging properties. The new anthradan, 9,10-bis(o-dimethoxy-phenyl)-anthradan, absorbs and emits at longer wavelengths than acedan, a well-known two-photon absorbing dye. It is also stable under two-photon excitation conditions and biocompatible, and thus used for two-photon imaging of mouse organ tissues to show bright, near-red fluorescence along with negligible autofluorescence. Such an anthradan thus holds promise as a new class of two-photon absorbing dyes for the development of fluorescent probes and tags for biological systems.

Synthesis and Characterization of Water-Soluble Conjugated Oligoelectrolytes for Near-Infrared Fluorescence Biological Imaging.

Near-infrared (NIR) fluorophores attract increasing attention as a molecular marker (or probe) for in vivo and in vitro biological fluorescence imaging. Three types of new NIR fluorescent conjugated oligoelectrolytes (COEs: Q-FlTBTTFl, Q-FlBBTFl, and Q-FlTBBTTFl) are synthesized with quaternized ammonium ionic groups in their side-chains for water solubility. The emission wavelength is modulated in the range 600-1300 nm, by adjusting the intramolecular charge transfer in the molecular backbone based on the electron-rich fluorene (and/or thiophene) and electron-deficient benzo[2,1,3]thiadiazole (or benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole) moieties. The COEs show a remarkably larger Stokes shift (147-276 nm) compared to commercial rhodamine and cyanine dyes in water, avoiding self-quenching and interference from the excitation backscattered light. The photoluminescence (PL) quantum efficiency is improved substantially by up to 27.8% in water by fabricating a vesicular complex, COE/v, with a block ionomer, poly[(ethylene oxide)-block-(sodium 2-acrylamido-2-methyl-1-propanesulfonate)]. In vitro cellular uptake images with the COEs are obtained with good biocompatibility by confocal single-photon and two-photon microscopy. The ex vivo and in vivo images of a mouse xenograft model treated with the Q-FlBBTFl/v exhibit a substantially stronger fluorescence signal at the tumor site than at the other organs, highlighting the potential of the COE/v as an NIR fluorescent imaging agent for the diagnosis of cancer.

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging.

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

In vivo longitudinal cellular imaging of small intestine by side-view endomicroscopy.

Visualization of cellular dynamics in the gastrointestinal tract of living mouse model to investigate the pathophysiology has been a long-pursuing goal. Especially, for chronic disease such as Crohn's disease, a longitudinal observation of the luminal surface of the small intestine in the single mouse is highly desirable to investigate the complex pathogenesis in sequential time points. In this work, by utilizing a micro-GRIN lens based side-view endomicroscope integrated into a video-rate confocal microscopy system, we successfully performed minimally-invasive in vivo cellular-level visualization of various fluorescent cells and microvasculature in the small intestinal villi. Also, with a transgenic mouse universally expressing photoconvertible protein, Kaede, we demonstrated repetitive cellular-level confocal endoscopic visualization of same area in the small intestinal lumen of a single mouse, which revealed the continuous homeostatic renewal of the small intestinal epithelium.

In vivo wide-field reflectance/fluorescence imaging and polarization-sensitive optical coherence tomography of human oral cavity with a forward-viewing probe.

We report multimodal imaging of human oral cavity in vivo based on simultaneous wide-field reflectance/fluorescence imaging and polarization-sensitive optical coherence tomography (PS-OCT) with a forward-viewing imaging probe. Wide-field reflectance/fluorescence imaging and PS-OCT were to provide both morphological and fluorescence information on the surface, and structural and birefringent information below the surface respectively. The forward-viewing probe was designed to access the oral cavity through the mouth with dimensions of approximately 10 mm in diameter and 180 mm in length. The probe had field of view (FOV) of approximately 5.5 mm in diameter, and adjustable depth of field (DOF) from 2 mm to 10 mm by controlling numerical aperture (NA) in the detection path. This adjustable DOF was to accommodate both requirements for image-based guiding with high DOF and high-resolution, high-sensitivity imaging with low DOF. This multimodal imaging system was characterized by using a tissue phantom and a mouse model in vivo, and was applied to human oral cavity. Information of surface morphology and vasculature, and under-surface layered structure and birefringence of the oral cavity tissues was obtained. These results showed feasibility of this multimodal imaging system as a tool for studying oral cavity lesions in clinical applications.

Microneedle biosensor for real-time electrical detection of nitric oxide for in situ cancer diagnosis during endomicroscopy.

A dual-diagnostic system of endom-icroscope and microneedle sensor is developed to demonstrate high-resolution imaging combined with electrical real-time detection of NO released from cancer tissues. The dual-diagnostic system can be a new platform for facile, precise, rapid, and accurate detection of cancers in various biomedical applications.