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π-Extended donor-acceptor (D-A)-type molecules, which bear both electron-donor and electron-acceptor substituents on the backbone, exhibit unique optical properties, such as bathochromic shifts in absorption and emission, large Stokes shifts, solvatochromic behavior, and fluorescence quenching in polar solvents. These unique properties are attributed to intramolecular charge transfer (ICT) or twisted intramolecular charge transfer (TICT) in the ground and excited states. This review article introduces three types of D-A-type molecules that are used as detection reagents for (1) methanol, (2) amino acids during solid-phase peptide synthesis (SPPS), and (3) amines present in the biological environment. For methanol detection, D-A-type fluorophores with basic guanidine moieties were developed to differentiate between methanol (MeOH) and ethanol (EtOH) based on the small difference in their pKa values (ΔpKa=0.4). Selective protonation of the guanidine moiety in methanol disrupts the D-A structure, allowing emission in the resultant polar environment. Similarly, an acid-base reaction between the hydrogen chloride (HCl) salts of the D-A-type molecules and amines is applied to detect amines during SPPS. In this method, a colorless solution of an HCl salt of the D-A-type molecule is deprotonated by amines, forming a yellow solution. This is the first reported quantitative and non-destructive colorimetric method for detecting amines. Finally, a turn-on-type amine-labeling reagent was developed for the nucleophilic aromatic substitution (S N Ar) reaction. This new reagent enables protein staining of living cells with a large Stokes shift and without solvent-polarity-dependent fluorescence quenching.

Fluorescent imaging sensors based on genetically-encoded and biocompatible proteins have become important tools in medical and biological research due to their high spatiotemporal resolution and ease of use. Protein engineering has led to the development of imaging sensors that visualize changes in the concentration of various target molecules/ions, such as calcium ions. In addition, the development of chemigenetic sensors based on complexes of proteins and synthetic molecules has been gaining momentum in recent years. In this article, the latest research trends in the development of these imaging sensors are introduced, with focus on the sensors developed by our group.

Dendrites receive excitatory synaptic inputs from upstream cell ensembles to trigger action potentials at the cell body. The efficiency of excitatory synaptic inputs on neuronal output depends on the spatiotemporal pattern of synaptic inputs. However, technical limitations still make it unclear how synaptic inputs are organized along dendrites in both space and time. Spine calcium imaging, which records synaptic inputs as calcium transients at individual spines using calcium ion-sensitive fluorophores, is a unique method for studying the spatiotemporal patterns of synaptic input. We developed a functional multiple-spine calcium imaging (fMsCI) that combines whole-cell patch-clamp recording and spinning-disk confocal imaging to observe hundreds of synaptic inputs simultaneously. Using this method, we discovered sequential synaptic inputs that accompanied sharp wave ripple oscillations. In this review, I will discuss the function of sequential synaptic inputs and the potential uses of fMsCI to better understand neurological disorders.

Ice cores recovered from ice sheets contain microorganisms that were transported to the surface of the ice sheet along with airborne dust. The abundance of microorganisms in ice cores, as measured by direct cell count under a fluorescence microscope, is generally lower than that in other aquatic environments. It is possible that the abundance of microorganisms in ice cores is underestimated due to the degradation of fluorescent dye or because non-specific fluorescence from mineral particles inhibits the fluorescence signal from microorganisms. To investigate this possibility, we tested 4 types of antifade solution and 19 types of fluorescence dye, and selected the best combination in terms of prolonging the fluorescent signal from microorganisms and distinguishing biological signals from noise produced by non-specific particles. The results show that the optimal combination of solution and dye for cell counts of scarce microorganisms in ice cores is the nuclear acid dye "YOYO-1" (Molecular Probes®) and the antifade solution "EverBrite Mounting Medium" (Biotium). [ABSTRACT FROM AUTHOR]

Glutamate receptors mediate excitatory neurotransmission in the central nervous system, which have essential roles in our learning and memory. Recent studies have revealed that the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptors (AMPA receptors) are dynamically regulated during synaptic plasticity, the cellular basis of learning and memory. Conventionally, biochemical methods such as surface-biotin labeling or genetic incorporation of fluorescent proteins have been utilized to analyze the AMPA receptors dynamics. However, conflicting findings have been reported because of serious issues in these conventional methods. As the alternative, we have developed a new method for labeling AMPA receptors endogenously expressed in neurons by chemical approaches. This is based on a covalent chemical labeling strategy driven by selective ligand-protein recognition to tether small fluorophores to the target receptors, termed ligand-directed acyl imidazole chemistry. This method has successfully visualized AMPA receptors endogenously expressed in neurons. However, the original method required several hours for fluorophore labeling, which hampered analyzing the dynamics of AMPA receptors in detail. As the alternative, we have recently developed an improved strategy for rapid and selective labeling of chemical probes to cell-surface AMPA receptors by combining ligand-directed chemistry and bio-orthogonal click chemistry. This method allowed to quantify their trafficking, which revealed unique features of AMPA receptors such as long lifetime and rapid recycling in neurons. Notably, this method can be expanded to other receptors. Thus, the two-step labeling method would be a useful tool for understanding the physiological or pathophysiological roles of glutamate receptors in neurons.

In this paper, novel rapid cancer imaging techniques using activatable fluorescent probes are showcased, whose fluorescence characteristics are significantly altered to distinguish between cancer sites, which was developed by using unique probe precision design methods established by the authors. The strategy is to develop probes that target enzymes whose activity has been reported to be enhanced in cancer sites, or to find the most suitable probes from a group of developed probes by screening using actual clinical specimens. Several medical technologies have been developed that enable selective detection of cancer sites within minutes by simply spraying the probes on suspected cancer sites. In addition, it has recently become clear that simultaneous imaging of multiple target enzyme activities can not only visualize the lesion site but also distinguish between malignant and benign lesions. It is highly expected that the day will soon come when surgeons will be able to clearly determine the cancer site to be treated and perform precise endoscopic or open-stomach surgery.

Matrix metalloproteinases (MMPs) regulate various cellular functions, such as motility, invasion, differentiation, and apoptosis. Precise in vivo quantification of MMPs in disease can provide beneficial information for both basic and clinical research studies. To this end, various types of probes have been developed for imaging MMPs in vivo. In this review, representative MMP-targeted probes, such as binding probes and activatable probes, are outlined, including highlights of our own research. In addition, strategies for the development of probes that apply "theranostics," a concept that integrates therapy and diagnostics, are elucidated with reference to [ 18 F]IPFP, a new probe developed in our laboratory. [ 18 F]IPFP was prepared by iodination of a known MMP inhibitor to enhance its affinity and labeled with the compact prosthetic agent 4-nitrophenyl 2-[ 18 F]fluoropropionate ([ 18 F]NFP) for MMP-targeted positron-emission tomography (PET) and other therapeutic properties. IPFP demonstrated high inhibitory activity toward MMP-12 (IC 50 value=1.5 nM). Radioactivity accumulation in the lungs 90 min after administration of [ 18 F]IPFP was 4-fold higher in chronic obstructive pulmonary disease (COPD) mice overexpressing MMPs compared with normal mice. Ex vivo PET confirmed the radioactivity distribution in tissues, and autoradiography analysis demonstrated accumulation differences between COPD and normal mice. Consequently, [ 18 F]IPFP showed potent inhibitory activities against MMPs and suitable pharmacokinetics for imaging pulmonary disease. Thus, [ 18 F]IPFP is a promising theranostic probe for pulmonary disease and is expected to be applied to various other MMP-related diseases. Strategies for MMP probe development introduced in this review are anticipated to lead to the development of superior imaging probes in the future.

The author has developed several methodological approaches that use nanophotonic and microfluidic devices to accelerate pharmaceutical research and development. Here, the author describes two of these approaches and provides practical examples. The first is a nanophotonic approach to break the concentration limit of diffusing fluorophore-labeled molecules in single-molecule imaging. Although single-molecule imaging is highly useful in characterizing the kinetics of biomolecular interactions, it requires nanomolar concentrations of labeled molecules in solution. Zero-mode waveguides are nanophotonic structures that reduce the illumination volume by more than three orders of magnitude relative to conventional fluorescence microscopy, thereby allowing single-molecule investigations at micromolar to millimolar concentrations of fluorescent molecules i.e., under near-physiological conditions. The second approach is microfluidic microdroplet-based, allowing the discovery of novel biomolecules with the desired activities. Microfluidics allows the ultrarapid production of monodisperse microdroplets such as water-in-oil microdroplets. Each microdroplet serves as a nano/picoliter-volume test tube, which increases assay sensitivity by increasing the effective concentration of molecules and decreasing the time required to reach detection thresholds. I hope you find this review helpful in your research.

Gastric proton pump (H + ,K + -ATPase) which is responsible for H + secretion of gastric acid (HCl) in gastric parietal cells is the major therapeutic target for treatment of acid-related diseases. H + ,K + -ATPase consists of two subunits, a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). N-glycosylation of βHK is essential for trafficking and stability of αHK in apical membrane of gastric parietal cells. Terminal sialic acid residues on sugar chains have an important role in various cellular functions. Recently, we succeeded in visualizing the sialylation and desialylation dynamics of βHK using a fluorescence bioimaging nanoprobe consisting of biocompatible polymers conjugated with lectins for detecting sialic acid. In H + ,K + -ATPase-expressing cell lines, rat gastric mucosa, and primary culture of rat gastric parietal cells, fluorescence imaging of sialic acid with the nanoprobe showed that sialylation of βHK is regulated by intragastric pH and that inhibition of gastric acid secretion induces desialylation of βHK. In biochemical and pharmacological studies, we revealed that enzyme activity of αHK is negatively regulated by desialylation of βHK. Our studies uncovered a novel negative-feedback mechanism of H + ,K + -ATPase in which sialic acids of βHK positively regulates H + ,K + -ATPase activity, and acidic pH decreases the pump activity by cleaving sialic acids of βHK. In this topic, we introduce the overview of our research using the bioimaging nanoprobe.

Target identification (target-ID) is an important step in elucidating the mechanisms of action of bioactive small molecules. In the past few decades, a number of target-ID methods have been developed. Among these, affinity labeling has been reliably used for specific modifications, as well as for the identification of weakly interacting protein targets, membrane-associated protein targets, and target-interacting proteins under native cellular conditions, which are generally difficult to achieve by conventional pull-down methods. In general, affinity labeling utilizes chemical probes composed of a bioactive small molecule, a reactive group, and a detection unit. However, the design and synthesis of highly functionalized chemical probes is often time-consuming. To address this issue, we have recently developed some simple affinity labeling methods using small fluorogenic tags, such as 4-alkoxy-7-nitro-2,1,3-benzoxadiazole (O-NBD), 2,3-dichloromaleimide (diCMI), and 4-azidophthalimide (AzPI), and successfully achieved the specific fluorescent labeling of target proteins, even in living cells. These methods should be useful for target-ID in phenotypic drug discovery.

Hydrogen sulfide (H 2 S) has been reported to play an important role in biological systems. More recently, sulfane sulfur (sulfur with 0 or -1 charge) molecules have been also reported to be involved in various biological phenomena such as regulation of redox signaling and antioxidant functions. Fluorescent probes are one of the important chemical tools because it is easy to use and enable the real-time detection of the target molecules in living cells and tissues. We have successfully developed a highly selective H 2 S-detecting fluorescent probe, HSip-1. HSip-1 has been designed on the basis of the facts that the macrocyclic polyamine ligands form a stable complex with Cu 2+ , and Cu 2+ also reacts with H 2 S and make a stable CuS complex. SSip-1 is a fluorescent probe for detecting sulfane sulfur and this fluorescent probe is designed on the basis of the unique feature of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. SSip-1 is a highly selective fluorescent probe and can detect sulfane sulfur reversibly. Both HSip-1 and SSip-1 were able to be used for the live-cell fluorescence imaging. Further, we applied HSip-1 to the high-throughput screening (HTS) for the inhibitors of 3-mercaptopyruvate sulfurtransferase (3MST), one of the reactive sulfur species (RSS)-generating enzymes. We successfully found new 3MST inhibitors by screening of 174,118 compounds. We expect that these fluorescent probes and inhibitors would be useful to elucidate new functions of RSS and RSS-generating enzymes.

An 81-year-oldwoman with advancedgastric cancer was referredto our hospital. Preoperative contrast-enhancedCT revealeda roundcalcification of the splenic hilum with 15mm in diameter as a splenic artery aneurysm. She underwent transcatheter arterial embolization(TAE)for the splenic artery aneurysm. Celiac artery angiography showedcollateral arterial network of the spleen from left gastric artery. Surgery for the gastric cancer was performed1 4 days after TAE. We cut the right gastric andbilateral epigastric arteries. After the left gastric artery clamping, we performedintraoperative indocyanine green(ICG)fluorography. ICG fluorography confirmedthat the bloodflow of the upper thirdof the stomach andspleen were maintained. We safely performed distal gastrectomy, and the postoperative course was uneventful.

Nerve-preservation technique during surgery is important. Intraoperative nerve injury often causes permanent palsy or numbness and/or neurogenic functional disorders. To evade such intraoperative nerve injuries, we proposed a novel manner to specifically visualize peripheral nerve fibers. Low-toxic agents clinically available, amphotericin B(AmB)or fluorescein isothiocyanate(FITC)were used as neuro-indicators. In in vitro, we used Schwann cells as nerve models to basically confirm these agents effectively functioned as neural markers. In in vivo, we examined whether this novel method was clinically applicable. The Schwann cells reacted with AmB or FITC emitted blue or yellow-green fluorescence in a dark environment. The rat nerve models also fluorescently glimmered in blue-tone when each agent was given. These data suggested that we could clinically discriminate nerve fibers from the surrounding tissues. Our fluorescent-imaging methods warrant further studies for clinical applications.

Osteoclasts are bone-resorbing giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. We have originally established an advanced imaging system for visualizing in vivo behavior of osteoclasts and their precursors with intravital two-photon microscopy. By means of the system, we found that sphingosine-1-phosphate, a lipid mediator enriched in blood, controlled the migratory behavior of osteoclast precursors. We also developed pH-sensing chemical fluorescent probes to detect localized acidification by bone-resorbing osteoclasts on the bone surface in vivo, and identified two distinct functional states of differentiated osteoclasts, 'bone-resorptive' and 'non-resorptive'. In this review, we summarize our recent studies on the dynamics and functions of osteoclasts. Our intravital imaging techniques would be beneficial for studying the cellular dynamics in arthritic inflammation and bone destruction in vivo and would thus be useful for evaluating novel therapies targeting aspects of osteoclast dynamics in patients with bone-destructive diseases.

Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19 F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19 F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

Fluorescence imaging is one of the most powerful techniques currently available for continuous observation of intracellular processes in living cells and tissues, and so it would be useful to have a large toolset of fluorescence probes suitable for visualizing bioactive molecules or biological phenomena. However, design strategies for small molecule-based fluorescence probes have been limited. Therefore, we are interested in establishing novel rational design strategies for developing functional fluorescence probes. For this purpose, we have focused on the intramolecular spirocyclization reaction of rhodamine derivatives bearing intramolecular nucleophiles as a fluorescence switching mechanism; these derivatives exist in thermal equilibrium between a fluorescent open form and a non-fluorescent spirocyclic form. By utilizing this reaction, we have successfully developed various kinds of fluorescence probes. This review first describes the design of our recently developed fluorescence probes, and then introduces their applications for visualizing the activities of reporter enzymes, for in vivo cancer imaging, and for super-resolution imaging. We believe these probes can serve as key tools to provide unprecedented insights into a wide range of biological phenomena.

The oxidative modification of low-density lipoprotein (LDL) is believed to play an important role in the pathogenesis of atherosclerosis. Therefore, probes for detection of oxidized LDL (ox-LDL) in atherosclerotic plaques and plasma are expected to be useful for the diagnosis of atherosclerosis. Recently, we found that four fluorescein isothiocyanate (FITC)-labeled heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6)-(FITC)KP6 and (FITC-AC)KP6- and then substitution with D-Lys at the N-terminus-(FITC)dKP6 and (FITC-AC)dKP6- bind with high specificity and high affinity to two oxidized forms of LDL, heavily oxidized LDL and minimally modified LDL (MM-LDL), through binding to lysophosphatidylcholine and oxidized phosphatidylcholine, present abundantly in heavily oxidized LDL and MM-LDL. Moreover, (FITC)dKP6 and (FITC-AC)dKP6 were more stable than (FITC)KP6 and (FITC-AC)KP6 in plasma in vitro. (FITC)KP6 could detect foam cells in atherosclerotic aortic plaques of apoE-knockout mice. These results suggest that four fluorescence-labeled heptapeptides could be efficient fluorescent probes for the specific detection of ox-LDL, and can therefore contribute to the identification, diagnosis, prevention, and treatment of atherosclerosis.

There are several approaches to understanding the physiological roles of biomolecules: (1) by observing the localization or activities of biomolecules (based on microscopic imaging experiments with fluorescent proteins or fluorescent probes) and (2) by investigating the cellular response via activation or suppression of functions of the target molecule (by using inhibitors, antagonists, siRNAs, etc.). In this context, protein-labeling technology serves as a powerful tool that can be used in various experiments, such as for fluorescence imaging of target proteins. Recently, we developed a protein-labeling technology that uses a mutant β-lactamase (a bacterial hydrolase) as the tag protein. In this protein-labeling technology, also referred to as the BL-tag technology, various β-lactam compounds were used as specific ligands that were covalently labeled to the tag. One major advantage of this labeling technology is that various functions can be carried out by suitably designing both the functional moieties such as the fluorophore and the β-lactam ligand structure. In this review, we briefly introduce the BL-tag technology and describe our future outlook for this technology, such as in fluorescence imaging of biomolecules and functional regulation of cellular proteins in living cells.

Flow cytometry (FCM) has been introduced into clinical laboratories to determine the lineage and functional stage of differentiation of hematopoietic tumor cells using multiple immunophenotypic markers. Multi-parametric analysis, which simultaneously measures multiple cellular characteristics, not only provides a large amount of novel information for each sample studied, but also requires a reduced volume of the specimen and avoids redundancies of reagents within antibody panels. Through a series of novel developments in FCM hardware, software, and dye-chemistry, recently developed multicolor flow cytometers are capable of detecting 10 or more fluorochromes simultaneously and expeditiously, taking advantage of not only cluster analysis but also detecting rare cellular populations within complex materials. In this study, we applied the multicolor FCM technology for: i) T-cell lineage assessment in early T-cell precursor acute lymphoblastic leukemia, ii) detection of a rare neoplastic population in angioimmunoblastic T-cell lymphoma composed of a complex mixture of cells, iii) assessment of bone marrow infiltration of B-cell lymphoma, and iv) sensitive detection of multiple myeloma cells with the use of a combination of antibodies, which is applicable to monitoring the response to treatment.

Bone tissue is maintained by dynamic equilibrium of various types of cells. Thus, real time intravital imaging of movement and phenotypic changes of cells in the steady state and in pathophysiological state help us to understand molecular mechanism of bone tissue maintenance. Therefore, this review introduces Fucci-Tg mice for cell cycle imaging, caspase-3 indicator SCAT3.1 mice for cell death, and photoconvertible protein Kaede-Tg mice and cell-type specific KikGR mice for visualization of cell movement within intra-organ and between inter-organs.

Recent advances in intravital bone imaging technology has enabled us to grasp the real cellular behaviors and functions in vivo , revolutionizing the field of drug discovery for novel therapeutics against intractable bone diseases. In this chapter, I introduce various updated information on pharmacological actions of several antibone resorptive agents, which could only be derived from advanced imaging techniques, and also discuss the future perspectives of this new trend in drug discovery.

For the clinical diagnosis of osteoporosis, X-ray CT and biochemical bone metabolism markers, however, there is no method to monitor osteoclast activity in the living system. We have developed the fluorescent probe to monitor osteoclast activity, by the combination of pH sensitive fluorescent dye with bisphosphonate which is delivered to the bone surface by its high affinity to hydroxyl apatite, which is named BAp-E. In vivo mouse imaging of activated osteoclast was successfully demonstrated using two photon excitation laser microscope.

Bone homeostasis is maintained by bone formation and bone resorption. The traditional view of bone metabolism as a primarily endocrine regulation has been expanded in recent years following the identification of nervous system controlling bone metabolism. Especially, sympathetic and parasympathetic nervous system regulates bone formation and bone resorption. In addition, sensory nervous system also has been shown to be involved in the regulation of bone homeostasis. These studies demonstrated that nervous system is closely related to bone remodeling.

Impairment of macrophage phagocytosis is a major cause of chronic inflammation. Bisphosphonates (BPs) are widely used as anti-osteoclastic agents. The effects of BPs on monocyte-macrophage lineage cells are being increasingly reported; however, the detailed effects of BPs on macrophage phagocytic activity are still unclear. We examined the effects of four BPs: clodronate as a non-nitrogen containing BP (non-N-BP), and pamidronate, alendronate, and zoledronate as nitrogen-containing BP(N-BP), on macrophage phagocytic activity. The uptake of high fluorescence-labeled polystyrene beads by the human monocytic cell line THP-1 was investigated by flow cytometry. All three N-BPs suppressed the phagocytosis of macrophages more potently than the non-N-BP, clodronate. Pamidronate and zoledronate were more potent than alendronate. BP induced the apoptosis of THP-1. Pamidronate and zoledronate induced apoptosis more effectively than clodronate. The method described to observe phagocytosis was simple and quantitative, and might be useful in screening for the effects of drugs, such as N-BP and non-N-BP, on phagocytic activity.

Intravital optical imaging technique is a promising method that allows us to investigate complex vital phenomena in vivo . In particular, discovery and development of unique fluorescent proteins and smart fluorescent dyes in conjunction with appropriate equipment such as two-photon microscopy and image processing software allow visualization of the behavior and function of bone and cartilage-related cells as well as the microenvironment of the cells in bone and cartilage in living animals. Here we show recent technological development and issues of the intravital optical imaging and the application of the fluorescent imaging approaches to bone and cartilage biology.

For successful development of cell-based therapy, both the disposition and differentiation of transplanted cells are directly related to therapeutic effects. In vivo imaging is an attractive tool to obtain real-time information on the disposition of target cells. In various types of imaging methods such as positron emission tomography(PET) and magnetic resonance imaging(MRI), fluorescence imaging is suitable for visualizing the disposition of cells because it can visualize single cells both in vitro and in vivo. For the trafficking of stem cells after transplantation, it is necessary to label living cells for long time periods without disturbing the function or differentiation of the labeled cells. Recently, we have developed quantum dots modified with polyamidoamine(PAMAM) dendrimers. These can more rapidly escape from endosomes and sustain their fluorescence intensity compared with unmodified quantum dots in primary cultured mesenchymal stem cells (MSCs). Fluorescence intensity was also sustained after intravenous injection of MSCs labeled with PAMAM dendrimer-modified quantum dots. To study the dynamics of MSCs in vivo, we constructed a piggyBack transposon vector that can integrate the target gene into the genome in mammalian cells, and established primary MSCs with long-term expression of EmGFP. In addition, we also developed a suction device stabilizing tissue for in vivo real time imaging. In this section, I present our recent findings on long-term fluorescent labeling of MSCs and in vivo visualizing of cell dynamics in a living mouse.

Several versatile and rational design strategies for novel fluorescence probes including those based on photoinduced electron transfer have been successfully established. Indeed, based on these design strategies, various novel fluorescence probes were successfully developed including those for reactive oxygen species and reporter enzymes. Furthermore, we have succeeded to visualize tiny tumors in living mice by using cancer-specific antibodies tagged with acidic-pH activatable fluorescence probes. Here, I will describe the details of probe design as well as some imaging results with living cells and animals by applying our novel fluorescence probes.

There has been a growing interest in analyzing complex biology in living animals by in vivo fluorescent imaging. In vivo fluorescent imaging by using novel fluorescent molecular probes and advanced fluorescent microscopy e.g. two-photon microscopy allow us to analyze deep tissues in bone. In this review, we demonstrated our data and discussed about in vivo fluorescent imaging in bone research field.

Fluorescence-derivatization-HPLC methods are powerful tools for performing the analysis of bioactive compounds with high sensitivity and selectivity. In this paper, the author reviews the development of the following four types of novel fluorescence-derivatization-HPLC analytical systems: (1) simultaneous HPLC analysis of melatonin and its related compounds through post-column electrochemical demethylation and fluorescence derivatization, (2) HPLC analysis of 5-hydroxyindoles based on fluorescence derivatization by online pre-column photocatalytic oxidation with benzylamine, (3) reagent peak-free HPLC analysis for aliphatic amines and amino acids using F-trap pyrene as a fluorous tag-bound fluorescence derivatization reagent, and (4) reagent peak-free HPLC analysis for carboxylic acids using a fluorous scavenging-derivatization method. The authors have also successfully applied these systems to biological and pharmaceutical analyses.

Live-cell imaging of cells infected with recombinant viruses expressing fluorescently tagged structural viral proteins enables to visualize virion maturation pathways at a virion particle level in the same cells as infection progresses. This technology have gradually unveiled previously unreported aspects of the pathways. This article focuses on live-cell imaging technology of herpes simplex virus 1 (HSV-1) and reviews the up-to-date topics of HSV-1 maturation pathway including our recent progress.