A Small‐Molecule Two‐Photon Probe for Nitric Oxide in Living Tissues

Two‐photon microscopy (TPM) has become an indispensable tool in the study of biology and medicine due to the capability of this method for molecular imaging deep inside intact tissues. For the maximum utilization of TPM, a variety of two‐photon (TP) probes for specific applications are needed. In this article, we report a small‐molecule TP probe (ANO1) for nitric oxide (NO) that shows a rapid and specific NO response, a 68‐fold fluorescence enhancement in response to NO, and a maximum TP‐action cross‐section of 170 GM (GM: 10^?50 cm⁴ photon^?1) upon reaction with excess NO. This probe can be easily loaded into cells and tissues and can real‐time monitor NO in living tissues at 100–180 μm depth for longer than 1200 s through the use of TPM, with minimum interference from other biologically relevant species.     

Detection of nickel in fish organs with a two-photon fluorescent probe

Molecular imaging by two‐photon microscopy (TPM) has become indispensable to the study of biology/medicine owing to its capability of imaging deep inside intact tissues. To make TPM a more‐versatile tool, a large variety of two‐photon probes are needed. Herein, we report a new two‐photon fluorescent probe (ANi2) that can be excited by 750 nm femtosecond pulses and detect Ni²⁺ ions in fresh fish organs at 90–175 μm depth without interference from the pH value or from other biologically relevant species through the use of TPM. TPM images of fish organs labeled with ANi2 revealed that Ni²⁺ ions accumulate in fish organs in the order: kidney > heart > gill ≥ liver. Moreover, a linear relationship was found between the two‐photon‐excited fluorescence (TPEF) and the inductively coupled plasma mass spectrometry intensities (ICP‐MS), thereby allowing the quantitative measurement of Ni²⁺ ions in live tissue.     

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.     

A Glowing Trajectory between Bio‐ and Chemiluminescence: From Luciferin‐Based Probes to Triggerable Dioxetanes

Bioluminescent and chemiluminescent probes are widely used for noninvasive imaging applications because of their high sensitivity and the simplicity of the equipment required to perform the measurement. Synthetic luciferin‐analogue probes with in vivo imaging performance better than that of luciferin are now available. In addition, caged luciferin‐based bioluminogenic probes have been emerged as a general tool for the visualization of different enzymes and analytes in vivo. Recent discoveries have led to development of highly efficient chemiluminescent probes that are extremely bright under physiological conditions. As discussed in this Minireview, chemiluminescence is ready to realize its potential as a valuable tool for imaging in living systems.     

Iridium(III) Anthraquinone Complexes as Two‐Photon Phosphorescence Probes for Mitochondria Imaging and Tracking under Hypoxia

In the present study, four mitochondria‐specific and two‐photon phosphorescence iridium(III) complexes, Ir1 – Ir4 , were developed for mitochondria imaging in hypoxic tumor cells. The iridium(III) complex has two anthraquinone groups that are hypoxia‐sensitive moieties. The phosphorescence of the iridium(III) complex was quenched by the functions of the intramolecular quinone unit, and it was restored through two‐electron bioreduction under hypoxia. When the probes were reduced by reductase to hydroquinone derivative products under hypoxia, a significant enhancement in phosphorescence intensity was observed under one‐ (λ=405 nm) and two‐photon (λ=720 nm) excitation, with a two‐photon absorption cross section of 76–153 GM at λ=720 nm. More importantly, these probes possessed excellent specificity for mitochondria, which allowed imaging and tracking of the mitochondrial morphological changes in a hypoxic environment over a long period of time. Moreover, the probes can visualize hypoxic mitochondria in 3D multicellular spheroids and living zebrafish through two‐photon phosphorescence imaging.     

Dinuclear Ruthenium(II) Complexes as Two‐Photon,Time‐Resolved Emission Microscopy Probes for Cellular DNA

The first transition‐metal complex‐based two‐photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA‐bound probes display characteristic emission lifetimes of more than 160 ns, while shorter‐lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence‐free imaging.     

Dinuclear Ruthenium(II) Complexes as Two‐Photon,Time‐Resolved Emission Microscopy Probes for Cellular DNA

The first transition‐metal complex‐based two‐photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA‐bound probes display characteristic emission lifetimes of more than 160 ns, while shorter‐lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence‐free imaging.     

Mini‐Sized Carbon Nitride Nanosheets with Double Excitation‐ and pH‐Dependent Fluorescence Behaviors for Two‐Photon Cell Imaging

Synthesis of mini‐sized carbon nitride nanosheets (CNNSs) by traditional methods remains a challenge. Herein, size‐tunable and uniform mini‐sized CNNSs are synthesized by hydrothermal carbonization of a single polyethyleneimine (PEI) precursor. The as‐obtained mini‐sized CNNSs possess uniform size, good hydrophilicity and abundant nitrogen active sites, which not only exhibit double excitation‐ and pH‐dependent fluorescence behaviors, but also two‐photon excitation fluorescence. áThe resulting CNNSs display low toxicity and can be efficiently delivered into live cells for two‐photon fluorescence imaging, offering great potential as fluorescence probes in biochemical applications.     

Large Two‐Photon Absorption of Terpyridine‐Based Quadrupolar Derivatives: Towards their Applications in Optical Limiting and Biological Imaging

Developing organic chromophores with large two‐photon absorption (TPA) in both organic solvents and aqueous media is crucial owing to their applications in solid‐state photonic devices and biological imaging. Herein, a series of novel terpyridine‐based quadrupolar derivatives have been synthesized. The influences of electron‐donating group, type of conjugated bridge, as well as solvent polarity on the molecular TPA properties have been investigated in detail. In contrast to the case in organic solvents, bis(thienyl)‐benzothiadiazole as a rigid conjugated bridge will completely quench molecular two‐photon emission in aqueous media. However, the combination of alkylcarbazole as the donor and bis(styryl)benzene as a conjugation bridge can enlarge molecular TPA cross‐sections in both organic solvent and aqueous media. The reasonable two‐photon emission brightness for the organic nanoparticles of chromophores 3 – 5 in the aqueous media, prepared by the reprecipitation method, enables them to be used as probes for in vivo biological imaging.     

A Theoretical Investigation of Two Typical Two‐Photon pH Fluorescent Probes

Intracellular pH plays an important role in many cellular events, such as cell growth, endocytosis, cell adhesion and so on. Some pH fluorescent probes have been reported, but most of them are one‐photon fluorescent probes, studies about two‐photon fluorescent probes are very rare. In this work, the geometrical structure, electronic structure and one‐photon properties of a series of two‐photon pH fluorescent probes have been theoretically studied by using density functional theory (DFT) method. Their two‐photon absorption (TPA) properties are calculated using the method of ZINDO/sum‐over‐states method. Two types of two‐photon pH fluorescent probes have been investigated by theoretical methods. The mechanisms of the Photoinduced Charge Transfer (PCT) probes and the Photoinduced Electron Transfer (PET) probes are verified specifically. Some designed strategies of good two‐photon pH fluorescent probes are suggested on the basis of the investigated results of two mechanisms. For the PCT probes, substituting a stronger electron‐donating group for the terminal methoxyl group is an advisable choice to increase the TPA cross section. For the PET probes, the TPA cross sections increase upon protonation.     

Rational Design of Fluorescent Phthalazinone Derivatives for One‐ and Two‐Photon Imaging

Phthalazinone derivatives were designed as optical probes for one‐ and two‐photon fluorescence microscopy imaging. The design strategy involves stepwise extension and modification of pyridazinone by 1) expansion of pyridazinone to phthalazinone, a larger conjugated system, as the electron acceptor, 2) coupling of electron‐donating aromatic groups such as N,N‐diethylaminophenyl, thienyl, naphthyl, and quinolyl to the phthalazinone, and 3) anchoring of an alkyl chain to the phthalazinone with various terminal substituents such as triphenylphosphonio, morpholino, triethylammonio, N‐methylimidazolio, pyrrolidino, and piperidino. Theoretical calculations were utilized to verify the initial design. The desired fluorescent probes were synthesized by two different routes in considerable yields. Twenty‐two phthalazinone derivatives were synthesized and their photophysical properties were measured. Selected compounds were applied in cell imaging, and valuable information was obtained. Furthermore, the designed compounds showed excellent performance in two‐photon microscopic imaging of mouse brain slices.     

From One‐Photon to Two‐Photon Probes: “Caged” Compounds,Actuators, and Photoswitches

Molecular systems that can be remotely controlled by light are gaining increasing importance in cell biology, physiology, and neurosciences because of the spatial and temporal precision that is achievable with laser microscopy. Two‐photon excitation has significant advantages deep in biological tissues, but raises problems in the design of “smart” probes compatible with cell physiology. This Review discusses the chemical challenges in generating suitable two‐photon probes.     

Genetically Encoded Activators of Small Molecules for Imaging and Drug Delivery

Chemical biologists have developed many tools based on genetically encoded macromolecules and small, synthetic compounds. The two different approaches are extremely useful, but they have inherent limitations. In this Minireview, we highlight examples of strategies that combine both concepts to tackle challenging problems in chemical biology. We discuss applications in imaging, with a focus on super‐resolved techniques, and in probe and drug delivery. We propose future directions in this field, hoping to inspire chemical biologists to develop new combinations of synthetic and genetically encoded probes.     

Application of Nonlinear Optical Microscopy for Imaging Skin†

Recent advances in the use of nonlinear optical microscopy (NLOM) in skin microscopy are presented. Nonresonant spectroscopies including second harmonic generation, coherent anti‐Stokes Raman and two‐photon absorption are described and applications to problems in skin biology are detailed. These nonlinear techniques have several advantages over traditional microscopy methods that rely on one‐photon excitation: intrinsic 3D imaging with <1 μm spatial resolution, decreased photodamage to tissue samples and penetration depths up to 1000 μm with the use of near‐infrared lasers. Thanks to these advantages, nonlinear optical spectroscopy has become a powerful tool to study the physical and biochemical properties of the skin. Structural information can be obtained using the response of endogenous chemical species in the skin, such as collagen or lipids, indicating that optical biopsy may replace current invasive, time‐consuming traditional histology methods. Insertion of specific probe molecules into the skin provides the opportunity to monitor specific biochemical processes such as skin transport, molecular penetration, barrier homeostasis and ultraviolet radiation‐induced reactive oxygen species generation. While the field is quite new, it seems likely that the use of NLOM to probe structure and biochemistry of live skin samples will only continue to grow.     

光敏开关及其在化学生物学中的应用*

近年来随着生物医学技术的发展,人们需要越来越细致地在分子水平上研究各种生命过程。为了能够实现实时原位地观察活细胞或组织中的生命化学过程,需要使用以物理方法来选择性激活的分子探针。以共聚焦激光技术为基础的光敏开关能很好地解决这一问题。迄今,发展和用于光敏开关的光敏剂已成为化学生物学研究的重要方向。本文重点总结了各种可应用于共聚焦激光系统的单、双光子光敏基团（单光子的光敏基团主要有：硝基苯类、香豆素类等;双光子光敏基团主要有：香豆素类、喹啉类及吲哚衍生物类）以及这几类光敏基团在化学生物学中的应用。     

Organized Aggregation of Porphyrins in Lipid Bilayers for Third Harmonic Generation Microscopy

Nonlinear optical microscopy has become a powerful tool for high‐resolution imaging of cellular and subcellular composition, morphology, and interactions because of its high spatial resolution, deep penetration, and low photo‐damage to tissue. Developing specific harmonic probes is essential for exploiting nonlinear microscopic imaging for biomedical applications. We report an organized aggregate of porphyrins (OAP) that formed within lipidic nanoparticles showing fingerprint spectroscopic properties, structure‐associated second harmonic generation, and superradiant third harmonic generation. The OAP facilitated harmonic microscopic imaging of living cells with significantly enhanced contrast. The structure‐dependent switch between harmonic (OAP‐intact) and fluorescence (OAP‐disrupted) generation enabled real‐time multi‐modality imaging of the cellular fate of nanoparticles. Robustly produced under various conditions and easily incorporated into pre‐formed lipid nanovesicles, OAP provides a biocompatible nanoplatform for harmonic imaging.     

Optical molecular imaging for systems biology: from molecule to organism

The development of highly efficient analytical methods capable of probing biological systems at system level is an important task that is required in order to meet the requirements of the emerging field of systems biology. Optical molecular imaging (OMI) is a very powerful tool for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vivo. In this article, recent advances in OMI are reviewed extensively, such as the development of molecular probes that make imaging brighter, more stable and more informative (e.g., FPs and semiconductor nanocrystals, also referred to as quantum dots), the development of imaging approaches that provide higher resolution and greater tissue penetration, and applications for measuring biological events from molecule to organism level, including gene expression, protein and subcellular compartment localization, protein activation and interaction, and low-mass molecule dynamics. These advances are of great significance in the field of biological science and could also be applied to disease diagnosis and pharmaceutical screening. Further developments in OMI for systems biology are also proposed.     

The Remarkable Effect of Halogen Substitution on the Membrane Transport of Fluorescent Molecules in Living Cells

Small‐molecule‐based fluorescent probes have become important tools in biology for sensing and imaging applications. However, the biological applications of many of the fluorescent molecules are hampered by low cellular uptake and high toxicity. In this paper, we show for the first time that the introduction of halogen atoms enhances the cellular uptake of fluorescent molecules and the nature of halogen atoms plays a crucial role in the plasma membrane transport in mammalian cells. The remarkably higher uptake of iodinated compounds compared to that of their chloro or bromo analogues suggests that the strong halogen bonding ability of iodine atoms may play an important role in the membrane transport. This study provides a novel strategy for the transport of fluorescent molecules across the plasma membrane in living cells.     

Raman Spectroscopy and Fluorescence Photon Migration for Breast Cancer Diagnosis and Imaging

We are developing optical methods based on near infra-red Raman spectroscopy and fluorescence photon migration for diagnosis and localization of breast cancer. We demonstrate the ability of Raman spectroscopy to classify accurately normal, benign and malignant breast tissues, an important step in developing Raman spectroscopic needle probes as a tool for improving the accuracy of needle biopsy. We also show that photon migration imaging can be used to localize accurately small fluorescent objects imbedded in a thick turbid medium with realistic optical properties, thus demonstrating the potential of this technique for optical imaging.     

Development of molecular imaging tools to investigate protein functions by chemical probe design

Molecular imaging technologies, which enable the visualization of the behaviors or functions of biomolecules in living systems, have received considerable attention from life scientists. Novel imaging technologies that overcome the limitations of current imaging techniques are desired. In this review, two independent technologies that were recently developed by the authors are described. The first technology is for smart (19)F magnetic resonance imaging (MRI) probes that were developed for in vivo applications. These probes were developed by exploiting paramagnetic relaxation enhancement in order to detect hydrolase activity. With respect to cellular applications, gene expression in cells was visualized using one of the (19)F MRI probes. It was confirmed that this probe design principle is effective for various hydrolases, and broad applications are expected. The second technology is for practical protein labeling. This labeling method is based on a mutant β-lactamase and its specific labeling probes. Since the probe is fluorescence resonance energy transfer (FRET)-based, this labeling method achieves both specific and fluorogenic labeling of target proteins. In addition, derivatization of the probe enabled the labeling of intracellular proteins and the modification of various functional molecules.