Single-molecule monitoring in living cells by use of fluorescence microscopy

Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.     

Fluorescence-resonance energy transfer (FRET) within the fluorescent metallacycles

During past few years, the construction of fluorescent metallacycles featuring the fluorescence-resonance energy transfer behavior has attracted extensive attention due to their diverse applications such as real-time monitoring the dynamics of coordination-driven self-assembly, photoswitching fluorescence-resonance energy transfer, and light-controlled generation of singlet oxygen for cancer therapy. This review focuses on the recent advances on the design principles, preparation methods, optical properties, and the wide applications of fluorescent metallacycles with the FRET property.     

Fluorescence-resonance energy transfer (FRET) within the fluorescent metallacycles

During past few years, the construction of fluorescent metallacycles featuring the fluorescence-resonance energy transfer behavior has attracted extensive attention due to their diverse applications such as real-time monitoring the dynamics of coordination-driven self-assembly, photoswitching fluorescence-resonance energy transfer, and light-controlled generation of singlet oxygen for cancer therapy. This review focuses on the recent advances on the design principles, preparation methods, optical properties, and the wide applications of fluorescent metallacycles with the FRET property.     

Recent progress in gold nanoparticle-based biosensing and cellular imaging

Gold nanoparticles (AuNPs) have been extensively used in optical biosensing and bioimaging due to the unique optical properties. Biological applications including biosensing and cellular imaging based on optical properties of AuNPs will be reviewed in the paper. The content will focus on detection principles, advantages and challenges of these approaches as well as recent advances in this field.     

Nanometer Scale Imaging and Biochemical Sensing

This seminar will cover two research topics in our group. The first one is nanometer scale sensing with living biological cells. In the biomedical sciences and technologies, the greatest advances in the last decade have been inspired by the genome project. What comes after the deciphering of the genetic code? Certainly one next step is the biochemistry driven by the gene, from the cellular nucleus to its organelles, cytoplasm and beyond. One important goal is to follow in real time the biochemical kinetics and dynamics of the living cell, much of which is in terms of small ions and biomolecules. Using both optical microscopy/spectroscopy and scanning probe microscopy, we have imaged single living cells and probed single molecule interactions.     

Hybrid Rhodamine Fluorophores in the Visible/NIR Region for Biological Imaging

Fluorophores and probes are invaluable for the visualization of the location and dynamics of gene expression, protein expression, and molecular interactions in complex living systems. Rhodamine dyes are often used as scaffolds in biological labeling and turn‐on fluorescence imaging. To date, their absorption and emission spectra have been expanded to cover the entire near‐infrared region (650–950 nm), which provides a more suitable optical window for monitoring biomolecular production, trafficking, and localization in real time. This review summarizes the development of rhodamine fluorophores since their discovery and provides strategies for modulating their absorption and emission spectra to generate specific bathochromic‐shifts. We also explain how larger Stokes shifts and dual‐emissions can be obtained from hybrid rhodamine dyes. These hybrid fluorophores can be classified into various categories based on structural features including the alkylation of amidogens, the substitution of the O atom of xanthene, and hybridization with other fluorophores.     

Ultrafast UV spectroscopy: from a local to a global view of dynamical processes in macromolecules

The aim of this Perspective article is to cover recent developments in the application of femtosecond UV spectroscopy to understand molecular dynamics, and outlining potential future directions in this area. With several examples from recent literature the added-value of these techniques and their capability to study in real time changes in structure, dynamics and electrostatic fields of macromolecules in a site-specific fashion, as well as to uncover concerted dynamics in biomolecules, will be shown and discussed. The emerging fields of UV pulse-shaping techniques and UV optical nonlinear spectroscopies will be discussed to outline their potential to generate a novel family of coherent nonlinear spectroscopies for spectroscopic and microscopic applications.     

Advances in Medical Wearable Biosensors: Design,Fabrication and Materials Strategies in Healthcare Monitoring

In the past decade, wearable biosensors have radically changed our outlook on contemporary medical healthcare monitoring systems. These smart, multiplexed devices allow us to quantify dynamic biological signals in real time through highly sensitive, miniaturized sensing platforms, thereby decentralizing the concept of regular clinical check-ups and diagnosis towards more versatile, remote, and personalized healthcare monitoring. This paradigm shift in healthcare delivery can be attributed to the development of nanomaterials and improvements made to non-invasive biosignal detection systems alongside integrated approaches for multifaceted data acquisition and interpretation. The discovery of new biomarkers and the use of bioaffinity recognition elements like aptamers and peptide arrays combined with the use of newly developed, flexible, and conductive materials that interact with skin surfaces has led to the widespread application of biosensors in the biomedical field. This review focuses on the recent advances made in wearable technology for remote healthcare monitoring. It classifies their development and application in terms of electrochemical, mechanical, and optical modes of transduction and type of material used and discusses the shortcomings accompanying their large-scale fabrication and commercialization. A brief note on the most widely used materials and their improvements in wearable sensor development is outlined along with instructions for the future of medical wearables.     

Self‐Assembly of Functional Discrete Three‐Dimensional Architectures in Water

Construction of discrete, self‐assembled architectures in water has gained significant interest in recent years as a wide range of applications arises from their defined 3D structure. In this review we jointly discuss the efforts of supramolecular chemists and biotechnologists who previously worked independently, to tackle discipline‐specific challenges associated with construction of assemblies from synthetic and bio‐derived components, respectively. Going forward, a more interdisciplinary research approach will expedite development of complexes with real‐world applications that exploit the benefits of compartmentalisation. In support of this, we summarise advances made in the development of discrete, water‐soluble assemblies, with particular focus on their current and prospective applications. Areas where understanding and methodologies can be transferred from one sector to the adjacent field are highlighted in anticipation this will yield advances not possible from either field alone.     

Monitoring the Glutathione Redox Reaction in Living Human Cells by Combining Metabolic Labeling with Heteronuclear NMR

The glutathione (GSH) redox reaction is critical for defense against cellular reactive oxygen species (ROS). However, direct and real‐time monitoring of this reaction in living mammalian cells has been hindered by the lack of a facile method. Herein, we describe a new approach that exploits the GSH biosynthetic pathway and heteronuclear NMR. [U‐¹³C]‐labeled cysteine was incorporated into GSH in U87 glioblastoma cells, and the oxidation of GSH to GSSG by a ROS‐producing agent could be monitored in living cells. Further application of the approach to cells resistant to temozolomide (TMZ), an anti‐glioblastoma drug, suggested a possible new resistance mechanism involving neutralization of ROS. This result was corroborated by the observation of up‐regulation of glutathione peroxidase 3 (GPx3). This new approach could be easily applied to redox‐dependent signaling pathways and drug resistance involving ROS.     

A Multifunctional Nanomicelle for Real‐Time Targeted Imaging and Precise Near‐Infrared Cancer Therapy

Simultaneous targeted cancer imaging, therapy and real‐time therapeutic monitoring can prevent over‐ or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near‐infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH‐activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer‐specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real‐time imaging. Upon NIR irradiation, R16FP‐mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.     

Time‐Resolved Synchrotron Radiation Excited Optical Luminescence: Light‐Emission Properties of Silicon‐Based Nanostructures

The recent advances in the study of light emission from matter induced by synchrotron radiation: X‐ray excited optical luminescence (XEOL) in the energy domain and time‐resolved X‐ray excited optical luminescence (TRXEOL) are described. The development of these element (absorption edge) selective, synchrotron X‐ray photons in, optical photons out techniques with time gating coincide with advances in third‐generation, insertion device based, synchrotron light sources. Electron bunches circulating in a storage ring emit very bright, widely energy tunable, short light pulses (<100 ps), which are used as the excitation source for investigation of light‐emitting materials. Luminescence from silicon nanostructures (porous silicon, silicon nanowires, and Si–CdSe heterostructures) is used to illustrate the applicability of these techniques and their great potential in future applications.     

Light‐induced Modulation of Protein Dynamics During the Photocycle of Bacteriorhodopsin†

Knowledge about the dynamical properties of a protein is of essential importance for understanding the structure–dynamics–function relationship at the atomic level. So far, however, the correlation between internal protein dynamics and functionality has only been studied indirectly in steady‐state experiments by variation of external parameters like temperature and hydration. In the present study we describe a novel type of (laser‐neutron) pump‐probe experiment, which combines in situ optical activation of the biological function of a membrane protein with a time‐dependent monitoring of the protein dynamics using quasielastic neutron scattering. As a first successful application we present data obtained selectively in the ground state and in the M‐intermediate of bacteriorhodopsin (BR). Temporary alterations in both localized reorientational protein motions and harmonic vibrational dynamics have been observed during the photocycle of BR. This observation is a direct proof for the functional significance of protein structural flexibility, which is correlated with the large‐scale structural changes in the protein structure occurring during the M‐intermediate. We anticipate that functionally important modulations of protein dynamics as observed here are of relevance for most other proteins exhibiting conformational transitions in the time course of functional operation.     

A microfluidic system enabling Raman measurements of the oxygenation cycle in single optically trapped red blood cells

Using a lab-on-a-chip approach we demonstrate the possibility of selecting a single cell with certain properties and following its dynamics after an environmental stimulation in real time using Raman spectroscopy. This is accomplished by combining a micro Raman set-up with optical tweezers and a microfluidic system. The latter gives full control over the media surrounding the cell, and it consists of a pattern of channels and reservoirs defined by electron beam lithography that is moulded into rubber silicon (PDMS). Different buffers can be transported through the channels using electro-osmotic flow, while the resonance Raman response of an optically trapped red blood cell (RBC) is simultaneously registered. This makes it possible to monitor the oxygenation cycle of the cell in real time and to investigate effects like photo-induced chemistry caused by the illumination. The experimental set-up has high potential for in vivo monitoring of cellular drug response using a variety of spectroscopic probes.     

Magnetically and Near‐Infrared Light‐Powered Supramolecular Nanotransporters for the Remote Control of Enzymatic Reactions

Cancer is one of the primary causes of death worldwide. A high‐precision analysis of biomolecular behaviors in cancer cells at the single‐cell level and more effective cancer therapies are urgently required. Here, we describe the development of a magnetically‐ and near infrared light‐triggered optical control method, based on nanorobotics, for the analyses of cellular functions. A new type of nanotransporters, composed of magnetic iron nanoparticles, carbon nanohorns, and liposomes, was synthesized for the spatiotemporal control of cellular functions in cells and mice. Our technology will help to create a new state‐of‐the‐art tool for the comprehensive analysis of “real” biological molecular information at the single‐cell level, and it may also help in the development of innovative cancer therapies.     

Monitoring photopolymerization reactions with optical pyrometry

This article describes the development of optical pyrometry (OP) as a new analytical technique for the continuous monitoring of the progress of both free‐radical and cationic photopolymerizations. The method is rapid, reproducible, and very easy to implement. A temperature profile of a photopolymerization can be obtained. Preliminary studies have shown that the temperatures of some polymerizing monomers can easily reach temperatures in excess of 250 °C. The effects of the mass and reactivity of the monomer, light intensity, structures, and concentrations of the photoinitiators and monomers as well as the presence or absence of oxygen on various free‐radical and cationic photopolymerizations were examined with this method. Coupling of real‐time infrared spectroscopy with OP provides a convenient method for simultaneously monitoring both the chemical conversion and the temperature of a photopolymerization. This combined technique affords new insights into the effects of temperature‐induced autoacceleration on the course of photopolymerizations. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 579–596, 2003     

Recent advances in mitochondria-and lysosomes-targeted small-molecule two-photon fluorescent probes

This review summarized the recent advances in small-molecule two-photon fl uorescent probes for monitoring a wide variety of biomolecules and changes inside micro-environment in mitochondria and lysosomes, or served as mitotracker and lysotracker with the assistance of two-photon microscopy.     

Methods of Split Reporter Reconstitution for the Analysis of Biomolecules

Intracellular signaling inside living cells is controlled by the specific localization of biomolecules, including proteins, with timescales ranging from milliseconds to several hours. To elucidate the related spatial and temporal signal processes, development of optical probes for cellular events is a challenging task in present studies. Herein, we describe recent advances in the basic design of the optical probes, which have been inspired by luminescent creatures, and their practical application to visualize intracellular events in living cells and animals. A discussion of different probe designs reveals their benefits and shortcomings.     

Real‐Time NMR Spectroscopy for Studying Metabolism

Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in‐cell NMR spectroscopy experiments. We are able to monitor real‐time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer‐based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.     

Oxygen indicators and intelligent inks for packaging food

The detection of oxygen using optical sensors is of increasing interest, especially in modified atmosphere food packaging (MAP), in which the package, usually containing food, is flushed with a gas, such as carbon dioxide or nitrogen. This tutorial review examines the ideal properties of an oxygen optical sensor for MAP and compares them with those developed to date, including the most recent advances. The basic technologies underpinning the different indicator types are described, examples given and their potential for application in MAP assessed. This tutorial review should be of interest to the MAP industry and researchers in optical sensors and oxygen sensing.