Long-Term,Single-Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores

Single-molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long-term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single-molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single-molecule imaging of specific protein targets for exceptionally long times.     

A New Variant of Emissive RNA Alphabets

A new fluorescent ribonucleoside alphabet (^mthN) consisting of pyrimidine and purine analogues, all derived from methylthieno[3,4-d]pyrimidine as the heterocyclic core, is described. Large bathochromic shifts and high microenvironmental susceptibility of their emission relative to previous alphabets derived from thieno[3,4-d]pyrimidine (^thN) and isothiazole[4,3-d]pyrimidine (^tzN) scaffolds are observed. Subjecting the purine analogues to adenosine deaminase, guanine deaminase and T7 RNA polymerase indicate that, while varying, all but one enzyme tolerate the corresponding ^mthN/^mthNTP substrates. The robust emission quantum yields, high photophysical responsiveness and enzymatic accommodation suggest that the ^mthN alphabet is a biophysically viable tool and can be used to probe the tolerance of nucleoside/tide-processing enzymes to structural perturbations of their substrates.     

Recent Progress of Small-Molecule Ratiometric Fluorescent Probes for Peroxynitrite in Biological Systems

Peroxynitrite (ONOO⁻) as a major reactive oxygen species plays important roles in cellular signal transduction and homeostatic regulation. Precise detection of ONOO⁻ in biological systems is vital for exploring its physiological and pathological function. Among numerous detection methods, fluorescence imaging technology using fluorescent probes offers some advantages, including simple operation, high sensitivity and selectivity, as well as real-time and nondestructive detection. In particular, ratiometric fluorescent probes, in which the built-in calibration of the two emission bands prevents interference from the biological environment, have been extensively employed to monitor the fluctuation of bioactive species. In this review, we will discuss small-molecule ratiometric fluorescent probes for ONOO⁻ in live cells or in vivo, which involves chemical structures, response mechanisms, and biological applications. Moreover, the challenges and future prospects of ONOO⁻-responsive ratiometric fluorescent probe are also proposed.     

Give or Take: Effects of Electron-Accepting/-Withdrawing Groups in Red-Fluorescent BODIPY Molecular Rotors

Mapping microviscosity, temperature, and polarity in biosystems is an important capability that can aid in disease detection. This can be achieved using fluorescent sensors based on a green-emitting BODIPY group. However, red fluorescent sensors are desired for convenient imaging of biological samples. It is known that phenyl substituents in the β position of the BODIPY core can shift the fluorescence spectra to longer wavelengths. In this research, we report how electron-withdrawing (EWG) and -donating (EDG) groups can change the spectral and sensory properties of β-phenyl-substituted BODIPYs. We present a trifluoromethyl-substituted (EWG) conjugate with moderate temperature sensing properties and a methoxy-substituted (EDG) molecule that could be used as a lifetime-based polarity probe. In this study, we utilise experimental results of steady-state and time-resolved fluorescence, as well as quantum chemical calculations using density functional theory (DFT). We also explain how the energy barrier height (Ea) for non-radiative relaxation affects the probe’s sensitivity to temperature and viscosity and provide appropriate Ea ranges for the best possible sensitivity to viscosity and temperature.     

Dosimetric gelator probes and their application as sensors

Supramolecular gels formed by the self-assembly of organic molecules are useful in many areas from materials to medicine. Of the different applications, exploitation of gels for the visual detection of analytes is a fairly recent trend in gel chemistry. Most of the gel-based sensors rely on non-covalent interactions between the gelator molecules and the added chemical analytes and therefore, often suffer from less selectivity and long response time. In this context, dosimetric gelator probes are superior to other gel-based sensors with high selectivity and fast response time. Unlike non-covalent binding sites, dosimetric gelators typically contain a reaction centre and undergo a specific chemical reaction selective to an analyte resulting in either formation or rupturing of covalent bonds. In this review, we provide an up-to-date report of various reaction-based gel systems applied for the sensing of analytes. We elaborately discuss the concept, design principles, self-assembly properties, and reaction mechanisms of such gelators. We also highlight the limitations, challenges, and the necessity of further exploration of dosimetric gels in this domain.     

Environment‐sensitive Fluorescent Turn‐on Chemical Probe for the Specific Detection of O‐Methylguanine‐DNA Methyltransferase (MGMT) in Living Cells

MGMT protein, which has been associated with resistance to antitumor alkylation drugs for many patients, is a very useful prognostic marker to provide a guide for therapeutic decisions. Considering the large number of cellular samples that have to be handled daily at the hospitals, it is thus important to develop a rapid and simple analytical method to distinguish MGMT activity in different types of cells. In this paper, we describe a MGMT‐activated fluorescence turn‐on probe for the rapid no‐wash imaging of MGMT in living cells. The probe consists of a specific MGMT suicide pseudosubstrate, O⁶‐benzyl‐guanine and an environment‐sensitive fluorophore, SBD. In the presence of MGMT, the enzyme transfers SBD to the protein active site where the hydrophobic surrounding causes the fluorophore to exhibit more than 50‐fold fluorescence enhancement. With this probe, bright fluorescence was observed for MGMT‐positive, Hela S3 and MCF‐7 cells, while MGMT‐deficient CHO cells displayed no fluorescence. We believe that this fluorescence activation probe design can also be extended to detect other transferases, for which there are still no effective methods to image them in living cells.     

Ferrocene derivatives supported on poly(N-vinylpyrrolidin-2-one) (PVP): Synthesis of new water-soluble electrochemically active probes for biomolecules

Carboxy-terminated polyvinylpyrrolidin-2-one (PVP) has been used as a new water-soluble and biocompatible polymeric support for a series of ferrocene labeled amino acid and peptide nucleic acid (PNA) monomer derivatives 4-7. The organometallic polymer-conjugates thus obtained are new and potentially useful as water-soluble electrochemically active probes for biomolecules. In view of such application, their electrochemical activity has been evaluated and has proved very high notwithstanding the complexity and bulkiness of the molecule, affording detection limits down to 10⁻⁸ M in the aqueous medium.     

Development of a systems analysis approach for resolving the structure of biodegrading soil systems

An experimental and mathematical method is developed for the microbial systems analysis of polyaromatic hydrocarbon (PAH)-degrading mixed cultures in PAH-contaminated “town gas” soil systems. Frequency response is the primary experimental and data analysis tool used to probe the structure of these complicated systems. The objective is to provide a fundamental protocol for evaluating the performance of specific mixed microbial cultures on specific soil systems by elucidating the salient system variables and their interactions. Two well-described reactor systems, a constant volume stirred tank reactor (CSTR) and a plug flow differential volume reactor, are used in order to remove performance effects that are related to reactor type as opposed to system structure. These two reactor systems are well-defined systems that can be described mathematically and represent the two extremes of one potentially important system variable, macroscopic mass transfer. The experimental and mathematical structure of the protocol is described, experimental data is presented, and data analysis is demonstrated for the stripping, sorption, and biodegradation of napththalene.     

水溶性量子点的制备及应用

高量子产率的水溶性量子点在众多领域如发光晶体、薄膜光激发器件,尤其是生物荧光标记中展现出巨大的潜在应用价值.量子点荧光明亮、稳定,激发光谱宽,发射光谱窄,且发射波长可通过改变材料的粒径大小和组成来调控,因而在生物样本尤其是活组织的多色成像中极为有用,能有效避免因样本自身发光和光散射导致的信号干扰.量子点的研究已成为一门新兴的交叉科学,是目前最有吸引力的研究领域之一.近年来,水溶性量子点作生物荧光标记物的研究取得了长足的进展.本文简述了量子点的结构特征及光学特性,重点综述了水溶性量子点的制备方法及应用研究进展,特别是它在生物、医学中作荧光探针的最新研究.