超分辨显微成像技术在活细胞成像中的应用与发展

超分辨显微成像技术(super-resolution microscopy,SRM)可以绕过光学衍射极限对成像分辨率的限制,让以前观察不到的纳米级结构实现可视化,这一重大研究进展推动了现代生命科学和生物医学研究的进步与发展.细胞是生物体的基本组成单位,对活细胞内部的细微结构和动力学过程进行研究是掌握生命本质必不可少的途...     

Use of flavin photochemistry to probe intraprotein and interprotein electron transfer mechanisms

Photoexcitation of flavin analogs generates the lowest triplet state (via intersystem crossing from the first excited singlet state) in the nanosecond time domain and with high quantum efficiency. The triplet, being a strong oxidant, can abstract a hydrogen atom (or an electron) from a reduced donor in a diffusion-controlled reaction. If the donor is a redox protein, the oxidation process can be used to initiate an electron transfer sequence involving either intramolecular or intermolecular reactions. If the donor is an organic compound such as EDTA, the neutral flavin semiquinone will be produced by H atom abstraction; this is a strong reductant and can subsequently transfer a hydrogen atom (or an electron) to an oxidized redox protein, thereby again initiating a sequence of intramolecular or intermolecular processes. If flavin photoexcitation is accomplished using a pulsed laser light source, the initiation of these protein electron transfer reactions can be made to occur in the nanosecond to microsecond time domain, and the sequence of events can be followed by time-resolved spectrophotometry to obtain rate constants and thus mechanistic information. The present paper describes this technology, and selected examples of its use in the investigation of redox protein mechanisms are given.     

Light sheet microscopes: Novel imaging toolbox for visualizing life’s processes

Capturing dynamic processes in live samples is a nontrivial task in biological imaging. Although fluorescence provides high specificity and contrast compared to other light microscopy techniques, the photophysical principles of this method can have a harmful effect on the sample. Current advances in light sheet microscopy have created a novel imaging toolbox that allows for rapid acquisition of high-resolution fluorescent images with minimal perturbation of the processes of interest. Each unique design has its own advantages and limitations. In this review, we describe several cutting edge light sheet microscopes and their optimal applications.     

金属酞菁光敏剂的三阶非线性光学性能研究

从光动力治疗癌症的疗效着眼研究酞菁配合物的三阶非线性光学性能。用时间分辨简并四波混频方法测量苯硫基钛菁锌(C56H32S4Zn),苯硫基铝酞菁(C56H32AlN8O4)以及烷氧基铝酞菁(C56H32AlN8O4)的三阶非线性光极化率;测量四波混频响应的衰减过程;研究时间响应的超快过程和慢过程及其动力学机制,它们分别对应于单态和三线态的寿命。在荧光显微成像系统中观察三种酞菁光敏剂对人肝癌细胞杀伤的形态变化,并用MTT方法检测细胞存活率。对三种酞菁配合物的三线态量子产率和寿命进行测定,结果与它们对人肝癌细胞的光动力杀伤作用相关联。     

Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells

The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site-specific protein labeling.     

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin‐interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target‐search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.     

Detection of single quantum dots in model organisms with sheet illumination microscopy

Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 μm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.