Multiplexed stimulated emission depletion nanoscopy(mSTED)for 5-color live-cell long-term imaging of organelle interactome

Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.

Fabrication of Silicon Carbide Quantum Dots via Chemical-Etching Approach and Fluorescent Imaging for Living Cells

A simple chemical-etching approach is used to prepare the silicon carbide quantum dots (QDs). The raw materials of silicon carbide (SiC) with homogeneous nanoparticles fabricated via self-propagating combustion synthesis are corroded in mixture etchants of nitric and hydrofluoric acid. After sonication and chromatography in the ultra-gravity field for the etched products, aqueous solution with QDs can be obtained. The microstructure evolution of raw particles and optical properties of QDs were measured. Different organophilic groups on the surface like carboxyl, oxygroup, and hyfroxy were produced in the process of etching. Fluorescent labeling and imaging for living cells of Aureobasidium pulluans were investigated. The results indicated that SiC QDs were not cytotoxic and could stably label due to the conjugation between organophilic groups of QDs and specific protein of cells, it can be utilized for fluorescent imaging and tracking cells with in vivo and long-term-distance. Moreover, mechanism and specificity of mark were also analyzed.

Live-cell imaging:new avenues to investigate retinal regeneration

Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish（Danio rerio） possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.

3-82 Environmental Control of Live Cell Imaging System at Microbeam Facility

Live cell imaging has significantly changed our understanding to cell biology during the past 10 years. However, providing a suitable environment to keep cells heathy is still a challenge in live cell imaging experiments, and has great influence on the reliability of the experimental results. Many factors are needed to maintain cells healthy, such as temperature, pH, oxygen tension, CO2 and so on.

Live imaging of the effects of fucoidan on cell proliferation for laboratory instruction

The aim of this study is to introduce live cell imaging and its applications for the evaluation of the effects of fucoidan, a fucose-enriched sulfated polysaccharide, on the proliferation of cultured cells in vitro. In this study, long-term time- lapse observation （87 h） of the effects of fucoidan was conducted using BioStation CT, an integrated cell culture observation system. In contrast, the effects of heparin, which has a similar structure to fucoidan, were observed to distinguish the differences between the two chemicals. At the same time, the viability of the floating cells detached by fucoidan in the medium was measured by culturing them again in the absence of fucoidan. Finally, total internal reflection fluorescence microscopy （TIRF） was used to confirm when the detachment of the cells by fucoidan occurred. The results indicate that the inhibitory effects of fucoidan on the proliferation of cells are dose-dependent （from 0.125 mg/ml to 1.0 mg/ml）. Fucoidan also causes cell detachment without killing all the cells within 24 hours. The cell detachment did not occur until after half an hour, as observed under the TIRF microscope. Combined with our previous study, the findings suggest that the inhibition of calcium responses by fucoidan may be one of the mechanisms underlying its inhibition of cell proliferation, which is responsible for the death of cancer cells. Cell proliferation can be visualized in the real time and the images can provide important information regarding when and how the cells grow and proliferate.

3-79 Live Cell Imaging Study of XRCC1 Kinetics at the

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics (IMP) to study early and fast cellular response to DNA damage after high linear energy transfer (LET) ion radiation. HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online.

Application of Image Fusion Methods to Cell Imaging Processing

Aim To fuse the fluorescence image and transmission image of a cell into a single image containing more information than any of the individual image. Methods Image fusion technology was applied to biological cell imaging processing. It could match the images and improve the confidence and spatial resolution of the images. Using two algorithms, double thresholds algorithm and denoising algorithm based on wavelet transform,the fluorescence image and transmission image of a Cell were merged into a composite image. Results and Conclusion The position of fluorescence and the structure of cell can be displyed in the composite image. The signal-to-noise ratio of the exultant image is improved to a large extent. The algorithms are not only useful to investigate the fluorescence and transmission images, but also suitable to observing two or more fluoascent label proes in a single cell.

Recent advances in optical techniques for dynamically probing cellular mechanobiology

Cellular mechanotransduction characterized by the transformation of mechanical stimuli into biochemical signals,represents a pivotal and complex process underpinning a multitude of cellular functionalities.This process is integral to diverse biological phenomena,including embryonic development,cell migration,tissue regeneration,and disease pathology,particularly in the context of cancer metastasis and cardiovascular diseases.Despite the profound biological and clinical significance of mechanotransduction,our understanding of this complex process remains incomplete.The recent development of advanced optical techniques enables in-situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside a cell.In this review,we delved into the current state-of-the-art techniques utilized to probe cellular mechanobiology,their principles,applications,and limitations.We mainly examined optical methodologies to quantitatively measure the mechanical properties of cells during intracellular transport,cell adhesion,and migration.We provided an introductory overview of various conventional and optical-based techniques for probing cellular mechanics.These techniques have provided into the dynamics of mechanobiology,their potential to unravel mechanistic intricacies and implications for therapeutic intervention.

Interaction between Bax and Bcl-XL proteins confirmed by partial acceptor photobleaching-based FRET imaging

Exact interaction mechanism between Bax and Bcl-XL,two key Bcl-2 family proteins,is an interesting and controversial issue.Partial acceptor photobleaching-based quantitative fluores-cence resonance energy transfer(FRET)measurement,PbFRET,is a widely used FRET quantification method in living cells.In this report,we implemented pixel-to-pixel PbFRET imaging on a wide-field microscope to map the FRET efficiency(E)images of single living HepG2 cells co-expressing CFP-Bax and YFP-Bcl-XL.The E value between CFP-Bax and YFP-Bcl-XL was 4.59%in cytosol and 11.31%on mitochondria,conclusively indicating the direct interaction of the two proteins,and the interaction of the two proteins was strong on mitochondria and modest in cytosol.

DNA tetrahedron-based split aptamer probes for reliable imaging of ATP in living cells

Accurate detection and imaging of adenosine triphosphate(ATP)expression levels in living cells is of great value for understanding cell metabolism,physiological activities,and pathologic mechanisms.Here,we developed a DNA tetrahedron-based split aptamer probe(TD probe)for ratiometric fluorescence imaging of ATP in living cells.The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b)to a DNA tetrahedron assembled by four DNA oligonucleotides(T1,T2,T3 and T4).In the presence of ATP,the TD probe will alter its structure from the open to closed state,thus bringing the separated donor and acceptor fluorophores into close proximity for high fluorescence resonance energy transfer(FRET)signals.The TD probe exhibits low cytotoxicity,efficient cell internalization and good biological stability.Moreover,based on the FRET“off”to“on”signal output mode,the TD probe can effectively avoid false-positive signals from complex biological matrices,which is significant for long-term reliable imaging in living cells.In addition,by changing the split aptamers attached to DNA tetrahedron,the proposed strategy may be extended for detecting various intracellular targets.Collectively,this strategy provides a valuable sensing platform for biomarkers analysis in living cells,thus having great potential for early clinical diagnosis and therapeutic evaluation.

Effect of Probe Lifting Height in Jumping Mode AFM for Living Cell Imaging

Atomic force microscopy(AFM)is one of the effective methods for imaging the morphological and physical properties of living cells in a near-physiological environment.However,several problems caused by the adhesion of living cells and extension of the cell membranes seriously affect the image quality during living cell imaging,hindering the study of living cells.In this work,jumping mode AFM imaging was used to image living cells at varied probe lifting heights to meet image quality requirements,and image quality related to the probe lifting height is discussed in detail.The jumping mode was divided into three parts based on the varying heights of the lifted probe,namely near-contact mode,half-jumping mode,and full-jumping mode,and the causes of their imaging drawbacks were analyzed.At an appropriate lifting height,the probe can be completely free from the influence of cell adhesion and self-excited oscillation,thus avoiding the occurrence of“trail”phenomena and invalid points in the imaging of living cells and improving the image quality.Additionally,this work provides a new approach to calculating the lateral force through the adhesion of trace and retrace scanning at a low height,which is important for studying the extension characteristics of the cell membrane.

基于铁配合物的焦磷酸根离子荧光探针的构建及生物成像

以萘并[2,1-b]呋喃-2-碳酰肼和4-甲酰基-3-羟基-N-丁基-1,8-萘二甲酰亚胺为原料经缩合反应制备了一种新型配体ARC,配体ARC与Fe^(3+)按照1∶1络合制备了荧光探针ARC-Fe^(3+),通过高分辨率质谱(HR-MS)、核磁共振氢谱(1H NMR)对探针结构进行了表征。利用紫外-可见吸收光谱、荧光光谱等方法对ARC-Fe^(3+)的识别性能进行了研究。结果表明,探针ARC-Fe^(3+)可通过荧光“OFF-ON”高选择、高灵敏地识别焦磷酸根离子(PPi),探针ARC-Fe^(3+)识别PPi的检测限为1.12×10^(-8) mol/L,且可在60 s内完成对PPi的响应。探针ARC-Fe^(3+)已被成功应用于细胞和活鼠体内对PPi的荧光成像研究。

基于智能化控制的高效活细胞成像技术

为提升活细胞功能研究的效率与准确性,显微成像平台研发了1款智能化控制精准加样系统。自主研发的加样系统具备远程自动化控制功能,能实现精准的药物定时定点添加。经研究和测试,该系统展现出卓越的通用性,能与常见的光学成像设备搭载使用,并能满足从短期到长期活细胞功能研究的各种实验要求。在活细胞连续成像的过程中,采用智能化与精准化的控制方法,可显著提升实验的准确性、稳定性和效率,尤其是能确保关键数据的完整性。该研究工作有效促进了细胞功能等关键科研领域的深入探索,成功增强了多台光学成像仪器的测试能力和应用价值,同时也推动了创新人才的成长和高标准科研平台的搭建。

The Limbal Niche and Its Role in Maintaining Corneal Regeneration

In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .

一种用于过氧亚硝酸根检测的近红外比率荧光探针的研制

设计合成了一种基于半花菁的近红外比率荧光探针分子Cy-P,并将其用于活细胞中的外源性和内源性过氧亚硝酸根(ONOO-)荧光成像检测.研究结果表明:探针分子Cy-P自身具有较大的π共轭结构,其最大发射波长在近红外区域;加入ONOO-后,强氧化性的ONOO-会导致Cy-P的π共轭体系被破坏,并生成蓝色荧光物质,最大发射波长蓝移了268 nm;探针分子Cy-P对ONOO-线性检测范围为0~15μmol/L,检测下限为13 nmol/L;该探针分子对ONOO-的识别性能优于其他各种可能干扰的生物分析物;该探针分子具有低细胞毒性,适用于活细胞中的外源性和内源性ONOO-的成像检测.

基于图像识别的酵母活细胞率快速测定系统

酵母活细胞率作为评判活性干酵母质量优劣的重要指标之一,准确快速地检测出结果显得尤为重要。本文针对传统方法进行活细胞率检测存在效率低、过程复杂等问题开发了一款计数软件,并将血球计数板替换为96孔细胞培养板进行计数。对比人工计数和软件计数在检测酵母活细胞率的误差以及采用不同容器进行检验的效率,结果表明,相对于人工计数,软件计数耗时更短,且误差较小;采用96孔细胞培养板批量检测比采用血球计数板耗时更短。

Live cell imaging of genomic loci using dCas9-SunTag system and a bright fluorescent protein

Dear Editor,CRISPR-Cas9 （clustered regularly interspaced short palin- dromic repeats-CRISPR associated） systems have been harnessed for kinds of genome manipulation, including gene editing, transcription regulation, and chromosome loci imaging （Dominguez et al., 2016; Komor et al., 2017）. A typical engineered CRISPR-Cas9 system is composed of a Cas9 protein and a single guide RNA （sgRNA）, which could form a protein/RNA complex to recognize and cleave DNA sequence （Hsu et al., 2014; Wright et al., 2016）.

A selective and sensitive off–on probe for palladium and its application for living cell imaging

A new rhodamine B derivative T1 has been rationally synthesized and displayed selective Pd（Ⅱ）-amplified absorbance and fluorescence emission above 540 nm in methanol-water. Upon the addition of Pd（Ⅱ）, the spirolactam ring was unfolded and a 1：1 metal-ligand complex formed, which can be used for ＇＇naked-eyes＂ detection. In addition, fluorescence imaging experiments of Pd^（2＋） in HepG2 living cells showed its valuable application in biological systems.

Simultaneous dual-contrast three-dimensional imaging in live cells via optical diffraction tomography and fluorescence

We report a dual-contrast method of simultaneously measuring and visualizing the volumetric structural information in live biological samples in three-dimensional(3D) space. By introducing a direct way of deriving the 3D scattering potential of the object from the synthesized angular spectra, we obtain the quantitative subcellular morphology in refractive indices(RIs) side-by-side with its fluorescence signals. The additional contrast in RI complements the fluorescent signal, providing additional information of the targeted zones. The simultaneous dual-contrast 3D mechanism unveiled interesting information inaccessible with previous methods, as we demonstrated in the human immune cell(T cell) experiment. Further validation has been demonstrated using a Monte Carlo model.

Ratiometric and selective two-photon fluorescent probe based on PET-ICT for imaging Zn^(2+)in living cells and tissues

A two-photon fluorescent probe TPZn was developed for specific ratiometric imaging Zn2＋ in living cells and tissues. Significant ratiometric fluorescence change was based on photoinduced electron transfer and intramolecular charge transfer. The synthetic method of TPZn was simple. It was successfully used to selectively image Zn2＋ based on the higher binding affinity for Zn2＋ than for Cd2＋. TPZn was easily loaded into the living cell and tissues with high membrane permeability in a complex biological environment. TPZn could clearly visualize endogenous Zn2＋ by TP ratiometric imaging in hippocampal slices at a depth of 120 μm. Thus, TPZn is a useful tool to image of Zn2＋ in living cells and tissues without interference from Cd2＋.