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.

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.

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.

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＋.

A unique two-photon fluorescent probe based on ICT mechanism for imaging palladium in living cells and mice

Palladium(0)as one of the vital transition metals,is employed in numerous industries,such as drug synthesis,aerospace high-tech field and automobile industry.When the Pd(0)enter into the body,it will bind with thiol-containing amino acids,DNA,RNA,and other biomolecules damaging to human health.Thus,developing a novel tool for monitoring and imaging of Pd(0)in vivo is very urgent.In the work,based on a intramolecular charge transfer(ICT)mechanism a two-photon fluorescent probe NIPd had been designed and synthesized for the recognition Pd(0).In vitro experiments data displayed that probe NIPd exhibited a 13-fold fluorescent increase for Pd(0)in 30 min in the aqueous solution with a detection limit of 16 nmol/L.It also showed the outstanding selectivity and antijamming performance.More importantly,NIPd could be served as a two-photon fluorescent probe for real-time monitoring Pd(0)in living cells and mice.

AIEgen based light-up probes for live cell imaging

Fluorescent light-up probes comprising a tetraphenylethene unit with aggregation-induced emission(AIE)characteristics and a water-soluble peptide have been designed and synthesized which provide cell membrane and nuclear permeability to live cells.This strategy has offered new opportunities for the development of probes with light-up ability and good signal-to-noise ratio.The selectivity or targeting specificity is determined by the peptide sequence,i.e.a nuclear localization signal that leads to nucleus imaging and a cell biomarker targeting peptide that offers specific light-up imaging of HT-29 cells.

A 1,8-naphthalimide-derived turn-on fluorescent probe for imaging lysosomal nitric oxide in living cells

Nitric oxide has played an important role in many physiological and pathological processes as a kind of important gas signal molecules. In this work, a new fluorescent probe LysoNO-Naph for detecting NO in lysosomes based on 1,8-naphthalimide was reported. LysoNO-Naph has sub-groups of o-phenylene- diamine as a NO reaction site and 4-（2-aminoethyl）-morpholine as a lysosome-targetable group. This probe exhibited good selectivity and high sensitivity （4.57 μmol/L） toward NO in a wide pH range from 4 to 12. Furthermore, LysoNO-Naph can be used for imaging NO in lysosomes in living cells.

Live-Cell Imaging of Dual-Labeled Golgi Stacks in Tobacco BY-2 Cells Reveals Similar Behaviors for Different Cisternae during Movement and Brefeldin A Treatment

In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A （BFA）-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a re- markable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addi- tion, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.

Single Molecule Imaging in Living Cell with Optical Method

Significance, difficult, international developing actuality and our completed works for single molecules imaging in living cell with optical method are described respectively. Additionally we give out some suggestions for the technology development further.

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging,a powerful analytical method to study single-cell behavior,such as gene expression and protein profiling,provides an essential basis for modern medical diagnosis.The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years.Simultaneously,chip-based living single-cell imaging is also limited by complicated trapping steps,low cell utilization,and difficult high-resolution imaging.To solve these problems,an ultra-thin temperature-controllable microwell array chip(UTCMA chip)was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells.The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope(or confocal microscope)with a high magnification objective(100×oil lens),and the temperature of the chip can be controlled by combining it with a home-made temperature control device.High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall.The cell utilization rate,single-cell capture rate,and microwell occupancy rate are all close to 100%in the microwell array.This method will be useful in rare single-cell research,extending its application in the biological and medical-related fields,such as early diagnosis of disease,personalized therapy,and research-based on single-cell analysis.

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.