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.

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. .

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.

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.

Cloning and Functional Analysis of Porcine Cycling A

Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin A gene and verified its functionality in swine umbilicus vein endothelial cells （SUVEC）. The human cyclin A gene was used to probe databases to clone the pig cyclin A gene electronically. The identified porcine cDNA contained an open reading frame of 1,299 bp, encoding 432 amino acids, the same length as the human cyclin A protein. The porcine cyclin A gene comprises eight exons on chromosome 8. The sequence of the in silico clone and expression of this novel gene were confirmed in SUVEC by reverse transcription PCR. Western blotting of cell lysates from SUVEC transfected with a cyclin A enhanced green fluorescent protein （EGFP） fusion construct revealed a band at approximately 40 kDa. Confocal microscopy of CycA-EGFP-expressing cells showed that the fusion protein was expressed in the nucleus. Flow cytometry demonstrated that more stably expressing SUVEC-CycA-EGFP were in G1 phase （15% to 20% increase） and fewer were in S phase （18% decrease） compared with control ceils. MTS assays showed that the proliferative activity of SUVEC-Cy- cAG-EGFP was significantly higher than that of the control cells. In conclusion, we have cloned the pig cyclin A gene and demonstrated that its biological function is consistent with cyclin A in other mammali- an species. This provides a foundation for future research on the impact of virus infection on cyclin A.

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.

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.

A benzothiazole-based ratiometric fluorescent probe with large Stokes shifts for quantitative profiling of sulfite in real samples and living cells

Rapid,selective,and quantitative profiling of SO_(3)^(2-)is a prerequisite for investigating its effect on different physiopathological processes.To address this challenge,our team prepared a ratiometric fluorescent probe HBTCS,based on 4-(benzothiazol-2-yl)-3-hydroxybenzaldehyde for specific detection of SO_(3)^(2-)under a singlewavelength excitation.The solution of probe HBTC-S itself exhibited orange fluorescence(λ_(em)=614 nm),and became green fluorescent(λ_(em)=471 nm)after the addition of SO_(3)^(2-).The vitro experiments suggested that the probe displayed high sensitivity,excellent selectivity,and a low detection limit(0.15μM).Moreover,probe HBTC-S was successfully applied for qualitative and quantitative monitoring of SO_(3)^(2-)in realistic samples and living HeLa cells in a ratiometric manner.

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.

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.

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 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.

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.

Molecular mechanisms of neurite regeneration and repair:insights from C.elegans and Drosophila

The difficulties of injured and degenerated neurons to regenerate neurites and regain functions are more significant than in other body tissues,making neurodegenerative and related diseases hard to cure.Uncovering the secrets of neural regeneration and how this process may be inhibited after injury will provide insights into novel management and potential treatments for these diseases.Caenorhabditis elegans and Drosophila melanogaster are two of the most widely used and well-established model organisms endowed with advantages in genetic manipulation and live imaging to explore this fundamental question about neural regeneration.Here,we review the classical models and techniques,and the involvement and cooperation of subcellular structures during neurite regeneration using these two organisms.Finally,we list several important open questions that we look forward to inspiring future research.

NIR-II live imaging study on the degradation pattern of collagen in the mouse model

The degradation of collagen in different body parts is a critical point for designing collagen-based biomedical products.Here,three kinds of collagens labeled by second near-infrared(NIR-II)quantum dots(QDs),including collagen with low crosslinking degree(LC),middle crosslinking degree(MC)and high crosslinking degree(HC),were injected into the subcutaneous tissue,muscle and joints of the mouse model,respectively,in order to investigate the in vivo degradation pattern of collagen by NIR-II live imaging.The results of NIR-II imaging indicated that all tested collagens could be fully degraded after 35 days in the subcutaneous tissue,muscle and joints of the mouse model.However,the average degradation rate of subcutaneous tissue(k=0.13)and muscle(k=0.23)was slower than that of the joints(shoulder:k=0.42,knee:k=0.55).Specifically,the degradation rate of HC(k=0.13)was slower than LC(k=0.30)in muscle,while HC showed the fastest degradation rate in the shoulder and knee joints.In summary,NIR-II imaging could precisely identify the in vivo degradation rate of collagen.Moreover,the degradation rate of collagen was more closely related to the implanted body parts rather than the crosslinking degree of collagen,which was slower in the subcutaneous tissue and muscle compared to the joints in the mouse model.

Real-time in situ observation of P53-mediated cascade activation of apoptotic pathways with nucleic acid multicolor fluorescent probes based on symmetrical gold nanostars

T-2 toxin,one of the most dangerous natural pollutants,induces apoptosis through multiple pathways.Amongst,P53 mediated apoptosis pathway,an important collection of molecules,plays a key role in cell vital activity.Real-time monitoring of upstream and downstream activation relationships of P53 mRNA,Bax mRNA,and cytochrome c(Cyt c)in signaling pathways is of great significance for understanding the apoptotic machinery in human physiology.In this work,a novel nucleic acid multicolor fluorescent probe,based on silica-coated symmetric gold nanostars(S-AuNSs@SiO_(2)),was developed for highly sensitive in situ real-time imaging of P53 mRNA,Bax mRNA,and Cyt c during T-2 toxin-induced apoptosis.The nucleic acid chains modified with carboxyl groups were modified on the surface of S-AuNSs@SiO_(2)by amide reaction.The complementary chains of targeted mRNA and the aptamer of targeted Cyt c were modified with different fluorophores,respectively,and successfully hybridized on S-AuNSs@SiO_(2)surface.When targets were present,the fluorescent chains bound to the targets and detached from the material,resulting in the quenched fluorescence being revived.The probes based on S-AuNSs showed excellent performance is partly ascribed to the presence of 20 symmetric“hot spots”.Notably,the amide-bonded probe exhibited excellent anti-interference capability against biological agents(nucleases and biothiols).During the real-time fluorescence imaging of T-2 toxin-induced apoptosis,the corresponding fluorescence signals of P53 mRNA,Bax mRNA,and Cyt c were observed sequentially.Therefore,S-AuNSs@SiO_(2)probe not only provides a novel tool for real-time monitoring of apoptosis pathways cascade but also has considerable potential in disease diagnosis and pharmaceutical medical.

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）.

Glow in the Dark： Fluorescent Proteins as Cell and Tissue-Specific Markers in Plants

Since the hallmark discovery of Aequorea victoria＇s Green Fluorescent Protein （GFP） and its adaptation for efficient use in plants, fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types, to monitor dynamic cell fate selection processes, and to obtain cell type-specific transcriptomes. Fluorescent tagging enabled visualization in living tissues and the precise recordings of dy- namic expression pattern changes. The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms. In developmental studies, the use of fluorescent proteins has become critical, where morphological markers of tissues, cell types, or differentiation stages are either not known or not easily recognizable. In this review, we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

A one-step rectification of sperm cell targeting ensures the success of double fertilization

Successful fertilization in animals depends on competition among millions of sperm cells, whereas double fertilization in flowering plants usually involves just one pollen tube releasing two immobile sperm cells. It is largely a mystery how the plant sperm cells fuse efficiently with their female targets within an embryo sac. We show that the initial positioning of sperm cells upon discharge from the pollen tube is usually inopportune for gamete fusions and that adjustment of sperm cell targeting occurs through release and re-adhesion of one sperm cell, while the other connected sperm cell remains in stagnation. This enables proper adhesion of each sperm cell to a female gamete and coordinates the gamete fusions. Our 〈br〉 findings reveal inner embryo sac dynamics that ensure the reproductive success of flowering plants and suggest a requirement for sperm cell differentiation as the basis of double fertilization.

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

B lymphocyte cell senses and acquires foreign anti-gens through clonal distributed B cell receptors(BCRs)expressed on the surface of plasma membrane.The presentation formats of antigens are quite diverse.Based on their Brownian diffusion mobility,there are three forms:free mobile soluble antigens,lateral mobile membrane bound antigens,and fixed immobile anti-gens.Here,using high resolution high speed live cell imaging approaches,we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell’s encountering of antigens with each format of motion features.Through high speed live cell imaging,we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the anti-gens.We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.