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.

Encapsulation of live cells by metal-organic frameworks for viability protection

In this study, a yeast@ZIF-8 core–shell composite material was successfully synthesized under room temperature in aqueous solution. The ZIF-8 shell endowed the inner yeast cells with a considerably extended lifetime without any nutrients at 4°C. Compared with the bare yeast cells, most coated yeast cells were kept alive even when cultured in zymolyase solution for 3h. Furthermore, the encapsulated yeast cells could be reactivated and regrown by dissolving the ZIF-8 shell with competitive coordination interactions.

Recent advances in small molecule fluorescent probes for simultaneous imaging of two bioactive molecules in live cells and in vivo

The interrelationships and synergistic regulations of bioactive molecules play pivotal roles in physiological and pathological processes involved in the initiation and development of some diseases,such as cancer and neurodegenerative and cardiovascular diseases.Therefore,the simultaneous,accurate and timely detection of two bioactive molecules is crucial to explore their roles and pathological mechanisms in related diseases.Fluorescence imaging associated with small molecular probes has been widely used in the imaging of bioactive molecules in living cells and in vivo due to its excellent performances,including high sensitivity and selectivity,noninvasive properties,real-time and high spatial temporal resolution.Single organic molecule fluorescent probes have been successively developed to simultaneously monitor two biomolecules to uncover their synergistic relationships in living systems.Hence,in this review,we focus on summarizing the design strategies,classifications,and bioimaging applications of dual-response fluorescent probes over the past decade.Furthermore,future research directions in this field are proposed.

3D Bioprinting with Live Cells

In recent years,the shortage of available organs for transplant patients has grown exponentially across the globe.Consequently,the healthcare industry is in dire need of artificial substitutes.Many recent research studies and tissue engineering groups have decided to utilize 3D bioprinting to produce these artificial organs.This synthetic organ printing is made possible by advancements in the materials required for the constructs,the printing method-ologies used to produce them,and the final physical structures’varying properties.The cutting-edge research and technology related to 3D and 4D live cell bioprinting have recently allowed researchers to produce multiple types of artificial organs and tissues.These tissues can be utilized for drug screening and organ replacement applica-tions.This article provides an extensive review of all the pertinent 3D live cell bioprinting technologies.First,we describe scaffolding methods and their comparison with the traditional technologies.Second,we explain the 3D bioprinting technology,its evolution,and its multiple types.Moreover,we describe the pros and cons of each bioprinting method.Third,we have discussed the critical bioink properties and their impact on the formation of 3D bioprinting models.In addition,we also describe the mechanical properties of bioprinters.Fourth,we have thoroughly discussed the various types of hydrogels and their properties.Every kind of hydrogel is utilized in specific applications,and we have presented a comprehensive list of its advantages and disadvantages.Fifth,we have discussed various applications of 3D bioprinting technology.We have considered a case study of human or-gans and elaborated on how bioprinters can revolutionize the organ replacement industry.Finally,we evaluated the possibility of 4D printing in the future organ industry,incorporating temporal factors into the bioprinting process.

Illuminating single genomic loci in live cells by reducing nuclear background fluorescence

The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene interaction.CRISPR/dCas9(clustered regularly interspaced short palindromic repeats/deactivated Cas9)labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci.However,the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout.This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS(target module)and scFv-sfGFP-NLS(signal module).We removed the nuclear location sequence(NLS)of the signal module and inserted two copies of EGFP into the signal module.The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm(N/C ratio)was decreased by 71%,and the ratio of the signal to the background(S/B ratio)was increased by 1.6 times.The system can stably label randomly selected genomic loci with as few as 9 repeat sequences.

Recent advances in living cell nucleic acid probes based on nanomaterials for early cancer diagnosis

The early diagnosis of cancer is vital for effective treatment and improved prognosis. Tumor biomarkers, which can be used for the early diagnosis, treatment, and prognostic evaluation of cancer, have emerged as a topic of intense research interest in recent years. Nucleic acid, as a type of tumor biomarker, contains vital genetic information, which is of great significance for the occurrence and development of cancer. Currently, living cell nucleic acid probes, which enable the in situ imaging and dynamic monitoring of nucleic acids, have become a rapidly developing field. This review focuses on living cell nucleic acid probes that can be used for the early diagnosis of tumors. We describe the fundamental design of the probe in terms of three units and focus on the roles of different nanomaterials in probe delivery.

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.

Iron-Imprinted Single-Atomic Site Catalyst-Based Nanoprobe for Detection of Hydrogen Peroxide in Living Cells

Fe-based single-atomic site catalysts(SASCs),with the natural metalloproteases-like active site structure,have attracted widespread attention in biocatalysis and biosensing.Precisely,controlling the isolated single-atom Fe-N-C active site structure is crucial to improve the SASCs’performance.In this work,we use a facile ion-imprinting method(IIM)to synthesize isolated Fe-N-C single-atomic site catalysts(IIM-Fe-SASC).With this method,the ion-imprinting process can precisely control ion at the atomic level and form numerous well-defined single-atomic Fe-N-C sites.The IIM-Fe-SASC shows better peroxidase-like activities than that of non-imprinted references.Due to its excellent properties,IIM-Fe-SASC is an ideal nanoprobe used in the colorimetric biosensing of hydrogen peroxide(H_(2)O_(2)).Using IIM-Fe-SASC as the nanoprobe,in situ detection of H_(2)O_(2)generated from MDA-MB-231 cells has been successfully demonstrated with satisfactory sensitivity and specificity.This work opens a novel and easy route in designing advanced SASC and provides a sensitive tool for intracellular H_(2)O_(2)detection.

Real-time observation of integrin bending/unbending conformational changes on living cells

Introduction Integrins are a large family of adhesion molecules broadly expressed on the surface of a wide variety of cells as heterodimers. Binding of integrins to ligands provides not only mechanical anchorage for the cell to another cell or

The Electrochemical Impedance Behavior of the Living Cells Escherichia Coli

The semiconductive characteristics of clectron-transfrring proteins in living cells E coli was investigated by electrochemsical impedance spectroscopy(EIS). We found that the electrochemical impedance of living cells as a function of temprature followed the Arrhenius equation for semiconductors. This result shows a strong evidence to prove the semiconductive behavior of proteins

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.

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.

The emerging technology of biohybrid micro-robots:a review

In the past few decades,robotics research has witnessed an increasingly high interest in miniaturized,intelligent,and integrated robots.The imperative component of a robot is the actuator that determines its performance.Although traditional rigid drives such as motors and gas engines have shown great prevalence in most macroscale circumstances,the reduction of these drives to the millimeter or even lower scale results in a significant increase in manufacturing difficulty accompanied by a remarkable performance decline.Biohybrid robots driven by living cells can be a potential solution to overcome these drawbacks by benefiting from the intrinsic microscale self-assembly of living tissues and high energy efficiency,which,among other unprecedented properties,also feature flexibility,self-repair,and even multiple degrees of freedom.This paper systematically reviews the development of biohybrid robots.First,the development of biological flexible drivers is introduced while emphasizing on their advantages over traditional drivers.Second,up-to-date works regarding biohybrid robots are reviewed in detail from three aspects:biological driving sources,actuator materials,and structures with associated control methodologies.Finally,the potential future applications and major challenges of biohybrid robots are explored.

Research on Electric Impedance Spectroscopy of Living Cell Suspensions by a Chip with Microelectrodes

A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed.The substrate and the electrodes of the chip were made of glass and gold,respectively.The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline,culture medium,living cell suspension etc.) by scanning from 10Hz to 45kHz.A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension.An actual circuit was also built and tested to verify the 6-element circuit model proposed.The micro-EIS chip has several advantages including the use of small sample volumes,high resolution and ease of operation.It shows good application prospects in the areas of cellular electrophysioiogy,drug screening and bio-sensors etc.

Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles（GNPs） based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay. In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy（CLSM） and classic flow cytometry（FCM） assay, and satisfactory results were obtained.

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.

Highly-efficient quantitative fluorescence resonance energy transfer measurements based on deep learning

Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used technique needs complicated experimental process and manual image analyses to obtain precise results,which take a long time and restrict the application of quantitative FRET especially in living cells.In this paper,a simplified and automatic quanti-tative FRET(saqFRET)method with high efficiency is presented.In saqFRET,photo-activatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein(EGFP)are used to simplify FRET crosstalk elimination.Traditional manual image analyses are time consuming when the dataset is large.The proposed automatic image analyses based on deep learning can analyze 100 samples within 30 s and demonstrate the same precision as manual image analyses.

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.

Green emitted CdSe@ZnS quantum dots for FLIM and STED imaging applications

Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for bioimaging.In this study,CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope(FLIM)and stimulated emission depletion(STED)nanoscopy imaging.The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield(PLQY)was 64%.The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW.The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser(27.5 mW)irradiation.Furthermore,the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes.Meanwhile,Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM,and their fluorescence lifetime was around 3.1 ns,consistent with the in vitro value,suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.