Automated apoptosis identification in fluorescence imaging of nucleus based on histogram of oriented gradients of high-frequency wavelet coefficients

The automatic and accurate identification of apoptosis facilitates large-scale cell analysis.Most identification approaches using nucleus fluorescence imaging are based on specific morphological parameters.However,these parameters cannot completely describe nuclear morphology,thus limiting the identification accuracy of models.This paper proposes a new feature extraction method to improve the performance of the model for apoptosis identification.The proposed method uses a histogram of oriented gradient(HOG)of high-frequency wavelet coefficients to extract internal and edge texture information.The HOG vectors are classified using support vector machine.The experimental results demonstrate that the proposed feature extraction method well performs apoptosis identification,attaining 95:7% accuracy with low cost in terms of time.We confirmed that our method has potential applications to cell biology research.

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.

H_(2)O_(2) INDUCES APOPTOSIS OF RABBIT CHONDROCYTES VIA BOTH THE EXTRINSIC AND THE CASPASE-INDEP ENDENT INTRINSIC PATHW AYS

Ostcoarthritis(OA),one of the most common joint discases with unknown etiology,is charac-terized by the progressive destruction of articular cartilage and the apoptosis of chondrocytes.The purpose of this study is to elucidate the molecular mechanisms of H_(2)O_(2)-mediated rabbit chon-drocytes apoptosis.CCK-8 assay showed that H_(2)O_(2) treatment induced a remarkable reduction of cell viability,which was further verified by the remarkable phosphatidylserine extemalization after H_(2)O_(2) treatment for 1 h,the typical characteristics of apoptosis.H_(2)O_(2) treatment induced a signifcant dysfunction of mitochondrial membrane potential(△ψm),but did not induce casapse-9 activation,indicating that H_(2)O_(2) treat ment induced caspase independent intrinsic apoptosis that was further verified by the fact that silencing of AIF but not inhibiting caspase-9 potently pre-vented H_(2)O_(2)-induced apoptosis.H_(2)O_(2) treatment induced a signifcant increase of caspase8 and-3 activation,and inhibition of caspase-8 or-3 significantly prevented H_(2)O_(2)-induced apoptosis,suggesting that the extrinsic pathway played an important role.Collectively,our findings demonstrate that H_(2)O_(2) induces apoptosis via both the casapse8-mediated extrinsic and the caspaseindependent intrinsic apoptosis pathways in ra bbit chondrocytes.

LIVE IMAGING OF XIAO-AI-PING-INDUCED CELL DEATH IN HUMAN LUNG ADENOCARCINOMA CELLS

Xiao-Ai-Ping(XAP),a traditional oriental medicinal herb isolated from the stem of Marsdenia tenacissima(Roxb.)Wight et Arn.,has been shown to induce tumor cell apoptosis.In this study,we used confocal fluorescence microscopy and fluorescence resonance energy transfer(FRET)techniques to study the molecular mechanism of XAPinduced apoptosis in single living human lung adenocarcinoma(ASTC-a-1)cells.The efficacious apoptosis was observed at 6 h after of 100μl XAP treatment.Further monitoring the dynamics of caspase-3 activation using FRET imaging in single living ASTC-a-1 cell expressing stably with SCAT3,a FRET probe,showed that XAP activated the caspase-3 at about 2 h after XAP treatment.These data suggest that caspase-3 activation was involved in the XAP-induced apoptosis in ASTC-a-1 cells.

Rapid,artifact-reduced,image reconstruction for super-resolution structured illumination microscopy

Super-resolution structured ilumination microscopy(SR-SIM)is finding increasing application in biomedical research due to its superior ability to visualize subcellular dynamics in living cells.However,during image reconstruction artifacts can be introduced and when coupled with time-consuming postprocessing procedures,limits this technique from becoming a routine imaging tool for biologists.To address these issues,an accelerated,artifact-reduced reconstruction algorithm termed joint space frequency reconstruction-based artifact reduction algorithm(JSFR-AR-SIM)was developed by integrating a high-speed reconstruc tion framework with a high-fidelity optimization approach designed to suppress the sidelobe artifact.Consequently,JSFR-AR-SIM produces high-quality,super-resolution images with minimal artifacts,and the reconstruction speed is increased.We anticipate this algorithm to facilitate SR-SIM becoming a routine tool in biomedical laboratories.

Superresolution live-cell imaging reveals that the localization of TMEM106B to filopodia in oligodendrocytes is compromised by the hypomyelination-related D252N mutation

Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development.Initially identified as a lysosomal protein,the TMEM106B D252N mutant has recently been associated with hypomyelination.However,how lysosomal TMEM106B facilitates myelination and how the D252N mutation disrupts that process are poorly understood.We used superresolution Hessian structured illumination microscopy(Hessian-SIM)and spinning discconfocal structured illumination microscopy(SD-SIM)to find that the wild-type TMEM106B protein is targeted to the plasma membrane,filopodia,and lysosomes in human oligodendrocytes.The D252N mutation reduces the size of lysosomes in oligodendrocytes and compromises lysosome changes upon starvation stress.Most importantly,we detected reductions in the length and number of filopodia in cells expressing the D252N mutant.PLP1 is the most abundant myelin protein that almost entirely colocalizes with TMEM106B,and coexpressing PLP1 with the D252N mutant readily rescues the lysosome and filopodia phenotypes of cells.Therefore,interactions between TMEM106B and PLP1 on the plasma membrane are essential for filopodia formation and myelination in oligodendrocytes,which may be sustained by the delivery of these proteins from lysosomes via exocytosis.

An intravascular needle coated by ZnO nanoflowers for in vivo elimination of circulating tumor cells

Circulating tumor cells(CTCs)are recognized as the main source of tumor recurrence and metastasis.Eliminating the CTCs in peripheral blood provides a new strategy to reduce the probability of recurrence or metastasis.Here,we proposed a concept to eliminate CTCs by inserting a needle in the superficial blood vessel.Using the property of ZnO and the structure of nanoflowers,we designed a medical needle coated with ZnO nanoflowers(ZNFs),which killed about 90%of captured CTCs in vitro and prevented the injecting CTCs from spreading to lung tissue in BABL/c mouse model.Results in vitro and in vivo demonstrated that the CTCs not only were captured and killed,but also detached from the needle surface after dead,enabling the ZNFs needle continually eliminate CTCs.Furthermore,a theoretical model was presented to explain the penetration mechanism of cells by nanostructures,which indicated that nanoflowers structure can puncture CTCs more easily than vertical nanowire structure.The concept of inserting an intravascular needle provides a potential strategy to lower the concentration of CTCs in blood and reduce the probability of tumor recurrence or metastasis.

Structured illumination-based super-resolution live-cell quantitative FRET imaging

Forster resonance energy transfer(FRET)microscopy provides unique insight into the functionality of biological systems via imaging the spatiotemporal interactions and functional state of proteins.Distinguishing FRET signals from sub-diffraction regions requires super-resolution(SR)FRET imaging,yet is challenging to achieve from living cells.Here,we present an SR FRET method named SIM-FRET that combines SR structured illumination microscopy(SIM)imaging and acceptor sensitized emission FRET imaging for live-cell quantitative SR FRET imaging.Leveraging the robust co-localization prior of donor and accepter during FRET,we devised a mask filtering approach to mitigate the impact of SIM reconstruction artifacts on quantitative FRET analysis.Compared to wide-field FRET imaging,SIM-FRET provides nearly twofold spatial resolution enhancement of FRET imaging at sub-second timescales and maintains the advantages of quantitative FRET analysis in vivo.We validate the resolution enhancement and quantitative analysis fidelity of SIM-FRET signals in both simulated FRET models and live-cell FRET-standard construct samples.Our method reveals the intricate structure of FRET signals,which are commonly distorted in conventional wide-field FRET imaging.