A digital microfluidic single-cell manipulation systemoptimized by extending-depth-of-field device

Microfuidic systems have been widely utilized in high-throughput biology analysis,but thedificulties in iquid manipulation and cell cultivation limit its application.This work has developed a new digital microfluidic(DMF)system for on-demand droplet control.By adopting anextending-depth-of-field(EDoF)phase modulator to the optical system,the entire depth of themicrofluidic channel can be covered in one image without any refocusing process,ensuring that 95%of the particles in the droplet are captured within three shots together with shaking pro-cesses.With this system,suspension droplets are generated and droplets containing only oneyeast cll can be recognized,then each single cell is cultured in the array of the chip.Byobservingtheir growth in cell numbers and the green fluorescence protein(GFP)production via fluorescence imaging,the single cell with the highest production can be identified.The results haveproved the heterogeneity of yeast cells,and showed that the combined system can be applied forrapid single-cell sorting,cultivation,and analysis.

Label-free visualization of lignin deposition in loquats using complementary stimulated and spontaneous Raman microscopy

The lignification triggered by biotic or abiotic stresses hardens fruits and vegetables and eventually influences their consumer appeal.Extensive prior efforts have been made to unveil the underlying mechanism of flesh lignification,primarily focused on its physicochemical and molecular biological properties.Nevertheless,most of these studies used destroyed and homogenized bulk tissues as analytes;as a result,potentially valuable spatial information was lost.In this study,the deposition of lignin in loquat flesh during lignification was visualized from the tissue level to the singlecell level by combining the advantages of stimulated Raman scattering(SRS)and spontaneous Raman microscopy using label-free in situ molecular imaging.SRS has the advantages of being fast and providing large-area chemical imaging to reveal the spatial heterogeneity of lignin and cell wall polysaccharide distribution in loquat flesh.After 2 days of storage at 0℃,increased lignins were observed by large-area SRS imaging.In addition,microscopic SRS images of the flesh cells indicated that the increased lignins were trapped in the cell corner(CC)and middle lamella(ML).Furthermore,the compositional and structural features of lignified cells(LCs),CC and ML of loquat flesh were investigated by spontaneous Raman microscopy,and the results showed that the LCs were a combination of lignin,cellulose,and hemicellulose,whereas CC and ML showed only deposited lignin and pectin without cross-linked cellulose and hemicellulose.This result further suggests that the lignins in the CC and ML regions of loquats were later synthesized alone during postharvest storage.This innovative combination of SRS and spontaneous Raman microscopy allows the label-free macroscale and fine chemical imaging of plant cell walls and will enhance our fundamental understanding of the structures and functions of the plant cell wall.

Structural and dynamical mechanisms of a naturally occurring variant of the human prion protein in preventing prion conversion

Prion diseases are associated with the misfolding of the normal helical cellular form of prion protein (PrPC) into the β-sheet-rich scrapie form (PrPSc) and the subsequent aggregation of PrPSc into amyloid fibrils. Recent studies demonstrated that a naturally occurring variant V127 of human PrPC is intrinsically resistant to prion conversion and aggregation, and can completely prevent prion diseases. However, the underlying molecular mechanism remains elusive. Herein we perform multiple microsecond molecular dynamics simulations on both wildtype (WT) and V127 variant of human PrPC to understand at atomic level the protective effect of V127 variant. Our simulations show that G127V mutation not only increases the rigidity of the S2–H2 loop between strand-2 (S2) and helix-2 (H2), but also allosterically enhances the stability of the H2 C-terminal region. Interestingly, previous studies reported that animals with rigid S2–H2 loop usually do not develop prion diseases, and the increase in H2 C-terminal stability can prevent misfolding and oligomerization of prion protein. The allosteric paths from G/V127 to H2 C-terminal region are identified using dynamical network analyses. Moreover, community network analyses illustrate that G127V mutation enhances the global correlations and intra-molecular interactions of PrP, thus stabilizing the overall PrPC structure and inhibiting its conversion into PrPSc. This study provides mechanistic understanding of human V127 variant in preventing prion conversion which may be helpful for the rational design of potent anti-prion compounds.

Polymorphism of Kdo-Based Glycolipids:The Elaborately Determined Stable and Dynamic Bicelles

Understanding how the diversity of glycolipids,including how their chemical structures and composition affect their biological functions,is a remarkable fundamental challenge.In this work,we employed a rare monosaccharide,3-deoxy-Dmanno-2-octulosonic acid(Kdo)to build a simple and biomimetic model to understand the diversity of glycolipids from the viewpoint of supramolecular chemistry.Kdo was chosen not only because its unusual 8-carbon acidic carbohydrate backbone is very different from common hexoses,but also because of its key structural role in lipopolysaccharides and prevalence in bacteria,plant life,and algae.It was found that although both of the two Kdo-lipids S-Kdo and Kdo-S derived from the same carbohydrate backbone and gave bicelles as their self-assembled morphology,experimental results revealed that the self-assembly showed pathway complexity.Bicelle is the thermodynamic product of S-Kdo,while for Kdo-S,the bicelle is only a kinetically trapped state,which finally transforms to a ribbon.Molecular simulation clearly revealed the different packing of Kdo-lipids in the bicelles with different contribution from hydrogen bonds and electrostatic interactions.

Engineering single-molecule fluorescence withasymmetric nano-antennas

As a powerful tool for studying molecular dynamics in bioscience,single-molecule fluorescence detection providesdynamical information buried in ensemble experiments.Fluorescence in the near-infrared(NIR)is particularly usefulbecause it offers higher signal-to-noise ratio and increased penetration depth in tissue compared with visiblefluorescence.The low quantum yield of most NIR fluorophores,however,makes the detection of single-moleculefluorescence difcult.Here,we use asymmetric plasmonic nano-antenna to enhance the fluorescence intensity ofAlEE1000,a typical NIR dye,by a factor up to 405.The asymmetric nano-antenna achieve such an enhancement mainlyby increasing the quantum yield(to~80%)rather than the local field,which degrades the molecules'photostability.Our coupled-mode-theory analysis reveals that the enhancements stem from resonance-matching between antennaand molecule and,more importantly,from optimizing the coupling between the near-and far-ield modes withdesigner asymmetric structures.Our work provides a universal scheme for engineering single-molecule fluorescence inthe near-infrared regime.

AgInS_(2)/ZnS quantum dots for noninvasive cervical cancer screening with intracellular pH sensing using fluorescence lifetime imaging microscopy

Intracellular pH plays a critical role in biological functions,and abnormal pH values are related to various diseases.Here,we report on an intracellular pH sensor AgInS_(2)(AIS)/ZnS quantum dots(QDs)that show long fluorescence lifetimes of hundreds of nanoseconds and low toxicity.Fluorescence lifetime imaging microscopy(FLIM)combined with AIS/ZnS QDs is used for the imaging of live cells in different pH buffers and different cell lines.The FLIM images of AIS/ZnS QDs in live cells demonstrate different intracellular pH values in different regions,such as in lysosomes or cytoplasm.This method can also distinguish cancer cells from normal cells,and the fluorescence lifetime difference of the AIS/ZnS QDs between the two types of cells is 100±7 ns.Most importantly,the exfoliated cervical cells from 20 patients are investigated using FLIM combined with AIS/ZnS QDs.The lifetime difference value between the normal and cervical cancer(CC)groups is 115±9 ns,and the difference between the normal and the precancerous lesion group is 64±9 ns.For the first time,the noninvasive method has been used for cervical cancer screening,and it has shown great improvement in sensitivity compared with a clinical conventional cytology examination.