Deep-learning based flat-fielding quantitative phase contrast microscopy.

Quantitative phase contrast microscopy (QPCM) can realize high-quality imaging of sub-organelles inside live cells without fluorescence labeling, yet it requires at least three phase-shifted intensity images. Herein, we combine a novel convolutional neural network with QPCM to quantitatively obtain the phase distribution of a sample by only using two phase-shifted intensity images. Furthermore, we upgraded the QPCM setup by using a phase-type spatial light modulator (SLM) to record two phase-shifted intensity images in one shot, allowing for real-time quantitative phase imaging of moving samples or dynamic processes. The proposed technique was demonstrated by imaging the fine structures and fast dynamic behaviors of sub-organelles inside live COS7 cells and 3T3 cells, including mitochondria and lipid droplets, with a lateral spatial resolution of 245 nm and an imaging speed of 250 frames per second (FPS). We imagine that the proposed technique can provide an effective way for the high spatiotemporal resolution, high contrast, and label-free dynamic imaging of living cells.

Klebsiella pneumoniae in the intestines of Musca domestica larvae can assist the host in antagonizing the poisoning of the heavy metal copper.

BACKGROUND: Musca domestica larvae are common saprophytes in nature, promoting the material-energy cycle in the environment. However, heavy metal pollution in the environment negatively affects their function in material circulation. Our previous research found that some intestinal bacteria play an important role in the development of housefly, but the responses of microbial community to heavy metal stresses in Musca domestica is less studied. RESULTS: In this study, CuSO4, CuSO4-Klebsiella pneumoniae mixture and CuSO4-K. pneumoniae phage mixture were added to the larval diet to analyze whether K. pneumoniae can protect housefly larvae against Cu2+ injury. Our results showed that larval development was inhibited when were fed with CuSO4, the bacterial abundance of Providencia in the intestine of larvae increased. However, the inhibition effects of CuSO4 was relieved when K. pneumoniae mixed and added in larval diets, the abundance of Providencia decreased. Electron microscope results revealed that K. pneumoniae showed an obvious adsorption effect on copper ion in vitro. CONCLUSIONS: Based on the results we assume that K. pneumoniae could adsorb Cu2+, reduce Cu2+ impact on gut community structure. Our study explains the role of K. pneumoniae antagonizing Cu2+, which could be applied as a probiotic to saprophytic bioantagonistic metal contamination.

Multi-harmonic structured illumination-based optical diffraction tomography.

Imaging speed and spatial resolution are key factors in optical diffraction tomography (ODT), while they are mutually exclusive in 3D refractive index imaging. This paper presents a multi-harmonic structured illumination-based optical diffraction tomography (MHSI-ODT) to acquire 3D refractive index (RI) maps of transparent samples. MHSI-ODT utilizes a digital micromirror device (DMD) to generate structured illumination containing multiple harmonics. For each structured illumination orientation, four spherical spectral crowns are solved from five phase-shifted holograms, meaning that the acquisition of each spectral crown costs 1.25 raw images. Compared to conventional SI-ODT, which retrieves two spectral crowns from three phase-shifted raw images, MHSI-ODT enhances the imaging speed by 16.7% in 3D RI imaging. Meanwhile, MHSI-ODT exploits both the 1st-order and the 2nd-order harmonics; therefore, it has a better intensity utilization of structured illumination. We demonstrated the performance of MHSI-ODT by rendering the 3D RI distributions of 5 µm polystyrene (PS) microspheres and biological samples.

Panoramic quantitative phase imaging of adherent live cells in a microfluidic environment.

Understanding how cells respond to external stimuli is crucial. However, there are a lack of inspection systems capable of simultaneously stimulating and imaging cells, especially in their natural states. This study presents a novel microfluidic stimulation and observation system equipped with flat-fielding quantitative phase contrast microscopy (FF-QPCM). This system allowed us to track the behavior of organelles in live cells experiencing controlled microfluidic stimulation. Using this innovative imaging platform, we successfully quantified the cellular response to shear stress including directional cellular shrinkage and mitochondrial distribution change in a label-free manner. Additionally, we detected and characterized the cellular response, particularly mitochondrial behavior, under varying fluidic conditions such as temperature and drug induction time. The proposed imaging platform is highly suitable for various microfluidic applications at the organelle level. We advocate that this platform will significantly facilitate life science research in microfluidic environments.

Structured illumination phase and fluorescence microscopy for bioimaging.

This study presents a dual-modality microscopic imaging approach that combines quantitative phase microscopy and fluorescence microscopy based on structured illumination (SI) to provide structural and functional information for the same sample. As the first imaging modality, structured illumination digital holographic microscopy (SI-DHM) is implemented along the transmission beam path. SI-DHM acts as a label-free, noninvasive approach and provides high-contrast and quantitative phase images utilizing the refractive index contrast of the inner structures of samples against the background. As the second imaging modality, structured illumination (fluorescence) microscopy (SIM) is constructed along the reflection beam path. SIM utilizes fluorescent labeling and provides super-resolution images for specific functional structures of samples. We first experimentally demonstrated phase imaging of SI-DHM on rice leaves and fluorescence (SIM) imaging on mouse kidney sections. Then, we demonstrated dual-modality imaging of biological samples, using DHM to acquire the overall cell morphology and SIM to obtain specific functional structures. These results prove that the proposed technique is of great importance in biomedical studies, such as providing insight into cell physiology by visualizing and quantifying subcellular structures.

Pseudomonas aeruginosa Y12 play positive roles regulating larval gut communities when housefly encountered copper stress.

Heavy metal contamination has become a global concern that threatens the lives of animals and insects throughout the food chain. Pseudomonas is a commonly found genus of bacteria that colonizes the intestines of insects and constitutes a necessary part of the insect gut microbiota. This research analyzed the influence of different concentrations of Cu2+ on housefly larval development, gut microbial structure and antioxidant defense system, and investigated the regulatory mechanism of P. aeruginosa Y12 on the gut microbiota when houseflies were exposed to Cu2+. We found that adding Cu2+ to the larval diet inhibited larval growth, while the mixed addition of P. aeruginosa Y12 and Cu2+ to the diet reduced the inhibitory effects of Cu2+ on larval growth. Oral administration of Cu2+ significantly changed the gut community structure and increased larval gut bacterial diversity. In vitro analysis revealed that P. aeruginosa Y12 showed Cu2+ adsorption effects and increased Cu2+ aggregation. The mixed addition of low concentrations of P. aeruginosa Y12 and Cu2+ to the larval diet caused a dynamic shift in the gut microbiota and resulted in a novel gut community structure with an increase in beneficial bacteria and a decrease in pathogenic bacteria. Furthermore, P. aeruginosa Y12 treatment influenced the activity of antioxidant enzymes in housefly larvae, indicating that the addition of P. aeruginosa Y12 to the larval diet could further influence the antioxidant system through P. aeruginosa Y12-Cu2+ interactions. In conclusion, our research revealed that intestinal flora dysbiosis was the essential reason why copper inhibits housefly larval growth. However, proper supplementation with P. aeruginosa Y12 played positive roles in regulating larval gut communities and protecting insects from toxic heavy metals.

Reconstruction algorithm using 2N+1 raw images for structured illumination microscopy.

This paper presents a structured illumination microscopy (SIM) reconstruction algorithm that allows the reconstruction of super-resolved images with 2N + 1 raw intensity images, with N being the number of structured illumination directions used. The intensity images are recorded after using a 2D grating for the projection fringe and a spatial light modulator to select two orthogonal fringe orientations and perform phase shifting. Super-resolution images can be reconstructed from the five intensity images, enhancing the imaging speed and reducing the photobleaching by 17%, compared to conventional two-direction and three-step phase-shifting SIM. We believe the proposed technique will be further developed and widely applied in many fields.

Molecular identification of the chitinase genes in Aedes albopictus and essential roles of AaCht10 in pupal-adult transition.

BACKGROUND: Aedes albopictus is an increasingly serious threat in public health due to it is vector of multiple arboviruses that cause devastating human diseases, as well as its widening distribution in recent years. Insecticide resistance is a serious problem worldwide that limits the efficacy of chemical control strategies against Ae. albopictus. Chitinase genes have been widely recognized as attractive targets for the development of effective and environmentally safe insect management measures. METHODS: Chitinase genes of Ae. albopictus were identified and characterized on the basis of bioinformatics search of the referenced genome. Gene characterizations and phylogenetic relationships of chitinase genes were investigated, and spatio-temporal expression pattern of each chitinase gene was evaluated using qRT-PCR. RNA interference (RNAi) was used to suppress the expression of AaCht10, and the roles of AaCht10 were verified based on phynotype observations, chitin content analysis and hematoxylin and eosin (H&E) stain of epidermis and midgut. RESULTS: Altogether, 14 chitinase-related genes (12 chitinase genes and 2 IDGFs) encoding 17 proteins were identified. Phylogenetic analysis showed that all these AaChts were classified into seven groups, and most of them were gathered into group IX. Only AaCht5-1, AaCht10 and AaCht18 contained both catalytic and chitin-binding domains. Different AaChts displayed development- and tissue-specific expression profiling. Suppression of the expression of AaCht10 resulted in abnormal molting, increased mortality, decreased chitin content and thinning epicuticle, procuticle and midgut wall of pupa. CONCLUSIONS: Findings of the present study will aid in determining the biological functions of AaChts and also contribute to using AaChts as potential target for mosquito management.

Bionic Plasmonic Nanoarrays Excited by Radially Polarized Vector Beam for Metal-Enhanced Fluorescence.

Metal-enhanced fluorescence (MEF) is an important fluorescence technology due to its ability to significantly improve the fluorescence intensity. Here, we present a new MEF configuration of the bionic nanorod array illuminated by radially polarized vector beam (RVB). The bionic nanorod array is fabricated via a nanoimprinting method by using the wings of the Chinese cicada "meimuna mongolica" as bio-templates, and later coating gold film by ion sputtering deposition method. The MEF performance of the prepared substrate is tested by a home-made optical system. The experiment results show that, in the case of RVB excitation, the intensity of fluorescence is more than 10 times stronger with the nano-imprinted substrate than that with glass. Using the bionic nanoarray as a substrate, the intensity of fluorescence is ~2 times stronger via RVB than that by the linearly polarized beam. In addition, the prepared substrate is verified to have good uniformity.

Aerobic and facultative anaerobic Klebsiella pneumoniae strains establish mutual competition and jointly promote Musca domestica development.

Introduction: The gut microenvironment in housefly harbors a rich and diverse microbial community which plays a crucial role in larval development. However, little is known about the impact of specific symbiotic bacteria on larval development as well as the composition of the indigenous gut microbiota of housefly. Methods: In the present study, two novel strains were isolated from housefly larval gut, i.e., Klebsiella pneumoniae KX (aerobe) and K. pneumoniae KY (facultative anaerobe). Moreover, the bacteriophages KXP/KYP specific for strains KX and KY were used to analyse the effects of K. pneumoniae on larval development. Results: Our results showed that dietary supplementation with K. pneumoniae KX and KY individually promoted housefly larval growth. However, no significant synergistic effect was observed when the two bacterial strains were administered in combination. In addition, using high-throughput sequencing, it was demonstrated that the abundance of Klebsiella increased whereas that of Provincia, Serratia and Morganella decreased when housefly larvae received supplementation with K. pneumoniae KX, KY or the KX-KY mixture. Moreover, when used combined, K. pneumoniae KX/KY inhibited the growth of Pseudomonas and Providencia. When the abundance of both bacterial strains simultaneously increased, a balance in total bacterial abundance was reached. Discussion: Thus, it can be assumed that strains K. pneumoniae KX and KY maintain an equilibrium to facilitate their development in housefly gut, by establishing competition but also cooperation with each other to maintain the constant composition of gut bacteria in housefly larvae. Thus, our findings highlight the essential role of K. pneumoniae in regulating the composition of the gut microbiota in insects.

Reflectional quantitative differential phase microscopy using polarized wavefront phase modulation.

Quantitative phase microscopy (QPM), as a label-free and nondestructive technique, has been playing an indispensable tool in biomedical imaging and industrial inspection. Herein, we introduce a reflectional quantitative differential phase microscopy (termed RQDPM) based on polarized wavefront phase modulation and partially coherent full-aperture illumination, which has high spatial resolution and spatio-temporal phase sensitivity and is applicable to opaque surfaces and turbid biological specimens. RQDPM does not require additional polarized devices and can be easily switched from reflectional mode to transmission mode. In addition, RQDPM inherits the characteristic of high axial resolution of differential interference contrast microscope, thereby providing topography for opaque surfaces. We experimentally demonstrate the reflectional phase imaging ability of RQDPM with several samples: semiconductor wafer, thick biological tissues, red blood cells, and Hela cells. Furthermore, we dynamically monitor the flow state of microspheres in a self-built microfluidic channel by using RQDPM converted into the transmission mode.

Whole-transcriptome profiling across different developmental stages of Aedes albopictus (Diptera: Culicidae) provides insights into chitin-related non-coding RNA and competing endogenous RNA networks.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, is one of the most invasive species and a vector of numerous arboviruses. The deleterious effects of long-term and inappropriate use of chemical pesticides have stimulated the exploration of new, environmentally friendly control strategies. Non-coding RNAs (ncRNAs) have been proven to participate in almost all biological processes of insects. METHODS: In this study, circular RNAs (circRNAs) and microRNAs (miRNAs) covering five developmental stages [egg, early larvae, late larvae, pupae, adult (female and male)] of A. albopictus were obtained using whole-transcriptome sequencing technology. Combined with long non-coding RNAs (lncRNAs) from previous research, circRNA/lncRNA‒miRNA‒mitochondrial RNA (mRNA) networks were constructed. RESULTS: A total of 1434 circRNAs and 208 miRNAs were identified. More differentially expressed circRNAs (DE circRNAs) and miRNAs (DE miRNAs) were found in the egg versus early larvae comparison group. Functional enrichment analysis demonstrated that most of the circRNA/lncRNA‒miRNA‒mRNA networks were involved in chitin metabolism. Hub genes of each circRNA/lncRNA‒miRNA‒mRNA network were screened out, which can be used as novel targets to disturb the molting process of A. albopictus. CONCLUSIONS: Regulatory relationships obtained from competing endogenous RNA (ceRNA) networks provide more information to manipulate the metamorphosis process and are helpful for developing effective and sustainable methods to control mosquitoes.

Chromosome-level assembly of Culex pipiens molestus and improved reference genome of Culex pipiens pallens (Culicidae, Diptera).

Culex pipiens molestus and Culex pipiens pallens are two distinct bioforms in the Culex pipiens complex that are important vectors of several pathogens and are widely distributed around the world. In the current study, we present a high-quality chromosome-level genome of Cx. pipiens f. molestus and describe the genetic characteristics of this genome. The assembly genome was 559.749 Mb with contig and scaffold N50 values of 200.952 Mb and 0.370 Mb, and more than 94.78% of the assembled bases were located on 3 chromosomes. A total of 19,399 protein-coding genes were predicted. Many gene families were expanded in the genome of Cx. pipiens f. molestus, particularly those of the chemosensory protein (CSP) and gustatory receptor (GR) gene families. In addition, utilizing Hi-C data, we improved the previously assembled draft genome of Cx. pipiens f. pallens, with scaffold N50 of 186.195 Mb and contig N50 of 0.749 Mb, and more than 97.02% of the assembled bases were located on three chromosomes. This reference genome provides a foundation for genome-based investigations of the unique ecological and evolutionary characteristics of Cx. pipiens f. molestus, and the findings in this study will help to elucidate the mechanisms involved in species divergence in the Culex pipiens complex.

Copper and Zinc Co-doped Titanium Dioxide Nanotubes Arrays on Controlling Nitric Oxide Releasing and Regulating the Inflammatory Responses for Cardiovascular Biomaterials.

BACKGROUND: Titanium dioxide (TiO2) nanotubes arrays have shown tremendous application foreground due to their unique characters of structure and performance. However, the single bio-function is still the limit on cardiovascular biomaterials. METHODS: The loadability function provides the possibility for the TiO2 nanotubes arrays to realize composite multifunction. The copper can catalyze the release of nitric oxide to promote the proliferation of endothelium cells and improve the anticoagulant. Also, zinc can adjust the inflammatory responses to improve anti-inflammation. RESULTS: In this patent work, we co-doped the copper and zinc onto TiO2 nanotubes arrays to estimate the hemocompatibility, cytocompatibility and responses of inflammation. The results showed that copper and zinc could introduce better multi-biofunctions to the TiO2 nanotubes arrays for the application in cardiovascular biomaterials. CONCLUSION: In summary, the NTs@Cu/Zn sample as a new composite material in this study had significant biocompatibility in vascular implantation and can be used as a potential material for polymer- free drug-eluting stents.

Large-field lattice structured illumination microscopy.

In this paper, we present large-field, five-step lattice structured illumination microscopy (Lattice SIM). This method utilizes a 2D grating for lattice projection and a spatial light modulator (SLM) for phase shifting. Five phase-shifted intensity images are recorded to reconstruct a super-resolution image, enhancing the imaging speed and reducing the photo-bleaching both by 17%, compared to conventional two-direction and three-shift SIM. Furthermore, lattice SIM has a three-fold spatial bandwidth product (SBP) enhancement compared to SLM/DMD-based SIM, of which the fringe number is limited by the SLM/DMD pixel number. We believe that the proposed technique will be further developed and widely applied in many fields.

Aberrant expression of transglutaminase 2 in pancreas and thymus of NOD mice underscores the importance of deamidation in neoantigen generation.

Post-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2, the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1ß and IFNγ in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD ß-cells underscores the role of deamidation in amplifying T-cell reactivity.

Factors driving the biomass and species richness of desert plants in northern Xinjiang China.

The desert ecosystem is an important part of the terrestrial ecosystem. Accurate estimations of the biomass and species richness of desert plants are of great value for maintaining ecosystem stability; however, current assessments remain a challenge due to the large spatial heterogeneity in biomass and species richness and difficulties posed by time-consuming field surveys, particularly in remote areas. In the present study, There were 527 sampling sites, and each sampling site contained approximately 9 quadrats. Approximately 4500 quadrats in total were taken from the Junggar Desert of northern Xinjiang, and the spatial distribution and factors driving the biomass and species richness of the desert ecosystem were quantitatively analyzed. The results showed that the average aboveground biomass, belowground biomass, litter, and the Patrick index of the Junggar Desert in northern Xinjiang were 115.42 gm-2, 924.77 gm-2, 13.06 gm-2, and 63, with values ranging from 2-708.12 gm-2, 120.25-3537.3 gm-2, 2-56.46 gm-2, and 0-377, respectively, The mean of the variation coefficient was 56.19%, 41.16%, 62.16% and 73.83%, suggesting moderate variation. The result is affected by the differences between the desert environment and climate. Climate factors had a relatively large impact on species richness, and the variation coefficient of species richness was large, indicating a large degree of dispersion of species richness. The direct influence of environmental and climatic factors on underground biomass (BGB) is relatively small, and its coefficient of variation is small. The spatial distribution of biomass and species richness in northern Xinjiang gradually decreased from west to east. Redundancy analysis showed that climate was the main factor driving desert biomass and species richness in northern Xinjiang, with an average independent explanatory power of 20.38% and 18.57%, respectively. Structural equation modeling indicated that climate factors, elevation, and community coverage had a direct positive effect on the aboveground biomass of the desert plants in northern Xinjiang and a direct negative effect on the belowground biomass. Moreover, climate factors and biological factors showed a direct positive effect on the species richness in northern Xinjiang.

Large-field structured illumination microscopy based on 2D grating and a spatial light modulator.

Structured illumination microscopy (SIM) has been widely used in biological research due to its merits of fast imaging speed, minimal invasiveness, super-resolution, and optical sectioning imaging capability. However, the conventional SIM that uses a spatial light modulator (SLM) for fringe projection often has a limited imaging field of view. Herein, we report a large-field SIM technique that combines a 2D grating for fringe pattern projection and an SLM for selecting fringe orientation and performing phase shifting digitally. The proposed SIM technique breaks the bottleneck of fringe number limited by the digital projection devices, while maintaining the advantage of high-speed (digital) phase shifting of conventional SIM. The method avoids the pixilation and dispersion effects of the SLMs. Finally, a 1.8-fold resolution enhancement in a large field of 690 × 517 µm2 under a 20×/NA0.75 objective is experimentally demonstrated. The proposed technique can be widely applied to biology, chemistry, and industry.

Direct axial plane imaging of particle manipulation with nondiffracting Bessel beams.

Optical manipulation with nondiffracting beams has been attracting great interest and finding widespread applications in many fields such as chemistry, physics, and biomedicine. Generally, optical manipulation is conducted in an optical microscopy system, which, in general, only allows for imaging motions of particles in the transverse plane, rendering the observation of dynamics processes occurring in the axial plane impractical. We propose and demonstrate an optical manipulation system that incorporates an axial plane imaging module. With this system, the trapping behavior in the transverse plane and the transportation process in the axial plane of a particle immersed in a Bessel beam were acquired simultaneously in real time.

An Injectable Nanocomposite Hydrogel for Potential Application of Vascularization and Tissue Repair.

In this contribution, an injectable hydrogel was developed with chitosan, gelatin, ß-glycerphosphate and Arg-Gly-Asp (RGD) peptide: this hydrogel is liquid in room temperature and rapidly gels at 37 °C; RGD peptide promises better growth microenvironment for various cells, especially endothelial cells (EC), smooth muscle cells (SMC) and mesenchymal stem cells (MSC). Both stromal cell-derived factor-1 (SDF-1) nanoparticle and vascular endothelial growth factor (VEGF) nanoparticles were loaded in the injectable hydrogel to simulate the natural nanoparticles in the extracellular matrix (ECM) to promote angiogenesis. In vitro EC/SMC and MSC/SMC co-culture experiment indicated that the nanocomposite hydrogel accelerated constructing embryonic form of blood vessels, and chick embryo chorioallantoic membrane model demonstrated its ability of improving cells migration and blood vessel regeneration. We injected this nanocomposite hydrogel into rat myocardial infarction (MI) model and the results indicated that the rats heart function recovered better compared control group. We hope this injectable nanocomposite hydrogel may possess wider application in tissue engineering.