Two-dimensional and three-dimensional dynamic imaging of live biofilms in a microchannel by time-of-flight secondary ion mass spectrometry

A vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface), was employed for in situ chemical imaging of live biofilms using time-of-flight secondary ion mass spectrometry (ToF-SIMS). Depth profiling by sputtering materials in sequential layers resulted in live biofilm spatial chemical mapping. Two-dimensional (2D) images were reconstructed to report the first three-dimensional images of hydrated biofilm elucidating spatial and chemical heterogeneity. 2D image principal component analysis was conducted among biofilms at different locations in the microchannel. Our approach directly visualized spatial and chemical heterogeneity within the living biofilm by dynamic liquid ToF-SIMS.Mapping how metabolic pathways are interconnected and controlled at the subcellular scale within dynamic living systems continues to present a grand scientific challenge. Biofilms, consisting of aggregations of bacterial cells and extracellular polymeric substance (EPS), present an important avenue for deciphering complex microbial communities. During biofilm formation, cells assemble in a secreted polymer milieu of polysaccharides, proteins, glycolipids, and DNA.^1,2 Microfluidics provides unprecedented control over flow conditions, accessibility to real-time observation, high-throughput testing, and mimics in vivo biological environments.³ An understanding of the mechanism underlying biofilm formation and the design of advanced microfluidic experiments could address challenges such as interpreting microbial community interactions, biofouling, and resistance to antimicrobial chemicals. However, only a handful of biofilm studies used microfluidic approaches that provided hydrated chemical imaging at high spatial resolution.^4–7 Most studies utilized confocal microscopy,⁴ FTIR spectroscopy,⁵ or other approaches (e.g., high density interdigitated capacitors⁷) for biofilm monitoring. Imaging mass spectrometry has been demonstrated in biofilm studies.^8,9 A coupled microfluidic-imaging mass spectrometry approach would provide the chemical molecular spatial mapping needed to better address the scientific challenge of biofilms.Recently, we developed a portable microfluidic reactor, System for Analysis at the Liquid Vacuum Interface (SALVI),^10,11 which overcame the grand challenge of studying liquids with high volatility and liquid interfaces using surface sensitive vacuum instruments. SALVI enables direct imaging of liquid surfaces using electron or ion/molecular based vacuum techniques. Our microfluidic approach used a polydimethylsiloxane (PDMS) microchannel fully enclosed with a thin silicon nitride (SiN) membrane (100 nm thick). For visualization, 2 μm diameter holes were opened in the SiN membrane in vacuo. These detection windows were dynamically drilled using the time-of-flight secondary ion mass spectrometry (ToF-SIMS) primary ion beam (e.g., Bi⁺).¹²Unlike liquid sample holders for transmission electron microscopy and scanning transmission electron microscopy, SALVI is self-contained and portable.¹³ As a result, it can potentially be used in many finely focused analytical tool with minimal adaptation.¹⁰ The analytical performance of SALVI has been demonstrated with a variety of analytes ranging from biology to material sciences.^14,15 Unlike most microfluidic applications that are only suitable under ambient conditions (e.g., separations, cell and small amount sample manipulation, and thermal flow-sensors),^16–18 SALVI is compatible with both in situ ambient and in vacuo spectroscopy analysis and imaging.¹⁹ Biofilms have been successfully cultivated inside the microfluidic channel and imaged using correlative confocal laser scanning microscopy (CLSM) and ToF-SIMS.²⁰Our approach opens a new avenue to study biological sample in their natural state. Although ToF-SIMS has been widely used for providing molecular signatures of organic and biological molecules in complex biological systems^21,22 or lipid spatial mapping,²³ the vacuum-based ToF-SIMS generally requires solid (either dried²⁴ or cryo treated²⁵) samples. Here, we report ToF-SIMS two dimensional (2D) and three dimensional (3D) chemical images of hydrated biofilms. In situ time and space-resolved identifications of fatty acid (FA) fragments characteristic of Shewanella are illustrated by 3D images reconstructed from the ToF-SIMS depth profile time series. Principal component analysis (PCA) further elucidates biofilm chemical and spatial heterogeneity and shows the key chemical component at different depth and location of the biofilm including the biofilm-surface attachment interface.For all growth experiments, two samples were cultured simultaneously. At days 5 and 6, one sample was harvested for immediate analysis, respectively, using a ToF-SIMS V spectrometer (IONTOF GmbH, Münster, Germany). Similar results were obtained from both samples, because the biofilm-attachment surface was probed. For consistency, only day 6 data are shown here, while additional data are provided in the supplementary material.²⁸ 2D and 3D image visualizations were obtained using the IONTOF instrument software. PCA was performed using MATLAB R2012a (MathWorks, Inc., Natick, MA, USA). 2D images of .bif format were converted and integrated into a matrix. Data were pretreated by normalization to total ions, square root transformation, and then mean centering.²⁶ For m/z spectra PCA, unit mass peaks from m/z 199 to m/z 255 were used (see Figure S-2²⁸). Unit mass peaks from m/z 1–300 were also used and results are comparable (see Figure S-3²⁸). Five characteristic FA peaks (m/z 199, 213, 227, 241, and 255, corresponding to C12, C13, C14, C15, and C16 FAs) were used in image PCA.²⁷ Images representing each PC were reconstructed from the score matrix using the red, green, and blue (RGB) color scale.Using depth profiling, we drilled through the SiN membrane and collected depth-resolved images of the live biofilm (Figure 1(a)). Our analysis of the negative ToF-SIMS spectra after SiN punch-through showed Shewanella FA fragments in the m/z 195–255 range.²⁰ From the depth profile time series, we selected five regions (highlighted as I, II, III IV, and V) within the FA m/z range to visualize 2D spatially resolved images collected for 46 s (1000 scans) before (I), during (II), or after (III, IV, V) SiN membrane punch-through.²⁰ When false color 2D images of FA fragments characteristic of Shewanella biofilms were selected from the dynamic depth profiling data, differences were observed (Figure 1(b)) among the five regions. Furthermore, the biofilm images after SiN membrane punch-through (III, IV, V) displayed variations across the 2 μm diameter surfaces, with C12 (m/z 199) being distributed across regions III, IV, and V and C15 (m/z 241) FAs mostly in region V (see Figure S-4 for additional FA images²⁸). This suggested that depth-resolved chemical heterogeneities were present in the biofilm. To illustrate, we reconstructed the 2D images from depth profiling data within the biofilm region (from the beginning of III through the end of V) and show spatially resolved 3D chemical images within the entire sample (Figure 1(c) and movies S1-S3²⁸). The reconstructed 3D images revealed the heterogeneous spatial distribution overlay for C12 (red) and C15 (green) FAs during 302 s biofilm depth profiling from day 5 (Figure S-5²⁸) and day 6 (Figure 1(c)).Open in a separate windowFIG. 1.(a) ToF-SIMS depth profiling of the day 6 biofilm attached to the SiN membrane in the microfluidic channel. Five regions representing sample before SiN punch-through (I) during punch-through (II) or within the biofilm region (III, IV, and V) are illustrated. (b) 2D false color images of day 6 biofilm FAs at the five time regions highlighted in (a). (c) Reconstructed 3D day 6 biofilm images showing FA fragment distributions within the entire biofilm region (III–V, 302 s). The time axis represents depth profiling from near the SiN surface into the biofilm. (d) Spectra PCA score plot of day 6 biofilm showing the differences and similarities among selected five regions (m/z 199–255). A 95% confidence limit for each region was defined by an ellipse with the same color to the corresponding region clusters. (e) Loadings of PC1 and PC2 corresponding to (d) and the plot of PC variance contributions.Spectral PCA was used to analyze the m/z spectra. The deepest region (V) into the biofilm was the most different from the other two biofilm regions (III and IV), further confirming the heterogeneities observed in the 2D images (e.g., C12 and C15 FA fragments) contributing most to this spatial difference. In addition, C12 FA fragments played a key role in the biofilms imaged near the SiN membrane attachment surface (III and IV). When inspected individually, C12 FAs were observed throughout the entire biofilm region, suggesting that C12 FA fragments may play a role in biofilm attachment to a surface and they may be main components of EPS throughout the biofilm. In contrast, C15 FAs were more abundant deeper within the biofilm, indicating that they may be more relevant to bacteria cells themselves.Uniform sputtering rate was assumed during depth profiling. To better determine the depth and shape of the SIMS ionization crater, AFM measurements were collected using an agarose sample in the SALVI reactor as a proxy for the biofilms (Figure S-6²⁸). The AFM results showed that the 100 nm SiN was drilled through and confirmed that the biofilm interface was probed by ToF-SIMS. Ideally, real-time correlative AFM and ToF-SIMS measurements will be needed due to the self-healing property of biofilms. However, such capability is currently under development.To further analyze chemical differences within biofilms, we performed ToF-SIMS depth profiling at three locations along the microchannel; namely, the inlet, center, and outlet as illustrated in Figure S-1(b).²⁸ At each location, we defined the five regions described in Figure 1(a), and 2D image PCA analysis was conducted on the biofilm region (from the beginning of III through the end of V) to visualize the chemical distributions on day 6. Figure 2(a) shows the loading plots for the m/z peaks that contribute to each PC image (Figure 2(b)). The first three PCs explained 93.79% of the variance within the data. For PC1, the strongest positive loading fragments were C12 and C15 FAs, which are the bright red areas in three PC1 images. The C12 FAs were the main contributor to the green regions in the PC2 image. The strongest loading for PC3 in blue was C14 FAs. Compared to PC1 and PC2, PC3 played a limited contribution to the overall spatial distribution discrimination. The merged images give a demonstration of chemical spatial distribution of key components of biofilms in the liquid microenvironment.Open in a separate windowFIG. 2.(a) Image PCA loading plots illustrating the contribution of each FA peak in the day 6 biofilm at three locations within the microfluidic channel. The variance contributions of each PC are shown at the bottom. (b) Reconstructed false-color 2D PCA images in RGB corresponding to each PC scores at these locations along the microfluidic channel. The RGB composite images of the three key PCs are depicted in the bottom. Only data within the 2 μm diameter circle were considered in analysis.Our results show that SALVI and liquid ToF-SIMS studies of live biofilms offer dynamic, depth-resolved chemical mapping and produce 2D and 3D visualizations of spatial heterogeneity within a biofilm. Chemical imaging of biofilms near the attachment interface can enhance our understanding of biofilm formation in environmental, medical, and industrial settings. Our approach provides a universal portable platform and enables in situ probing of complex living biological systems potentially across multiple time and space scales. Because of the portability and vacuum compatibility, SALVI offers a valuable linkage with proteomic mass spectrometry via microfluidics and a nondestructive package for integrative in situ analysis of live biological systems in system biology.     

Potential application of a knowledgebase of iron metabolism of Acidithiobacillus ferrooxidans as an alternative platform

BackgroundAcidithiobacillus ferrooxidans is a facultative anaerobe that depends on ferrous ion oxidation as well as reduced sulfur oxidation to obtain energy and is widely applied in metallurgy, environmental protection, and soil remediation. With the accumulation of experimental data, metabolic mechanisms, kinetic models, and several databases have been established. However, scattered data are not conducive to understanding A. ferrooxidans that necessitates updated information informed by systems biology.ResultsHere, we constructed a knowledgebase of iron metabolism of A. ferrooxidans (KIMAf) system by integrating public databases and reviewing the literature, including the database of bioleaching substrates (DBS), the database of bioleaching metallic ion-related proteins (MIRP), the A. ferrooxidans bioinformation database (Af-info), and the database for dynamics model of bioleaching (DDMB). The DBS and MIRP incorporate common bioleaching substrates and metal ion-related proteins. Af-info and DDMB integrate nucleotide, gene, protein, and kinetic model information. Statistical analysis was performed to elucidate the distribution of isolated A. ferrooxidans strains, evolutionary and metabolic advances, and the development of bioleaching models.ConclusionsThis comprehensive system provides researchers with a platform of available iron metabolism-related resources of A. ferrooxidans and facilitates its application.How to citeZhou Z, Ma W, Liu Y, et al. Potential application of a knowledgebase of iron metabolism of Acidithiobacillus ferrooxidans as an alternative platform. Electron J Biotechnol 2021;51; https://doi.org/10.1016/j.ejbt.2021.04.003     

Development of a method for automatic basso continuo playing

The purpose of this study is to develop an automatic basso continuo playing system. In order to find a musically appropriate sequence of chords, we propose the principle of “harmony cost”, which is defined as the sum of two different costs: one is the “local cost” which indicates the unlikelihood of a certain chord allocation and the other is the “transition cost”, which indicates the unlikelihood of a certain connection between two chords. Automatic basso continuo playing is realised by searching for the optimal chord sequence, which minimises the accumulated harmony cost. The proposed method is evaluated by three experiments. Each experiment verifies the effectiveness of the proposed method.     

Numerical simulation on the opto-electro-kinetic patterning for rapid concentration of particles in a microchannel

This paper presents a mathematical model for laser-induced rapid electro-kinetic patterning (REP) to elucidate the mechanism for concentrating particles in a microchannel non-destructively and non-invasively. COMSOL^®(v4.2a) multiphysics software was used to examine the effect of a variety of parameters on the focusing performance of the REP. A mathematical model of the REP was developed based on the AC electrothermal flow (ACET) equations, the dielectrophoresis (DEP) equation, the energy balance equation, the Navier-Stokes equation, and the concentration-distribution equation. The medium was assumed to be a diluted solute, and different electric potentials and laser illumination were applied to the desired place. Gold (Au) electrodes were used at the top and bottom of a microchannel. For model validation, the simulation results were compared with the experimental data. The results revealed the formation of a toroidal microvortex via the ACET effect, which was generated due to laser illumination and joule-heating in the area of interest. In addition, under some conditions, such as the frequency of AC, the DEP velocity, and the particle size, the ACET force enhances and compresses resulting in the concentration of particles. The conditions of the DEP velocity and the ACET velocity are presented in detail with a comparison of the experimental results.     

Modulation of rotation-induced lift force for cell filtration in a low aspect ratio microchannel

Cell filtration is a critical step in sample preparation in many bioapplications. Herein, we report on a simple, filter-free, microfluidic platform based on hydrodynamic inertial migration. Our approach builds on the concept of two-stage inertial migration which permits precise prediction of microparticle position within the microchannel. Our design manipulates equilibrium positions of larger microparticles by modulating rotation-induced lift force in a low aspect ratio microchannel. Here, we demonstrate filtration of microparticles with extreme efficiency (>99%). Using multiple prostate cell lines (LNCaP and human prostate epithelial tumor cells), we show filtration from spiked blood, with 3-fold concentration and >83% viability. Results of a proliferation assay show normal cell division and suggest no negative effects on intrinsic properties. Considering the planar low-aspect-ratio structure and predictable focusing, we envision promising applications and easy integration with existing lab-on-a-chip systems.     

一种平原区园地遥感信息提取的新方法

针对平原区园地与居民点落叶林不易区分的特点,提出适合于中分辨遥感影像的平原区园地信息提取特征指标——平原区园地指数.利用影像的多时相特征,结合面向对象的分类方法构建平原区园地信息提取模式.安徽省砀山县园地提取实验表明,该方法简单易行,有效避免了“椒盐现像”,提高了分类精度,对于准确地确定平原区园地面积及其分布情况具有重要的实际应用价值.     

生态文明视角下江西省旅游业发展浅探

相比于工业文明来说,生态文明是一种高级且更为先进的现代文明新形态,而且它的核心主张是从传统的人对自然的统治转变为人与自然的和谐发展。本文以江西省为例,结合其当前旅游发展的生态环境基础及影响,对未来江西旅游业发展的相关策略进行了探究。     

北京市高端制造业全产业链发展模式研究—以高性能碳纤维为例

以专利分析为视角,结合碳纤维产业数据并开展典型企业调研,分析了国内外及北京市的产业发展现状,并对国内及北京市碳纤维全产业链所存在的问题进行解读。碳纤维产业长期被日本和欧美所垄断,我国技术与产品与国外仍然存在代差。目前,我国已经初步构建出产学研用合作发展的模式,北京市研发端实力强劲,并涌现出航空航天领域等一批高端制造业企业对碳纤维发展提出了充足的牵引动力,具备整合国内优质资源的基础条件。根据北京市的资源和优势,以碳纤维为例,构建以高端需求为牵引的北京市高端制造业全产业链发展模式。     

西部传统工业城市转型发展策略研究——以广西百色市为例

就目前国内工业类型而言,从原料来源角度分析可分为资源型、来料加工型、混合型三种类型,我国西部地区多以资源型为主。由于本土资源难以为继和城市功能结构缺失,以及产业优化与提升等原因,将促使西部传统工业城市转型。而在转型过程中,功能结构调整与补充、产业联动发展、交通物流打造将是西部工业城市转型主要发展策略。     

区域中小企业成长发展水平综合评价研究——以珠三角地区为例

通过构造区域中小企业成长发展评价模型,从区域中小企业发展规模、盈利能力、偿债能力和营运能力等四个方面评价区域中小企业的发展水平、发展速度和增长水平,并以珠江三角洲地区2001—2009年度的中小企业发展数据为例,对珠三角地区中小企业的成长发展水平进行了综合评价。     

创新顾客参与新产品开发活动的激励机制——以JS科技公司为例

针对现有研究缺乏结合创新贡献率和关系强度对创新顾客推荐激励模型进行系统探索,基于创新顾客参与新产品开发活动的重要性及其与普通顾客的差异性,构建创新顾客参与新产品开发活动的产品推荐激励模型,确定企业需要设置推荐激励计划时的条件,并以JS公司为例,通过计算机模拟仿真对模型的推导结论进行验证。结果发现,企业给予创新顾客的推荐激励强度比给予普通顾客的低,但创新顾客带来的净利润比普通顾客高;当创新顾客只向与其为强关系的朋友推荐产品/服务,或与其为强关系朋友的占比较大时,企业不需要设置推荐激励计划;随着创新顾客的创新贡献率增大,企业应减小推荐激励强度,而随着创新顾客与被推荐者之间弱关系强度增大,企业应增加推荐激励强度。研究为企业提出管理启示：应积极引导创新顾客进行产品推荐,并结合创新顾客与被推荐者的关系强度优化推荐激励计划,同时应重视创新顾客进行产品推荐的真实性。     

Urinary Retention as a Cause of Hyponatremia in an Elderly Man

Hyponatremia is a common disorder in elderly and can result in changes in cognition, seizures, coma or even respiratory arrest if not recognised and treated. Syndrome of inappropriate anti diuretic hormone secretion (SIADH) is the most common cause of hyponatremia in elderly hospitalised patients and in most cases the etiology cannot be determined on routine investigations. We present a 76 year old male with symptomatic hyponatremia who had chronic urinary retention due to a urethral stricture. His sodium levels improved with catheterisation and worsened again after the catheter was removed. This supports the hypothesis that urinary retention and bladder distension can stimulate ADH release from the posterior pituitary, producing a picture similar to SIADH.     

竞争情报软件云时代发展分析——SaaS的应用

以云计算的SaaS模式为例,探讨了目前竞争情报软件在利用云计算技术方面的尝试,分析了软件SaaS化的诸多优点,最后结合现有状况提出了竞争情报软件在SaaS应用方面的未来发展趋势。     

科技人员“双创”政策执行现状及建议--以江西省省属科研院所为例

江西省鼓励科技人员"双创"的有关政策出台后,对省内25个省属独立科研院所共2 500多名科技人员开展政策执行效果跟踪调研,在国内现有政策执行模式以及修正的"模糊-冲突"模型基础上,从政策执行主体入手剖析"双创"政策执行进程,发掘影响"双创"政策执行成效的关键性因素,提出提高"双创"政策执行成效的对策建议。     

加快地区经济发展 着力构建和谐社会——以乌兰察布市地方经济为例

文章针对乌兰察布市经济发展的制约因素,提出了促进地区经济发展、构建社会主义和谐社会的对策。     

新时代高校快递物流的发展思路——以山东农业大学为例

电子商务的高速发展、各大快递企业之间的激烈竞争和后疫情时代的到来,对新时代高校快递的发展产生了深远影响。面对高校快递发展出现的新情况、新要求、新问题,我们亟须开展新的研究,从加强校企合作、提高系统运作效率、提升服务水平、优化利益分配结构、发展绿色环保快递业务等方面出发,全面提升高校的快递业务水平。