Marker-free Method Research Articles

The Immunoprotection of OmpH Gene Deletion Mutation of Pasteurella multocida on Hemorrhagic Sepsis in Qinghai Yak.

OmpH is among the most important virulence factors of Pasteurella multocida, which mediates septicemia in yaks (Bos grunniens I) after infection with the bacteria. In the present study, yaks were infected with wild-type (WT) (P0910) and OmpH-deficient (ΔOmpH) P. multocida strains. The mutant strain was generated through the reverse genetic operation system of pathogens and proteomics technology. The live-cell bacterial count and clinical manifestations of P. multocida infection in Qinghai yak tissues (thymus, lung, spleen, lymph node, liver, kidney, and heart) were analyzed. The expression of differential proteins in the yak spleen under different treatments was analyzed using the marker-free method. We found that compared with the mutant strain, the titer of wild-type strains was significantly higher in tissues. Additionally, compared with other organs, the bacteria titer was significantly higher in the spleen. Compared with the WT p0910 strain, the mutant strain generated milder pathological changes in the tissues of yak. Proteomics analysis revealed that 57 of the 773 proteins expressed in P. multocida were significantly differentially expressed between the ΔOmpH and P0910 groups. Of the 57, 14 were over-expressed, whereas 43 were under-expressed. The differentially expressed proteins in the ΔompH group regulated the ABC transporter (ATP-powered translocation of many substrates across membranes) system, the two-component system, RNA degradation, RNA transcription, glycolysis/gluconeogenesis, biosynthesis of ubiquinone and other terpenoid-quinones, oxidative phosphorylation (citrate cycle) as well as fructose and mannose metabolism. The relationship among 54 significantly regulated proteins was analyzed using STRING. We found that WT P0910 and ΔOmpH of P. multocida infection activated the expression of ropE, HSPBP1, FERH, ATP10A, ABCA13, RRP7A, IL-10, IFN-γ, IL-17A, EGFR, and dnaJ. Overall, deletion of the OmpH gene weakened the virulence but maintained the immunogenicity of P. multocida in yak. The findings of this study provide a strong foundation for the pathogenesis of P. multocida and the management of related septicemia in yaks.

A Microvision-Based Motion Measurement System for Nanopositioners Using the Feature-to-Phase Method

The development of automation at the nanoscale has been calling for precision motion sensing for robotic nanopositioners. This study presents a microvision-based measurement system for the accurate and efficient motion sensing of three-degree-of-freedom (DOF) nanopositioners. In this measurement system, an optimized target searching (OTS) scheme is proposed for automatic tracking target selection. A strategy is designed to combine feature matching with phase correlation to balance the measurement accuracy and efficiency. By proposing a multiple target tracking scheme, high-precision angular measurement is achieved, and velocity estimation is also provided in this microvision-based measurement system. Subsequently, offline simulations and online experiments are performed to comprehensively evaluate the performances of the microvision-based system with comparisons to traditional instrumentation. The simulation and experimental results demonstrate that the proposed system and marker-free method can realize excellent extensibility, strong robustness, and high precision of motion tracking for the nanopositioners, with measurement accuracy higher than 93 nm and 96 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu $ </tex-math></inline-formula> rad.

A selection marker-free method for gene deletion and editing in baculovirus genomes

Here, we develop a simple, efficient, bacmid-based, selection marker-free method for gene deletion and editing in baculovirus genomes. Specifically, based on pFastbac1, a donor plasmid with long left and right homology arms but without a reporter was constructed for disrupting ie1, an essential baculovirus gene. Instead of ligating with a plasmid, the homology arms were introduced to the polyhedrin locus of BmNPV bacmid using the BmNPV bac-to-bac expression system. Two viruses generated from the modified bacmid and unmodified BmNPV bacmid were then used to co-infect BmN cells in order that recombination takes place at the ie1 locus between them. Finally, without multiple rounds of purification, total cellular DNA was isolated, transformed into Cacl2-treated competent DH10B cells, and then blue colonies were selected for PCR screening. Remarkably, the proportion of blue colonies containing ie1-disrupted bacmid was found to be around 7 %. Moreover, using primers flanking the homology arms further confirmed that all these positive recombinants were double crossovers. These findings indicate that our method is also capable of gene modification if inverse PCR or seamless cloning is used to construct the donor plasmid and sequencing is employed to select positive colonies.

3D Registration Accuracy of a Novel Marker-Free Technique for Double-Scan Protocol.

The aim of this retrospective study was to introduce a novel technique of a double-scan protocol with markerfree registration and compare it to the already-used techniques regarding the accuracy of registration. Fifty-nine fully edentulous patients underwent double-scan procedures by three different methods: the barium sulfate method for 11 patients; the gutta-percha method for 26 patients; and the marker-free method for 22 patients. Point-to-point registration of the two scans was followed by a voxel-based surface "best fit" registration. The mean registration error of each case was digitally recorded. Differences in registration error between groups were evaluated using one-way analysis of variance (ANOVA). The accuracy of the registration showed no significant differences according to the method (P = .719). The marker-free procedure was presented as a novel technique for registration of the scans in the double-scan protocol. There was no significant difference in the accuracy of the registration between the three techniques: marker-free, gutta-percha markers, and fully radiopaque barium sulfate scan appliance. Therefore, the marker-free technique might be a viable option for the double-scan protocol.

A Selection Marker-Free Method for Gene Deletion and Editing in Baculovirus Genomes

A Selection Marker-Free Method for Gene Deletion and Editing in Baculovirus Genomes

NADH Fluorescence Lifetime Imaging Microscopy Reveals Selective Mitochondrial Dysfunction in Neurons Overexpressing Alzheimer's Disease-Related Proteins.

Alzheimer’s disease (AD), the most prevalent form of dementia, affects globally more than 30 million people suffering from cognitive deficits and neuropsychiatric symptoms. Substantial evidence for the involvement of mitochondrial dysfunction in the development and/or progression of AD has been shown in addition to the pathological hallmarks amyloid beta (Aβ) and tau. Still, the selective vulnerability and associated selective mitochondrial dysfunction cannot even be resolved to date. We aimed at optically quantifying mitochondrial function on a single-cell level in primary hippocampal neuron models of AD, unraveling differential involvement of cell and mitochondrial populations in amyloid precursor protein (APP)-associated mitochondrial dysfunction. NADH lifetime imaging is a highly sensitive marker-free method with high spatial resolution. However, deciphering cellular bioenergetics of complex cells like primary neurons has still not succeeded yet. To achieve this, we combined highly sensitive NADH lifetime imaging with respiratory inhibitor treatment, allowing characterization of mitochondrial function down to even the subcellular level in primary neurons. Measuring NADH lifetime of the same neuron before and after respiratory treatment reveals the metabolic delta, which can be taken as a surrogate for cellular redox capacity. Correlating NADH lifetime delta with overexpression strength of Aβ-related proteins on the single-cell level, we could verify the important role of intracellular Aβ-mediated mitochondrial toxicity. Subcellularly, we could demonstrate a higher respiration in neuronal somata in general than dendrites, but a similar impairment of somatic and dendritic mitochondria in our AD models. This illustrates the power of NADH lifetime imaging in revealing mitochondrial function on a single and even subcellular level and its potential to shed light into bioenergetic alterations in neuropsychiatric diseases and beyond.

Quantitative phase microscopy imaging based on fractional spiral phase plate

Quantitative phase imaging (QPI), which combines phase imaging with optical microscopy technology, provides a marker-free, fast, non-destructive, and high-resolution imaging method for observing transparent biological samples. It is widely used in life science, biomedicine, etc. As an emerging QPI technology, spiral phase contrast microscopy (SPCM) uses a spiral phase filter to achieve edge enhancement of amplitude or phase objects. Using the multi-step phase-shifting technology, a complex sample can be measured quantitatively, which has the advantages of high stability, high sensitivity and high precision. However, the SPCM requires at least three-step phase-shifted spiral phase filtered images to achieve the quantitative reconstruction of the amplitude and phase of a sample, and the image acquisition process and the reconstruction process are relatively complicated, which require high stability of system, and the SPCM has low temporal resolution. In order to further improve the performance of SPCM and increase the system stability, sensitivity and temporal resolution, in this paper a quantitative phase imaging method and system based on a fractional spiral phase plate is proposed. Through a sample intensity image filtered by a fractional spiral phase plate, the modified Gerchberg-Saxton iterative phase retrieval algorithm is used to quantitatively reconstruct the phase of a pure phase sample, which simplifies the experimental process and phase reconstruction steps of spiral phase contrast microsocopy. In the computer simulation experiments, the phase imaging process and the reconstruction process of spiral phase plates based on different topological charges are studied, the feasibility of which is analyzed. Finally, through imaging and phase reconstruction of the phase grating and biological cell sample, it is verified that the phase contrast microscopy method based on the fractional spiral phase plate can effectively improve the contrast of spiral phase contrast microscopy and can obtain a quantitative reconstruciton of a weak phase object. The phase information of a sample has significance in research and application for developing the spiral phase contrast microscopy.

A marker-free method for structural dynamic displacement measurement based on optical flow

Information on dynamic displacement is an effective indicator for structures condition evaluation and provides a quantised insight into the structural analysis. This paper proposes a low-cost system based on computer vision using a smartphone to measure dynamic displacement, and then identify the dynamic properties of structures. Conventional sensors like linear variable differential transformers (LVDT), GPS, and accelerometer in monitoring systems have limitations of high price, inaccessibility, and accuracy. However, some new technologies eliminate these disadvantages. For example, a smartphone with a high-resolution camera becomes more affordable, which is regarded as appropriate equipment for structural health monitoring (SHM). Based on the optical flow, this method allows users to track points with a specific interval in the chosen region and reduces the displacement drift induced by the Kanade–Lucas–Tomasi (KLT) method. With the method applied, the region of interest is relocated according to pixel motion, and feature points are reselected. The accuracy of the system is verified on a laboratory suspension bridge model, and the results of modal frequency are confirmed with an accelerometer and FEM simulations.

A marker-free method for registering multi-scan terrestrial laser scanning data in forest environments

Terrestrial laser scanning (TLS) has been recognized as an accurate means for non-destructively deriving three-dimensional (3D) forest structural attributes. These attributes include but are not limited to tree height, diameter at breast height, and leaf area density. As such, TLS has become an increasingly important technique in forest inventory practices and forest ecosystem studies. Multiple TLS scans collected at different locations are often involved for a comprehensive characterization of 3D canopy structure of a forest stand. Among which, multi-scan registration is a critical prerequisite. Currently, multi-scan TLS registration in forests is mainly based on a very time-consuming and tedious process of setting up hand-crafted registration targets in the field and manually identifying the common targets between scans from the collected data. In this study, a novel marker-free method that automatically registers multi-scan TLS data is presented. The main principle underlying our method is to identify shaded areas from the raw point cloud of a single TLS scan and to use them as the key features to register multi-scan TLS data. The proposed method is tested with 17 pairs of TLS scans collected in six plots across China with various vegetation characteristics (e.g., vegetation type, height, and understory complexity). Our results showed that the proposed method successfully registered all 17 pairs of TLS scans with equivalent accuracy to the manual registration approach. Moreover, the proposed method eliminates the process of setting up registration targets in the field, manually identifying registration targets from TLS data, and processing raw TLS data to extract individual tree attributes, which brings it the advantages of high efficiency and robustness. It is anticipated that the proposed algorithms can save time and cost of collecting TLS data in forests, and therefore improves the efficiency of TLS forestry applications.

Marker-free optical stereo motion tracking for in-bore MRI and PET-MRI application.

Prospective motion correction is arguably the "silver bullet" solution for magnetic resonance imaging (MRI) studies impacted by motion, applicable to almost any pulse sequence and immune from the spin history artifacts introduced by a moving object. In prospective motion correction, the magnetic field gradients and radio frequency waveforms are adjusted in real time in response to measured head motion so as to maintain the head in a stationary reference frame relative to the scanner. Vital for this approach are accurate and rapidly sampled head pose measurements, which may be obtained optically using cameras. However, most optical methods are limited by the need to attach physical markers to the skin, which leads to decoupling of head and marker motion and reduces the effectiveness of correction. In this work we investigate the feasibility and initial performance of a stereo-optical motion tracking method which does not require any attached markers. The method relies on detecting distinctive natural features or amplified features (using skin stamps) directly on the forehead in multiple camera views, and then deriving pose estimates via a 3D-2D registration between the skin features and a database of forehead landmarks. To demonstrate the feasibility and potential accuracy of the marker-free method for discrete (step-wise) head motion, we performed out-of-bore and in-bore experiments using robotically and manually controlled phantoms in addition to in-bore testing on human volunteers. We also developed a convenient out-of-bore test bed to benchmark and optimize the motion tracking performance. For out-of-bore phantom tests, the pose estimation accuracy (compared to robotic ground truth) was 0.14mm and 0.23degrees for incremental translation and rotation, respectively. For arbitrary motion, the pose accuracy obtained using the smallest forehead feature patch was equivalent to 0.21±0.11mm positional accuracy in the striatum. For in-bore phantom experiments, the accuracy of rigid-body motion parameters (compared to wireless MR-sensitive markers) was 0.08-0.41±0.18mm/0.05-0.3±0.12deg and 0.14-0.16±0.12mm/0.08-0.17±0.08deg for the small and large feature patches, respectively. In vivo results in human volunteers indicated sub-millimeter and sub-degree pose accuracy for all rotations and translations except the depth direction (max error 1.8mm) when compared to a registration-based approach. In both bench-top and in vivo experiments we demonstrate the feasibility of using very small feature patches directly on the skin to obtain high accuracy head pose measurements needed for motion-correction in MRI brain studies. The optical technique uses in-bore cameras and is consistent with the limited visibility of the forehead afforded by head coils used in brain imaging. Future work will focus on optimization of the technique and demonstration in prospective motion correction.

Temporal comparison of construction sites using photogrammetric point cloud sequences and robust phase correlation

Registration of multi-temporal data is an important task when conducting construction monitoring and analysis using 3D point clouds acquired at different time points. However, due to the complexity of scenes in construction sites and the intrinsic attributes of the datasets (e.g., noise, outliers, and uneven densities), the registration of multi-temporal datasets is a challenging task. To this end, in this paper, we propose a fast and marker-free method for coarse registration of multi-temporal point clouds by integrating a projection-based strategy with Fourier-based signal matching methods, consisting of three major steps: decoupling of 3D transformation with projection-based dimensionality reduction, estimation of horizontal transformation by matching 2D horizontally projected images, and estimation of vertical translation by matching 1D vertical signals. In the decoupling step, the principal projection plane is firstly identified through a robust plane fitting method. Then, each point cloud is decomposed into two independent parts according to the estimated projection plane, namely a 2D image and a 1D signal. The 2D image is obtained by mapping 3D points to the principal projection plane along the vertical direction. Correspondingly, the 1D signals are attained through projecting 3D points to the principal axis in the horizontal direction. In the following step, the rotation, scaling, and translation parameters between 2D images of different point clouds are decoupled with Fourier-Mellin transform. These transformation parameters are then solved with a robust 2D phase correlation algorithm. Simultaneously, 1D signals of non-registered point clouds are matched with a robust 1D phase correlation approach. Finally, combining estimated transformation parameters from both 2D images and 1D signals, the multi-temporal point clouds can be efficiently registered. Our registration method is tested using one TLS benchmark dataset and two multi-temporal point cloud sequences from different construction sites to validate the versatility and effectiveness of the proposed workflow. In terms of the registration accuracy, our proposed method can achieve satisfying performance, with accurate registration for both rotation and translation estimation.

Joint Iterative Fast Projection Matching for Fully Automatic Marker-free Alignment of Nano-tomography Reconstructions

Highly accurate, fully automatic marker-free image alignment plays an important role in nano-tomographic reconstruction, particularly in cases where the spatial resolution of the tomographic system is on the nanometer scale. However, highly accurate marker-free methods such as the projection matching method are computationally complex and time-consuming. Achieving alignment accuracy with reduced computational complexity remains a challenging problem. In this study, we propose an efficient method to achieve marker-free fully automatic alignment. Our method implements three main alignment procedures. First, the frequency-domain common line alignment method is used to correct the in-plane rotational errors of each projection. Second, real-space common line alignment method is used to correct the vertical errors of the projections. Finally, a single layer joint-iterative reconstruction and re-projection method is used to correct the horizontal projection errors. This combined alignment approach significantly reduces the computational complexity of the classical projection matching method, and increases the rate of convergence towards determining the accurate alignment. The total processing time can be reduced by up to 4 orders of magnitude as compared to the classical projection matching method. This suggests that the algorithm can be used to process image alignment of nano-tomographic reconstructions on a conventional personal computer in a reasonable time-frame.

Examination of the dielectrophoretic spectra of MCF7 breast cancer cells and leukocytes.

The detection of circulating tumor cells (CTCs) in blood is crucial to assess metastatic progression and to guide therapy. Dielectrophoresis (DEP) is a powerful cell surface marker-free method that allows intrinsic dielectric properties of suspended cells to be exploited for CTC enrichment/isolation from blood. Design of a successful DEP-based CTC enrichment/isolation system requires that the DEP response of the targeted particles should accurately be known. This paper presents a DEP spectrum method to investigate the DEP spectra of cells without directly analyzing their membrane and cytoplasmic properties in contrast to the methods in literature, which employ theoretical assumptions and complex modeling. Integrating electric field simulations based on DEP theory with the experimental data enables determination of the DEP spectra of leukocyte subpopulations, polymorphonuclear and mononuclear leukocytes, and MCF7 breast cancer cells as a model of CTC due to their metastatic origin over the frequency range 100kHz-50MHz at 10 Vpp . In agreement with earlier findings, differential DEP responses were detected for mononuclear and polymorphonuclear leukocytes due to the richness of the cell surface features and morphologies of the different leukocyte types. The data reveal that the strength of the DEP force exerted on MCF7 cells was particularly high between 850kHz and 20MHz. These results illustrate that the proposed technique has the potential to provide a generic platform to identify DEP responses of different biological particles.

CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) system is being used successfully for efficient and targeted genome editing in various organisms, including the nematode Caenorhabditis elegans. Recent studies have developed a variety of CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: nonhomologous end joining and homologous recombination. Here, we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning (canonical marker-free and cassette selection methods), as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the germline. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Guide RNA preparation Alternate Protocol 1: sgRNA cloning using fusion PCR Basic Protocol 2: Preparation of a repair template for homologous recombination Alternate Protocol 2: Preparation of repair template donors for the cassette selection method Basic Protocol 3: Injecting animals Basic Protocol 4: Screening transgenic worms with marker-free method Alternate Protocol 3: Screening transgenic worms with cassette selection method.

Abstract LB-326: Full-Length mRNA transcriptome analysis of matched circulating tumor and immune cells from breast cancer subjects

Abstract Background Understanding the genetic heterogeneity of circulating immune and tumors cells at single-cell resolution will enable one to decipher the interplay between cancer and immune cells, and ultimately lead to better design of immunotherapy assays. Marker-free methods for isolation of cells are attractive because they provide an opportunity to analyze a larger set of these cells that may otherwise be missed due to variable or no expression of protein (marker) markers. Integration of the Biolidics ClearCell® FX System and the Fluidigm® Polaris™ system enabled us not only to develop a marker-free workflow to isolate immune and circulating tumor cells (CTCs) but also to seamlessly prepare amplified cDNA from immune and CTCs for full-length mRNA-seq analysis. Method and Results The ClearCell FX System processes blood samples and isolates CTCs in a marker-free manner. During the CTC isolation process the immune cells are separated from CTCs on the microfluidic device. To differentiate larger blood cells from CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Different enrichment strategies on Polaris allowed us to either specifically select for CTCs (live+, universal cell marker+, CD45-, CD31-) or immune cells (live+, universal cell marker+, CD45+). The enriched cells were then lysed, mRNA were reverse-transcribed, and cDNA were preamplified on a Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated (off-IFC) using the Nextera® XT DNA library prep protocol and sequenced on Illumina® MiSeq™ systems. We successfully profiled full-length mRNA of 81 CTCs from stage III and IV subjects. Based on hormone receptor status, these six subjects were categorized as three types: (1) ER-/PR-/HER2- (triple-negative breast cancer; n=1), (2) ER+/PR+/HER2- (n=3), and (3) ER+/PR+/HER2- (n=2). For immune cell comparative analysis, we included CD45+ (FACS-sorted) from healthy donor. Unsupervised hierarchal clustering of gene expression data showed clustering by subject with a unique set of genes expressed by triple-negative breast cancer only. Heterogeneity was noted in immune cells from subjects at baseline (pretreatment), under treatment with drugs and a healthy subject without cancer. Using TraCeR computational method, we successfully reconstructed full-length, paired T cell receptor (TCR) sequences from CD45+ single-cell RNA sequence data from breast cancer subjects. Conclusion We present the feasibility of integrating two microfluidics platforms to isolate circulating immune and tumor cells for transcriptome and functional study. Our data suggests that the heterogeneity of immune and cancer cells can be elucidated from single-cell mRNA sequencing data and full-length, paired T cell receptor sequences can be reconstructed from immune cells of breast cancer subjects. Citation Format: Naveen Ramalingam, Yifang Lee, Lukasz Szpankowski, Anne Leyrat, Brian Fowler, Ninez Delos Angeles, Chad Sanada, Andrew Wu, Yoon Sim Yap, Jay West, Ali Asgar Bhagat, Kyle Hukari, Mark Lynch, David King. Full-Length mRNA transcriptome analysis of matched circulating tumor and immune cells from breast cancer subjects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-326.

Modal refractive index measurement in nanowire lasers—a correlative approach

We present a method to correlate multimodal measurements—namely optical spectroscopy and electron microscopy—over large ensembles of randomly distributed single nano-objects. Using an algorithmic approach derived from astrometry, a marker-free method of uniquely associating nano-objects characterised using multiple techniques is described. This approach is applied to nanolasers, enabling an experimental calculation of modal refractive index in sub-micron diameter nanowires. By matching the lasing spectrum and electron microscopy image of 13 nanowire lasers, the refractive index of the TE01 mode in GaAs/AlGaAs multiple-quantum well nanolasers is determined to be ng = 4.7 ± 0.3.

Knockout of rapC Improves the Bacillomycin D Yield Based on De Novo Genome Sequencing of Bacillus amyloliquefaciens fmbJ.

Bacillus amyloliquefaciens, a Gram-positive and soil-dwelling bacterium, could produce secondary metabolites that suppress plant pathogens. In this study, we provided the whole genome sequence results of B. amyloliquefaciens fmbJ, which had one circular chromosome of 4 193 344 bp with 4249 genes, 87 tRNA genes, and 27 rRNA genes. In addition, fmbJ was found to contain several gene clusters of antimicrobial lipopeptides (bacillomycin D, surfactin, and fengycin), and bacillomycin D homologues were further comprehensively identified. To clarify the influence of rapC regulating the synthesis of lipopeptide on the yield of bacillomycin D, rapC gene in fmbJ was successfully deleted by the marker-free method. Finally, it was found that the deletion of rapC gene in fmbJ significantly improved bacillomycin D production from 240.7 ± 18.9 to 360.8 ± 30.7 mg/L, attributed to the increased the expression of bacillomycin D synthesis-related genes through enhancing the transcriptional level of comA, comP, and phrC. These results showed that the production of bacillomycin D in B. amyloliquefaciens fmbJ might be regulated by the RapC-PhrC system. The findings are expected to advance further agricultural application of Bacillus spp. as a promising source of natural bioactive compounds.

Marker-free method for accurate alignment between correlated light, cryo-light, and electron cryo-microscopy data using sample support features.

Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100 nm.

Hfq regulates antibacterial antibiotic biosynthesis and extracellular lytic‐enzyme production inLysobacter enzymogenes OH11

Lysobacter enzymogenes is an important biocontrol agent with the ability to produce a variety of lytic enzymes and novel antibiotics. Little is known about their regulatory mechanisms. Understanding these will be helpful for improving biocontrol of crop diseases and potential medical application. In the present study, we generated an hfq (encoding a putative ribonucleic acid chaperone) deletion mutant, and then utilized a new genomic marker-free method to construct an hfq-complemented strain. We showed for the first time that Hfq played a pleiotropic role in regulating the antibacterial antibiotic biosynthesis and extracellular lytic enzyme activity in L. enzymogenes. Mutation of hfq significantly increased the yield of WAP-8294A2 (an antibacterial antibiotic) as well as the transcription of its key biosynthetic gene, waps1. However, inactivation of hfq almost abolished the extracellular chitinase activity and remarkably decreased the activity of both extracellular protease and cellulase in L. enzymogenes. We further showed that the regulation of hfq in extracellular chitinase production was in part through the impairment of the secretion of chitinase A. Collectively, our results reveal the regulatory roles of hfq in antibiotic metabolite and extracellular lytic enzymes in the underexplored genus of Lysobacter.

Refractive index measurements of single, spherical cells using digital holographic microscopy.

In this chapter, we introduce digital holographic microscopy (DHM) as a marker-free method to determine the refractive index of single, spherical cells in suspension. The refractive index is a conclusive measure in a biological context. Cell conditions, such as differentiation or infection, are known to yield significant changes in the refractive index. Furthermore, the refractive index of biological tissue determines the way it interacts with light. Besides the biological relevance of this interaction in the retina, a lot of methods used in biology, including microscopy, rely on light-tissue or light-cell interactions. Hence, determining the refractive index of cells using DHM is valuable in many biological applications. This chapter covers the main topics that are important for the implementation of DHM: setup, sample preparation, and analysis. First, the optical setup is described in detail including notes and suggestions for the implementation. Following that, a protocol for the sample and measurement preparation is explained. In the analysis section, an algorithm for the determination of quantitative phase maps is described. Subsequently, all intermediate steps for the calculation of the refractive index of suspended cells are presented, exploiting their spherical shape. In the last section, a discussion of possible extensions to the setup, further measurement configurations, and additional analysis methods are given. Throughout this chapter, we describe a simple, robust, and thus easily reproducible implementation of DHM. The different possibilities for extensions show the diverse fields of application for this technique.