DeepSeeded: Volumetric Segmentation of Dense Cell Populations with a Cascade of Deep Neural Networks in Bacterial Biofilm Applications.

Accurate and automatic segmentation of individual cell instances in microscopy images is a vital step for quantifying the cellular attributes, which can subsequently lead to new discoveries in biomedical research. In recent years, data-driven deep learning techniques have shown promising results in this task. Despite the success of these techniques, many fail to accurately segment cells in microscopy images with high cell density and low signal-to-noise ratio.

Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake.

Single-molecule tracking to determine the abundances and stoichiometries of freely-diffusing protein complexes in living cells: Past applications and future prospects.

Most biological processes in living cells rely on interactions between proteins. Live-cell compatible approaches that can quantify to what extent a given protein participates in homo- and hetero-oligomeric complexes of different size and subunit composition are therefore critical to advance our understanding of how cellular physiology is governed by these molecular interactions. Biomolecular complex formation changes the diffusion coefficient of constituent proteins, and these changes can be measured using fluorescence microscopy-based approaches, such as single-molecule tracking, fluorescence correlation spectroscopy, and fluorescence recovery after photobleaching.

Dimerization of iLID optogenetic proteins observed using 3D single-molecule tracking in live E. coli.

3D single-molecule tracking microscopy has enabled measurements of protein diffusion in living cells, offering information about protein dynamics and cellular environments. For example, different diffusive states can be resolved and assigned to protein complexes of different size and composition. However, substantial statistical power and biological validation, often through genetic deletion of binding partners, are required to support diffusive state assignments.

BCM3D 2.0: accurate segmentation of single bacterial cells in dense biofilms using computationally generated intermediate image representations.

Accurate detection and segmentation of single cells in three-dimensional (3D) fluorescence time-lapse images is essential for observing individual cell behaviors in large bacterial communities called biofilms. Recent progress in machine-learning-based image analysis is providing this capability with ever-increasing accuracy. Leveraging the capabilities of deep convolutional neural networks (CNNs), we recently developed bacterial cell morphometry in 3D (BCM3D), an integrated image analysis pipeline that combines deep learning with conventional image analysis to detect and segment single biofilm-dwelling cells in 3D fluorescence images.

Distinct Cytosolic Complexes Containing the Type III Secretion System ATPase Resolved by Three-Dimensional Single-Molecule Tracking in Live Yersinia enterocolitica.

The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by Gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations and freely diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear.

3D GAN image synthesis and dataset quality assessment for bacterial biofilm.

Motivation: Data-driven deep learning techniques usually require a large quantity of labeled training data to achieve reliable solutions in bioimage analysis. However, noisy image conditions and high cell density in bacterial biofilm images make 3D cell annotations difficult to obtain. Alternatively, data augmentation via synthetic data generation is attempted, but current methods fail to produce realistic images.

Optically Accessible Microfluidic Flow Channels for Noninvasive High-Resolution Biofilm Imaging Using Lattice Light Sheet Microscopy.

Imaging platforms that enable long-term, high-resolution imaging of biofilms are required to study cellular level dynamics within bacterial biofilms. By combining high spatial and temporal resolution and low phototoxicity, lattice light sheet microscopy (LLSM) has made critical contributions to the study of cellular dynamics. However, the power of LLSM has not yet been leveraged for biofilm research because the open-on-top imaging geometry using water-immersion objective lenses is not compatible with living bacterial specimens; bacterial growth on the microscope's objective lenses makes long-term time-lapse imaging impossible and raises considerable safety concerns for microscope users.

Graph-Theoretic Post-Processing of Segmentation With Application to Dense Biofilms.

Recent deep learning methods have provided successful initial segmentation results for generalized cell segmentation in microscopy. However, for dense arrangements of small cells with limited ground truth for training, the deep learning methods produce both over-segmentation and under-segmentation errors. Post-processing attempts to balance the trade-off between the global goal of cell counting for instance segmentation, and local fidelity to the morphology of identified cells.

Non-invasive single-cell morphometry in living bacterial biofilms.

Fluorescence microscopy enables spatial and temporal measurements of live cells and cellular communities. However, this potential has not yet been fully realized for investigations of individual cell behaviors and phenotypic changes in dense, three-dimensional (3D) bacterial biofilms. Accurate cell detection and cellular shape measurement in densely packed biofilms are challenging because of the limited resolution and low signal to background ratios (SBRs) in fluorescence microscopy images.

Facile Synthesis and Metabolic Incorporation of -DAP Bioisosteres Into Cell Walls of Live Bacteria.

Bacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes cross-linked by cell wall transpeptidases (TP). While synthetic PG fragments containing l-lysine in the third position on the stem peptide are easier to access, those with -diaminopimelic acid (-DAP) pose a severe synthetic challenge.

Structural Similarity Image Analysis for Detection of Adenosine and Dopamine in Fast-Scan Cyclic Voltammetry Color Plots.

Fast-scan cyclic voltammetry (FSCV) is widely used for in vivo detection of neurotransmitters, but identifying analytes, particularly mixtures, is difficult. Data analysis has focused on identifying dopamine from cyclic voltammograms, but it would be better to analyze all the data in the three-dimensional FSCV color plot. Here, the goal was to use image analysis-based analysis of FSCV color plots for the first time, specifically the structural similarity index (SSIM), to identify rapid neurochemical events.

Reprogramming fatty acyl specificity of lipid kinases via C1 domain engineering.

C1 domains are lipid-binding modules that regulate membrane activation of kinases, nucleotide exchange factors and other C1-containing proteins to trigger signal transduction. Despite annotation of typical C1 domains as diacylglycerol (DAG) and phorbol ester sensors, the function of atypical counterparts remains ill-defined. Here, we assign a key role for atypical C1 domains in mediating DAG fatty acyl specificity of diacylglycerol kinases (DGKs) in live cells.

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit.

Enabling technologies in super-resolution fluorescence microscopy: reporters, labeling, and methods of measurement.

Super-resolution fluorescence microscopy continues to experience a period of extraordinary development. New instrumentation and fluorescent labeling strategies provide access to molecular and cellular processes that occur on length scales ranging from nanometers to millimeters and on time scales ranging from milliseconds to hours. At the shortest length scales, single-molecule imaging methods now allow measurement of nanoscale localization, motion, and binding kinetics of individual biomolecules.

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking.

The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate well-sampled distributions of experimental measurements and if experimental biases are taken into account during data analysis.

Single-molecule tracking in live Yersinia enterocolitica reveals distinct cytosolic complexes of injectisome subunits.

In bacterial type 3 secretion, substrate proteins are actively transported from the bacterial cytoplasm into the host cell cytoplasm by a large membrane-embedded machinery called the injectisome. Injectisomes transport secretion substrates in response to specific environmental signals, but the molecular details by which the cytosolic secretion substrates are selected and transported through the type 3 secretion pathway remain unclear. Secretion activity and substrate selectivity are thought to be controlled by a sorting platform consisting of the proteins SctK, SctQ, SctL, and SctN, which together localize to the cytoplasmic side of membrane-embedded injectisomes.

Bacterial scaffold directs pole-specific centromere segregation.

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system.

Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging.

The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.

Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions.

We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.

Structure of isolated biomolecules by electron diffraction-laser desorption: uracil and guanine.

We report the structure of isolated biomolecules, uracil and guanine, demonstrating the capability of a newly developed electron diffraction apparatus augmented with surface-assisted IR laser desorption. This UED-4 apparatus provides a pulsed, dense molecular beam, which is stable for many hours and possibly days. From the diffraction patterns, it is evident that the plume composition is chemically pure, without detectable background from ions, fragmentation products, or molecular aggregates.

Ultrashort electron pulses for diffraction, crystallography and microscopy: theoretical and experimental resolutions.

Pulsed electron beams allow for the direct atomic-scale observation of structures with femtosecond to picosecond temporal resolution in a variety of fields ranging from materials science to chemistry and biology, and from the condensed phase to the gas phase. Motivated by recent developments in ultrafast electron diffraction and imaging techniques, we present here a comprehensive account of the fundamental processes involved in electron pulse propagation, and make comparisons with experimental results. The electron pulse, as an ensemble of charged particles, travels under the influence of the space-charge effect and the spread of the momenta among its electrons.