Search bioRxiv⌕ Search

Biology subjects

Bible, A. N.

Publications and source records attributed to Bible, A. N..

7 recordsLinked to original sources

TileBac: A Benchmark CryoEM Dataset of Bacteria in Ultralow-Dose Montage Tiles

Current segmentation models are capable of routine identification of biological features in noisy cryogenic electron microscopy (cryoEM) images. However, there are still challenges with complete segmentation of high boundary, thin objects such as bacterial cell envelopes and flagella. Moreover, ultralow-dose cryoEM images pose as an additional challenge to boundary distinctions between the object and background. Here, we present TileBac, a benchmark dataset of ultralow-dose montage tiles of Pantoea sp. YR343 to segment bacterial inner and outer membranes for evaluation of model effectiveness. We show that foundation models outperform convolutional neural networks at continuous bacterial cell envelope segmentation despite having lower performance metrics. We release the TileBac benchmark dataset on Hugging Face for further insights into model architecture development.

microbiology↗

Top Model Decision Tree: Selecting Segmentation Models for Reliable Quantitative Analysis in Low- and Ultralow-Dose CryoEM

Deep learning neural networks provide a powerful approach for segmenting low-contrast cryogenic electron microscopy (cryoEM) images. However, model performance can vary significantly across imaging conditions and may hinder downstream quantitative analyses. Here, we present a structured evaluation workflow to systematically screen segmentation models based on performance, inference speed, robustness across imaging conditions, and reliability of downstream quantitative measurements. Using the Bacterial Cell Envelope Thickness Tool (BCET) as a test case, we evaluate multiple architectures (YOLOv11, YOLO26, U-Net, Detectron2, and SAM3) under low-dose and ultralow-dose cryoEM conditions. While several models achieve high metrics, model choice strongly influences downstream measurements of envelope thickness. Models optimized for high F1-scores may produce unreliable segmentation masks from object crowding, interpolation artifacts or imaging conditions. Our results reveal distinct trade-offs between performance, speed, and robustness amongst models. YOLOv11 provides the highest fidelity membrane segmentation for quantitative measurements and the Meta-based model SAM3 offers improved robustness under ultralow-dose conditions with competitive inference performance. This work provides practical guidance for model selection in cryoEM workflows, emphasizing that optimal choice depends on experimental priorities and downstream analysis requirements rather than metrics alone. These findings are broadly relevant to cryoEM workflows as AI-based analysis expands beyond the biological sciences. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/730486v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@f29df4org.highwire.dtl.DTLVardef@601d6eorg.highwire.dtl.DTLVardef@2c5023org.highwire.dtl.DTLVardef@1413f76_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

Facility-Scale Workflows for Data Acquisition, Standardization, Machine Learning Analysis, and Reproducible Science

Scientific user facilities routinely generate large-scale microscopy datasets across diverse instruments and vendors, differing substantially in file formats, dimensionality, and resolution. Beyond these inconsistencies, datasets are frequently fragmented living across isolated instruments and constrained by security policies and uneven metadata practices. Consequently, tracking, standardizing, processing, and visualizing these datasets in a manner compatible with modern machine learning and autonomous experimentation workflows remains a major challenge. While existing initiatives address data archiving, standardization, or analysis individually, few provide integrated solutions that bridge instrument-level acquisition and scalable ML workflows within heterogeneous, security-constrained user facilities. Here, we establish a deployable, facility-scale infrastructure that bridges instrument-level data generation with cloud-based ML analytics while remaining compliant with institutional network constraints. Our framework integrates on-premises cloud computing, the in-house Pycroscopy ecosystem, and an open-source metadata management platform to transform heterogeneous microscopy datasets into standardized, ML-ready representations. We demonstrate this approach across distinct microscopy modalities through end-to-end workflows encompassing metadata capture, format harmonization, automated database ingestion, segmentation-based ML inference, and interactive visualization. By structurally separating acquisition from cloud-based analysis services, the framework enables scalable model deployment and iterative refinement without direct connectivity to instrument computers. Together, this work provides a reproducible blueprint for facility-scale data and AI infrastructure, enabling ML-ready analytics, metadata traceability, and future autonomous experimentation workflows in microscopy-driven research.

microbiology↗

Leveraging CryoEM and AI-Driven Morphological Feature Analysis for Insights on Bacterial Structures

Bacteria adapt by undergoing dynamic structural changes in response to environmental cues, which are often indicative of deeper phenotypic shifts in physiology and behavior. Understanding these changes across length scales is crucial for elucidating bacterial lifecycles, informing antifouling surface design, enhancing pathogen detection, and advancing renewable energy applications. Cryogenic electron microscopy (cryoEM) enables high-resolution imaging of bacterial structures in hydrated, biologically relevant conditions. However, current quantitative analysis strategies that extract structural information from bacterial samples remain labor-intensive. This work presents an AI-driven segmentation workflow tailored to low-dose cryoEM datasets to rapidly analyze bacterial ultrastructural features from Pantoea sp. YR343, a Gram-negative bacterium isolated from the rhizosphere of Populus deltoides that forms robust biofilms along plant roots. YOLOv11 image segmentation quantifies inner and outer membrane thickness and flagella length, enabling automated analysis of bacterial ultrastructure. The workflow reliably distinguishes membranes from carbon edges of the TEM grid and contaminant crystalline ice while matching manual measurements and increasing throughput. Flagella detection routines additionally quantify nearest-neighbor proximity between bacterial envelopes and flagella. A field-of-view module further detects bacteria at low magnification for rapid screening. Together, these tools provide a scalable and automated framework for high-throughput, quantitative analysis of bacterial ultrastructure in cryoEM data. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=39 SRC="FIGDIR/small/692658v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@18eac2org.highwire.dtl.DTLVardef@1dc67a1org.highwire.dtl.DTLVardef@1179499org.highwire.dtl.DTLVardef@11d08f8_HPS_FORMAT_FIGEXP M_FIG AI-based tools enable rapid characterization of bacterial ultrastructure in low-dose cryoEM. The envelope thickness tool quantifies membrane thickness and anisotropy. The flagella module analyzes filament morphology and detects cell-flagella contacts. The field-of-view (FOV) module C_FIG

microbiology↗

Identification of gene products involved in plant colonization by Pantoea sp. YR343 using a diguanylate cyclase expressed in the presence of plants

Microbial colonization of plant roots is a highly complex process that requires the coordination and regulation of many gene networks, yet the functions of many of these gene products remain poorly understood. Pantoea sp. YR343, a gamma-proteobacterium isolated from the rhizosphere of Populus deltoides, forms robust biofilms along the root surfaces of Populus and possesses plant growth-promoting characteristics. The mechanisms governing biofilm formation along plant roots by bacteria, including Pantoea sp. YR343, are not fully understood and many genes involved in this process have yet to be discovered. In this work, we identified three diguanylate cyclases in the plant-associated microbe Pantoea sp. YR343 that are expressed in the presence of plant roots, One of these diguanylate cyclases, DGC2884 localizes to discrete sites in the cells and its overexpression results in reduced motility and increased EPS production and biofilm formation. We then performed a genetic screen by expressing this diguanylate cyclase from an inducible promoter in order to identify candidate downstream effectors of c-di-GMP signaling which may be involved in root colonization by Pantoea sp. YR343. Further, we demonstrate the importance of other domains in DGC2884 to its activity, which in combination with the genes identified by transposon mutagenesis, may yield insights into activity and regulation of homologous enzymes in medically and agriculturally relevant microbes.

microbiology↗

Quantifying biofilm propagation on chemically modified surfaces

Conditions affecting biofilm formation differ among bacterial species and this presents a challenge to studying biofilms in the lab. This work leverages functionalized silanes to control surface chemistry in the study of early biofilm propagation, quantified with a semi-automated image processing algorithm. These methods support the study of Pantoea sp. YR343, a gram-negative bacterium isolated from the poplar rhizosphere. We found that Pantoea sp. YR343 does not readily attach to hydrophilic surfaces but will form biofilms with a "honeycomb" morphology on hydrophobic surfaces. Our image processing algorithm described here was used to quantify this honeycomb morphology over time and displayed a logarithmic behavior in the propagation of the honeycomb biofilm. This methodology was repeated with a flagella-deficient fliR mutant of Pantoea sp. YR343 which resulted in reduced surface attachment. Quantifiable differences between Pantoea WT and {Delta}fliR biofilm morphologies were captured by the image processing algorithm, further demonstrating the insight gained from these methods.

microbiology↗

Pore-scale hydrodynamics influence the spatial evolution of bacterial biofilms in a microfluidic porous network

Bacteria occupy heterogeneous environments, attaching and growing within pores in materials, living hosts, and matrices like soil. Systems that permit high-resolution visualization of dynamic bacterial processes within the physical confines of a realistic and tractable porous media environment are rare. Here we use microfluidics to replicate the particle shape and packing density of natural sands in a 2D platform to study the flow-induced spatial evolution of bacterial biofilms underground. We discover that initial bacterial dispersal and particle attachment is a stochastic process driven by bacterial rheotactic transport across pore space velocity gradients. Over time, we find that gravity-driven flow conditions activate different cell-clustering phentoypes in EPS producing and EPS defective bacteria strains, which subsequently changes the overall spatial distribution of cells across the porous media network as colonies grow and alter the fluid dynamics of their microenvironment.

microbiology↗