Automated cell counting for Trypan blue-stained cell cultures using machine learning

Cell counting is a vital practice in the maintenance and manipulation of cell cultures. It is a crucial aspect of assessing cell viability and determining proliferation rates, which are integral to maintaining the health and functionality of a culture. Additionally, it is critical for establishing the time of infection in bioreactors and monitoring cell culture response to targeted infection over time. However, when cell counting is performed manually, the time involved can become substantial, particularly when multiple cultures need to be handled in parallel. Automated cell counters, which enable significant time reduction, are commercially available but remain relatively expensive. Here, we present a machine learning (ML) model based on YOLOv4 that is able to perform cell counts with a high accuracy (>95%) for Trypan blue-stained insect cells. Images of two distinctly different cell lines, Trichoplusia ni (High FiveTM; Hi5 cells) and Spodoptera frugiperda (Sf9), were used for training, validation, and testing of the model. The ML model yielded F1 scores of 0.97 and 0.96 for alive and dead cells, respectively, which represents a substantially improved performance over that of other cell counters. Furthermore, the ML model is versatile, as an F1 score of 0.96 was also obtained on images of Trypan blue-stained human embryonic kidney (HEK) cells that the model had not been trained on. Our implementation of the ML model comes with a straightforward user interface and can image in batches, which makes it highly suitable for the evaluation of multiple parallel cultures (e.g. in Design of Experiments). Overall, this approach for accurate classification of cells provides a fast, bias-free alternative to manual counting.BN/Nynke Dekker La

A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint

Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.BN/Nynke Dekker La

Global correction of optical distortions in multicolor single-molecule microscopy using Zernike polynomial gradients

Accurate image alignment is critical in multicolor single-molecule fluorescence microscopy. Global alignment using affine transformations leaves residual errors due to the nonlinearity of the distortions, which decreases the effective field of view. Subsequent local refinement demands either large amounts of reference data and processing time or specialized imaging techniques like active stabilization. Here, we present a global alignment method, S/T polynomial decomposition, that uses sums of Zernike polynomial gradients to decompose the distortion between two images, correcting both linear and nonlinear distortions simultaneously. With minimal reference data, we gain diagnostic information about the distortion and achieve a colocalization accuracy comparable to local registration methods across the entire field of view. BN/Nynke Dekker LabTeam Carlas SmithImPhys/Computational ImagingDelft Center for Systems and Contro

DNA replication origins retain mobile licensing proteins

DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.BN/Nynke Dekker La

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy

Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force-fluorescence spectroscopy studies on a large variety of biological systems.BN/Nynke Dekker La