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bioRxiv · 10.1101/2025.03.18.643916

Using Beads as a Focus Fiduciary to Aid Software-based Autofocus Accuracy in Microscopy

Gibson, I.·Osterlund, E. J.·Truant, R.
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Abstract

Brightfield microscopy is an ideal application for studying live cell systems in a minimally invasive manner. This is advantageous in long-term experiments to study dynamic cellular processes such as stress response. Depending on the sample type and preparation, the inherent qualities of brightfield microscopy, being very low contrast, can contribute to technical issues such as focal drift, sequencing lags, and complete failure of software autofocus systems. Here, we describe the use of microbeads as a focus aid for long-term live cell imaging to address these autofocus issues. This protocol is inexpensive to implement, without extensive additional sample preparation and can be used to capture focused images of transparent cells in a label-free manner. To validate this protocol, a widefield inverted microscope was used with software-based autofocus to image overnight in time-lapse format, demonstrating the use of the beads to prevent focal drift in long-term experiments. This improves autofocus accuracy on relatively inexpensive microscopes without using hardware-based focus aids. To validate this protocol, the KNIME logistics software was used to train a random forest model to perform binary image classification. Key featuresO_LIlabel-free live cell imaging in time-lapse format C_LIO_LITroubleshooting software autofocus for brightfield mode C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/643916v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1038c25org.highwire.dtl.DTLVardef@1e5f175org.highwire.dtl.DTLVardef@14f394corg.highwire.dtl.DTLVardef@3abca3_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Gibson, I., Osterlund, E. J., Truant, R.. 2025-03-19. Using Beads as a Focus Fiduciary to Aid Software-based Autofocus Accuracy in Microscopy. https://doi.org/10.1101/2025.03.18.643916

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