Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy
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...
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Bio-protocol LLC
2025-07-01
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| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5388&type=0 |
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| author | Isabel Gibson Elizabeth Osterlund Ray Truant |
| author_facet | Isabel Gibson Elizabeth Osterlund Ray Truant |
| author_sort | Isabel Gibson |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-95b2b937cc954cbe9e60c560c5a995ff |
| institution | DOAJ |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-95b2b937cc954cbe9e60c560c5a995ff2025-08-20T02:46:01ZengBio-protocol LLCBio-Protocol2331-83252025-07-01151410.21769/BioProtoc.5388Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in MicroscopyIsabel Gibson0Elizabeth Osterlund1Ray Truant2Department of Biochemistry and Biomedical Science, McMaster University, Hamilton, ON, CanadaDepartment of Biochemistry and Biomedical Science, McMaster University, Hamilton, ON, CanadaDepartment of Biochemistry and Biomedical Science, McMaster University, Hamilton, ON, CanadaBrightfield 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.https://bio-protocol.org/en/bpdetail?id=5388&type=0 |
| spellingShingle | Isabel Gibson Elizabeth Osterlund Ray Truant Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy Bio-Protocol |
| title | Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy |
| title_full | Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy |
| title_fullStr | Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy |
| title_full_unstemmed | Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy |
| title_short | Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy |
| title_sort | using beads as a focus fiduciary to aid software based autofocus accuracy in microscopy |
| url | https://bio-protocol.org/en/bpdetail?id=5388&type=0 |
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