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

Area of Science:

• Microscopy
• Cell Biology
• Biotechnology

Background:

• Brightfield microscopy is valuable for live cell studies but suffers from low contrast, leading to autofocus issues like focal drift.
• Long-term live cell imaging requires stable focus to observe dynamic cellular processes, such as stress responses.
• Existing autofocus systems can fail with transparent samples, hindering extended experiments.

Purpose of the Study:

• To develop and validate an inexpensive, label-free method to improve autofocus accuracy for long-term live cell imaging using brightfield microscopy.
• To address technical challenges associated with focal drift and software autofocus failures in time-lapse microscopy.
• To provide a practical solution for researchers studying dynamic cellular processes in transparent cells.

Main Methods:

• Utilized microbeads as a focus aid in conjunction with a widefield inverted microscope and software-based autofocus.
• Implemented a protocol requiring minimal additional sample preparation for live cell imaging.
• Employed KNIME software to train a random forest model for binary image classification to validate the protocol.

Main Results:

• Demonstrated successful prevention of focal drift during overnight time-lapse imaging using microbeads.
• Showcased improved autofocus accuracy on relatively inexpensive microscopes without hardware modifications.
• Validated the protocol's effectiveness for capturing focused, label-free images of transparent cells.

Conclusions:

• Microbeads serve as an effective and economical focus aid for long-term, label-free live cell imaging in brightfield microscopy.
• This protocol enhances the reliability of software autofocus systems, overcoming limitations with transparent samples.
• The method offers a practical solution for troubleshooting autofocus issues in time-lapse microscopy experiments.