PAAQ: Paired Alternating AcQuisitions for virtual high frame rate multichannel cardiac fluorescence microscopy

Area of Science:

• Developmental biology
• Biomedical imaging
• Microscopy techniques

Background:

• In vivo fluorescence microscopy is crucial for studying early heart development.
• High frame rates are needed for fast cardiac contractions, but sensitive cameras are often slow.
• Imaging multiple fluorophores simultaneously exacerbates these challenges.

Purpose of the Study:

• To present a novel method, Paired Alternating AcQuisitions (PAcQu), for high-frame-rate, multi-channel in vivo imaging.
• To overcome limitations of slow cameras in capturing rapid cyclic biological processes.
• To enable simultaneous imaging of distinct tissues using different fluorophores.

Main Methods:

• Paired Alternating AcQuisitions (PAcQu) uses variable temporal illumination patterns.
• Alternates between channel-specific fluorescence and a motion-encoding brightfield reference in odd/even frames.
• Employs image-based sorting and regularized curve fitting to estimate cardiac cycle positions.
• Assembles multichannel videos with virtually increased frame rates.

Main Results:

• Demonstrated quantitative and visual improvements in reconstructed videos compared to existing methods.
• Successfully generated a 100 fps multi-channel video using a 15 Hz camera in zebrafish embryos.
• Validated the method on both synthetic and experimental imaging data.

Conclusions:

• PAcQu significantly enhances the capabilities of in vivo fluorescence microscopy for studying dynamic biological systems.
• The method provides a cost-effective solution for high-speed, multi-channel imaging without specialized fast cameras.
• This technique opens new avenues for detailed investigation of embryonic development and other rapid cellular processes.