Multi-plane remote refocusing epifluorescence microscopy to image dynamic Ca 2 + events

Area of Science:

• Biomedical Engineering
• Optical Microscopy
• Cell Biology

Background:

• Studying endothelial and smooth muscle cells in blood vessels requires high-speed imaging of distinct layers.
• Rapid communication between these cell layers is crucial for vascular function.
• Existing microscopy techniques may not offer sufficient speed or resolution for this purpose.

Purpose of the Study:

• To develop and implement a novel remote refocusing imaging system for simultaneous, high-speed imaging of multiple focal planes.
• To enable independent imaging of endothelial and smooth muscle cell layers in blood vessels.
• To provide a cost-effective alternative to complex confocal microscopy for rapid biological imaging.

Main Methods:

• An epifluorescence-based remote refocusing system was designed and implemented.
• The system utilizes different dyes and excitation light for each imaging plane.
• A novel triggering system synchronizes laser activation and image acquisition for each focal plane.
• High numerical aperture (NA) objectives were used for imaging and reimaging.

Main Results:

• The system achieves imaging speeds of up to 20 frames per second (fps) per layer.
• Axial plane separations down to 15 micrometers were achieved.
• Excellent lateral stability with a mean displacement of less than or equal to 0.32 micrometers was demonstrated.
• Independent imaging and quantification of endothelial and smooth muscle cell calcium (Ca2+) activity were successfully performed for several minutes.

Conclusions:

• The developed remote refocusing system offers a cost-effective solution for rapid, dual-plane imaging without optical sectioning.
• It provides high frame rates, good axial resolution, and excellent lateral stability for studying cell layer interactions.
• This technique facilitates the analysis of dynamic cellular processes, such as calcium activity, in vascular tissues.