Modified inverted selective plane illumination microscopy for sub-micrometer imaging resolution in...

Area of Science:

• Biomedical Engineering
• Optical Microscopy
• Cellular Biology

Background:

• Polydimethylsiloxane (PDMS) microdevices are valuable for in vitro 3D cellular studies.
• Optical path differences (OPD) in PDMS distort light sheet microscopy, limiting imaging resolution.
• Existing methods struggle with sub-micrometer imaging in microfluidic devices due to optical aberrations.

Purpose of the Study:

• To develop a method for removing optical path differences in PDMS microdevices for enhanced light sheet microscopy.
• To achieve sub-micrometer imaging resolution in 3D cellular networks within microfluidic environments.
• To demonstrate the utility of the improved imaging technique for observing dynamic cellular processes.

Main Methods:

• Adapted a commercial inverted selective plane illumination microscope (iSPIM).
• Implemented refractive index matching using sucrose solution to correct external OPD.
• Utilized an electrically tunable lens (ETL) to correct internal OPD and par-focus the light sheet.
• Integrated a single-mode fiber (SMF) illuminator for refractive index matching monitoring.

Main Results:

• Achieved sub-micrometer imaging resolution (0.6 ± 0.04 μm to 0.91 ± 0.03 μm).
• Demonstrated a >3-fold improvement in imaging resolution by removing OPD.
• Successfully visualized single platelet recruitment to an aggregate under flow at 150 μm depth within a microfluidic channel.

Conclusions:

• The modified inverted selective plane illumination microscopy (m-iSPIM) effectively overcomes OPD in PDMS microdevices.
• m-iSPIM enables high-resolution, spatiotemporal 3D imaging of biological samples in microfluidic systems.
• This technique facilitates the study of subcellular responses in physiologically relevant microenvironments.