High content 3D imaging by dual-view oblique plane microscopy

Area of Science:

• Biophysics
• Microscopy
• Optical Engineering

Background:

• Oblique plane microscopy (OPM) is a light-sheet fluorescence microscopy (LSFM) technique using a single objective for excitation and detection.
• Dual-view OPM (dOPM) is an optically folded variant of OPM.

Purpose of the Study:

• To present an improved dOPM system with enhanced spatial resolution and fluorescence collection efficiency.
• To evaluate system performance using different illumination angles and compare experimental data with simulations.
• To demonstrate the system's capabilities for live imaging, multifield-of-view 3D imaging, and dynamic event observation in biological samples.

Main Methods:

• Developed an improved dOPM system utilizing a 60×/1.2NA water immersion objective.
• Measured spatial resolution (FWHM) and optical sectioning strength at 35° and 45° illumination angles.
• Compared fluorescence collection efficiency with vectorial raytracing simulations.
• Conducted time-lapse imaging of organoids and cells in collagen gel, including FUCCI cell-cycle reporter quantification.
• Performed multifield-of-view imaging of biological samples in 96-well plates.

Main Results:

• The 35° illumination angle yielded slightly better lateral resolution and collection efficiency.
• Fused dOPM views achieved median bead FWHM of 0.29 µm (x), 0.31 µm (y), and 0.83 µm (z) with optical sectioning of 2.45-3.00 µm.
• Demonstrated reduced photobleaching in dOPM compared to widefield epi-fluorescence imaging for live organoids.
• Successfully imaged cells in collagen gel, quantified FUCCI reporter for drug dose-response curves in spheroids.
• Observed dynamic biological events including organoid lumen dynamics and migration in ex vivo tissue slices.

Conclusions:

• The improved dOPM system provides high-resolution, low-photobleaching 3D imaging capabilities.
• dOPM is suitable for time-lapse studies of dynamic biological processes and multifield-of-view screening.
• The system facilitates quantitative analysis of cellular and organoid behavior in various biological contexts.