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

Oblique plane microscopy (OPM) is a form of light-sheet fluorescence microscopy (LSFM) employing a single microscope objective at the sample for both fluorescence excitation and detection. Dual-view OPM (dOPM) is an optically folded form of OPM. We present an improved dOPM system employing a 60×/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35° and 45° with respect to the coverslip. Illumination at 35° provides slightly better lateral resolution and collection efficiency. Collection efficiency measurements are compared to a full vectorial raytracing simulation of the system. Using a light-sheet angle of 35°, the median bead FWHM for 100 nm diameter fluorescent beads in x, y, and z and the optical sectioning strength were measured over a volume of 100 × 100 × 100 μm3 to be 0.29, 0.31, 0.83, and 2.45–3.00 μm, respectively when the two dOPM views are fused. We demonstrate less photobleaching in time-lapse dOPM of live mEmerald-expressing organoids compared to widefield epi-fluorescence z-stack imaging under the condition of equal detected fluorescence signal from a point object in focus. We demonstrate dOPM for multifield-of-view 3D imaging of biological samples in 96-well plates and apply it to imaging cells in collagen gel and quantifying the FUCCI cell-cycle reporter to provide drug dose–response curves in spheroids. We also use it to perform time-lapse multifield-of-view imaging and demonstrate the detection of organoid lumen closure and reopening, organoid migration within a collagen gel and observing dynamic events in arrays of ex vivo tissue slices.