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

Abstract

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 60x/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35 degrees and 45 degrees with respect to the coverslip. Illumination at 35 degrees 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 degrees, 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 x 100 x 100 mu m3 to be 0.29, 0.31, 0.83, and 2.45-3.00 mu m, 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.