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Fluorescent two-photon selective-plane illumination microscopy (2P-SPIM) enables deep imaging of cellular information such as proliferation, type identification, and signaling using fluorescence. Optical coherence tomography (OCT) can capture complementary structural information based on intrinsic optical scattering. We developed a specialized multimodal high-resolution embryonic imaging system combining the benefits of OCT with 2P-SPIM. The OCT and 2P-SPIM beams were optically co-aligned and scanned using the same scanners and the same objective lens. The resulting light sheet thickness was ~13 µm with a transverse resolution of ~2.1 µm. The OCT system was based on a 1050 nm centered swept source laser with a bandwidth of ~100 nm and a sweep rate of 100 kHz. The OCT system utilized a Michelson-style interferometer and had a lateral resolution of ~15 µm and an axial resolution of ~7 µm. The capabilities of the multimodal imaging system were demonstrated using images of fluorescent microbeads and a fluorescently tagged mouse embryo at gestational day 9.5. Due to the co-alignment of the OCT and 2P-SPIM systems, image registration was simple and allowed for high-throughput multimodal imaging without the use of sophisticated registration methods.

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A high-resolution imaging system combining optical coherence tomography (OCT) and light sheet fluorescence microscopy (LSFM) was developed. LSFM confined the excitation to only the focal plane, removing the out of plane fluorescence. This enabled imaging a murine embryo with higher speed and specificity than traditional fluorescence microscopy. OCT gives information about the structure of the embryo from the same plane illuminated by LSFM. The co-planar OCT and LSFM instrument was capable of performing co-registered functional and structural imaging of mouse embryos simultaneously.