Zebrafish are a widely used developmental model because of their transparent embryos and external development. These distinctive characteristics provide valuable insights into embryonic development. Optical coherence tomography (OCT) offers label-free structural imaging and has emerged as a preferred tool for embryonic imaging. On the other hand, light sheet fluorescence microscopy (LSFM) enables time-lapse molecular imaging of multi-hour to multi-day developmental processes due to its low phototoxicity and photobleaching compared to traditional confocal fluorescence microscopy. We developed a multimodal imaging system to obtain concurrent structural and molecular information by combining OCT and LSFM for embryonic imaging. A Michelson-type swept-source OCT system with a central wavelength of 1050 nm, the bandwidth of 100 nm, and sweep rate of 100 kHz captured the structural information with a lateral resolution of ~15 μm and an axial resolution of ~7 μm. The LSFM system captured the molecular information with a transverse resolution of ~2.1 μm and an axial resolution of ~13 μm. The optically co-aligned OCT and LSFM beams were scanned through the same scan head for trivial co-registration of the multimodal images. We imaged 1-5 μm green fluorescence microbeads to show the capability of this system. We then conducted imaging of zebrafish vasculature development with a transgenic line, Tg(kdrl:EGFP), where the erythroblasts express GFP. The results show that the multimodal system enables us to provide co-registered zebrafish structural and functional imaging.
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.
Optical coherence tomography (OCT) and light sheet fluorescence microscopy (LSFM) are well-established imaging techniques preferred in developmental biology, e.g., embryonic imaging. However, each technique has its own drawbacks, such as resolution and molecular specificity with OCT and field-of-view (FOV) and speed with LSFM. To overcome these limitations for small animal embryo imaging, we have developed a co-aligned multimodal imaging system combining OCT and LSFM. The OCT probe and LSFM excitation beams were combined and scanned with a galvanometer-mounted mirror through the same objective lens. The light sheet thickness was ~13 μm. The LSFM collection arm consisted of a 0.8 numerical aperture water immersion objective, tube lens, and CCD camera, resulting in a transverse resolution of ~2.1 μm. The OCT system was based on a 100 kHz swept-source laser with a central wavelength of 1050 nm and had a lateral resolution of ~15 µm and an axial resolution of ~7 μm. Images of fluorescent microbeads and a fluorescent-tagged mouse embryo at gestational day 9.5 showed the capabilities of the multimodal imaging system. Since the OCT system and LSFM system were co-aligned, image registration was straightforward and enabled high-throughput multimodal imaging without the need for complex registration techniques.
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