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.
Understanding the interactions between light and small samples at the diffraction limit is critical for solving inverse problems in microscopy. Several models for light and matter interactions have been proposed, including Born and Rytov approximations, Mie theory, T-matrix, Finite element methods, and coupled wave theory. Coupled wave approaches provide unique advantages for realistic samples by allowing refinement of the sample in the Fourier domain, where many realistic samples are considered sparse. However, this model still relies on computationally intensive operations as the sample and field resolution increases. In this paper, we develop an optimized open-source tool using established coupled-wave theory. This can be computationally efficient for realistic problems, since many practical samples are sparse in the Fourier domain. Then we analyze the computational complexity of the model and optimize the process.
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.
We developed a high-resolution multimodal system for mouse embryonic imaging that combines Optical Coherence Tomography (OCT) and Light Sheet Fluorescence Microscopy (LSFM). LSFM Illumination is restricted to fluorophores in the focal volume, and collecting the light using a microscope objective increases the signal from that plane and reduces the noise coming from outside of the plane. Colinearly aligning this modality with the OCT beam allows one to acquire the structural information from the same plane that is illuminated by the LSFM beam. A 3D image of 9.5 day mouse embryo was captured using this multimodal system.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.