Phase variance optical coherence microscopy for label-free imaging of the developing vasculature in zebrafish embryos

Abstract

, "Phase variance optical coherence microscopy for labelfree imaging of the developing vasculature in zebrafish embryos," J. Abstract. A phase variance optical coherence microscope (pvOCM) has been created to image blood flow in the microvasculature of zebrafish embryos, without the use of exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2.8 μm in tissue and imaging depth of more than 100 μm. Images of 2 to 5 days postfertilization zebrafish embryos identified the detailed anatomical structure based on OCM intensity contrast. Phase variance contrast offered visualization of blood flow in the arteries, veins, and capillaries. The pvOCM images of the vasculature were confirmed by direct comparisons with fluorescence microscopy images of transgenic embryos in which the vascular endothelium is labeled with green fluorescent protein. The ability of pvOCM to capture activities of regional blood flow permits it to reveal functional information that is of great utility for the study of vascular development. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

3D in vivo imaging with extended‐focus optical coherence microscopy

“…We increased the pixel dwell time by 5 times in xf‐OCM to have the same amount of light incident onto the sample as in standard OCM. As a result, the imaging speed was reduced, which may not be desired in time‐sensitive imaging experiments, such as detecting the blood flow with a phase variance OCM technique . However, in cases of the light source being ultrafast lasers, such as femtosecond lasers, the annular mask is no longer an issue for energy efficiency since these laser sources usually have a broad range of high output power levels .…”

“…In the reference arm, the light went through almost the same optical components to match the dispersion in the sample arm. Backscattered light from the sample arm and reference arm generated interference spectra, which were detected by a custom‐built spectrometer as described in our previous work . A custom LabVIEW (National Instruments) program controlled the scanning pattern of galvanometer scanners and data acquisition of the spectrometer.…”

“…During imaging experiments, zebrafish larvae were anesthetized with tricaine (0.02 %) to immobilize specimens. Sample mounting methods were described in our previous work , but with less need to avoid specular reflections of the bottom glass due to the reduced DOF in xf‐OCM compared with the previous system design.…”

Multi-shaping technique reduces sidelobe magnitude in optical coherence tomography

“…OCT imaging data of the human retina were acquired from a custom SD-OCT system (2048-pixel spectrometer) developed at Varocto Inc, as described in our previous work [29]. OCM imaging data of the microbead phantom and zebrafish embryo were acquired from custom spectral-domain OCM (SD-OCM) systems (4096-pixel spectrometer) described in our previous studies [30,31].…”

“…We applied the multi-shaping technique to in vivo imaging data from a 3 days-postfertilization (dpf) zebrafish embryo. The protocol for the animal experiments, such as mounting the specimen in phytagel, was described in our previous publication [30]. Images of the embryo were acquired using our SD-OCM system, with 2.8-µm lateral and axial resolution [30].…”