experimental preparation and ex ovo culture of chicken embryo for brillouin microscopy

Longitudinal Monitoring of Neural Plate Mechanics in Chick Embryo Development

Neural tube defects (NTDs) are severe birth defects of the central nervous system caused by failures in neural tube closure (NTC) during embryonic development1. The etiology of NTDs is complex. Studies have shown that NTC involves a sequence of morphogenetic processes, including convergent extension, bending of the neural plate (e.g., apical constriction), elevating the neural fold, and finally adhesion of the neural fold. These processes are regulated by multiple molecular and genetic mechanisms2,3, and any malfunction in these processes may result in NTDs4,5,6. As mounting evidence suggests that mechanical cues also play crucial roles during NTC3,7,8,9,10,11, and relationships have been found between genes and mechanical cues12,13,14, it becomes imperative to investigate the tissue biomechanics during neurulation.
Several techniques have been developed for measuring the mechanical properties of embryonic tissues, including laser ablation (LA)15, tissue dissection and relaxation (TDR)16,17, micropipette aspiration (MA)18, Atomic Force Microscopy (AFM)-based nanoindentation19, microindenters (MI)&#160;and microplates (MP)20, micro rheology (MR) with optical/magnetic tweezers21,22,23, and droplet-based sensors24. Existing methods can measure mechanical properties at spatial resolutions ranging from subcellular to tissue scales. However, most of these methods are invasive because they require contact with the sample (e.g., MA, AFM, MI, and MP), external material injection (e.g., MR and droplet-based sensors), or tissue dissection (e.g., LA and TDR). As a result, it is challenging for existing methods to monitor the mechanical evolution of neural plate tissue in situ25. Recently, reverberant optical coherence elastography has shown promise for non-contact mechanical mapping with high spatial resolution26.
Confocal Brillouin microscopy is an emerging optical modality that enables non-contact quantification of tissue biomechanics with subcellular resolution27,28,29,30. Brillouin microscopy is based on the principle of spontaneous Brillouin light scattering, which is the interaction between the incident laser light and the acoustic wave induced by thermal fluctuations within the material. Consequently, the scattered light experiences a frequency shift, known as the Brillouin shift &#969;R, following the equation31:
<img alt="Equation 1" src="/files/ftp_upload/66117/66117eq01.jpg" style="margin: auto;" /> (1)
Here, <img alt="Equation 2" src="/files/ftp_upload/66117/66117eq02.jpg" style="margin: auto;" /> is the refractive index of the material,&#160;&#955; is the wavelength of the incident light, M&#39; is the longitudinal modulus, &#961; is the mass density, and &#952; is the angle between the incident light and the scattered light. For the same type of biological materials, the ratio of refractive index and density <img alt="Equation 3" src="/files/ftp_upload/66117/66117eq03.jpg" style="margin: auto;" /> is approximately constant28,32,33,34,35,36. Thus, the Brillouin shift can be directly used to estimate relative mechanical changes in physiological processes. The feasibility of Brillouin microscopy has been validated in various biological samples29,37,38. Recently, time-lapse mechanical imaging of a live chick embryo was demonstrated by combining a Brillouin microscope with an on-stage incubation system39. This protocol provides detailed descriptions of sample preparation, experiment implementation, and data post-processing and analysis. We hope this effort will facilitate the widespread adoption of non-contact Brillouin technology for studying biomechanical regulation in embryo development and birth defects.

Author Spotlight: Non-Contact Measurement of Tissue Mechanics in Live Chick Embryos Using Brillouin Microscopy

Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo

The scope of this research is to develop a novel tool that can measure tissue mechanics of a live embryo. Using this tool, we want to understand how tissue mechanics evolves when the embryo is experiencing neuron tube closure. This can help us understand the mechanical regulations in embryonic development.To measure tissue stiffness, current methods such as atomic force microscopy, nanoidention, and micropipette aspiration need to contact sample physically and apply force to deform it. This is highly challenging for measuring tissue mechanics of live embryo in situ. Here, we employed an optical technology named Brillouin microscopy.Compared to other technologies, Brillouin microscopy only uses a focus beam to measure tissue mechanics. Thus, this is a non-contact and a noninvasive method and has some microspatial resolution. This allow us to capture time-lapse mechanical image of a live embryo in situ.In the future, we want to understand the role of tissue mechanics in embryonic development thoroughly. In specific, we want to study how genetic factors, biochemical signaling pathway, and tissue mechanics coordinate with each other in morphogenesis. This could provide us with new insight in the provision of birth disease such as neuron tube defects.

Experimental Preparation and Ex Ovo Culture of Chicken Embryo for Brillouin Microscopy

To begin, cut the filter paper into a rectangular shape of approximately 17 by 20 millimeters and remove the four corners. Then, create approximately seven by 10 millimeters of hollow center in the filter paper. Attach a commercially available ring to a flexible film layer and create a hollow center in the flexible film layer.Place a prepared ring into a 35-millimeter Petri dish. After pre-culturing, retrieve the egg from the incubator, and place it on its long axis on the egg carton tray. Clean the entire surface of the eggshell with 70%ethanol, and allow it to rest for 15 minutes.Hold the egg on its short axis and crack it at the bottom. Open the egg over a clean 100-millimeter Petri dish to extract its contents. Using a pipette, transfer approximately 10 milliliters of the thin albumin into a 15-milliliter tube for ex ovo culture.Fill the center well of the culture dish with thin albumin. Using tissue paper, gently remove the thick albumin attached to the vitelline membrane of the embryo. Now, carefully affix the filter paper to the vitelline membrane, ensuring the body axis of the embryo aligns with the center rectangle on the filter paper.Cut the membrane surrounding the filter paper with scissors. Using tweezers, gently pull the isolated filter paper away from the yoke in an oblique direction. Flip the filter paper upside down to position the embryo with its dorsal side down.Carefully immerse the entire filter paper from one side of the long axis into the 100-millimeter Petri dish containing wash medium. Using a clean pipette, gently spray the wash medium parallel to the filter paper to remove any residual yolk. Next, carefully remove the filter paper from the wash medium, and use tissue paper to absorb any excess medium from the edges of the embryo.Place the filter paper with the embryo onto the culture dish, ensuring that the dorsal side of the embryo is facing downward. Transfer the culture dish to the onstage incubator for ex ovo culture. Fill the culture dishes with a warm wash medium.When the embryo reaches a desired developmental stage, transfer the filter paper with the embryo to the culture dish filled with a wash medium. Place the culture dish into the onstage incubator of the Brillouin microscope. For the calibration process, measure the Brillouin signal of water.Then, measure the Brillouin signal of methanol. Under bright-field imaging with a four times objective lens, adjust the laser illumination point to the second pair of somites. Then, switch to a 40 times objective lens and perform fine adjustment of the laser point to the middle of the neural tube.Unblock the laser beam and adjust the focal plane. Scan the embryo using a two-dimensional translational stage and acquire a Brillouin image of the region of interest. After completing the scan, carefully remove the filter paper with the embryo.Use tissue paper to absorb any excess wash medium from the embryo. Place the embryo back into the culture dish filled with thin albumin for continuous culturing. For Brillouin image reconstruction, load the water and methanol signal data.Click set region and select the region with four dots for calculating calibration parameters. Save the calibration results. Load the embryo scanning data, and click start for the processing of the parameters.Finally, reconstruct the 2-D Brillouin image based on the Brillouin shifts of all pixels. Brillouin shift in the estimated longitudinal modulus of the neural plate against somite number and incubation time showed an increase in Brillouin shift of the tissue during neural tube closure.

Research

JoVE Journal

Developmental Biology

Neural tube closure (NTC) is a critical process during embryonic development. Failure in this process can lead to neural tube defects, causing congenital ...