This work is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (K25HD097288, R21HD112663).

The protocol has been approved by the Institutional Animal Care and Use Committee of Wayne State University.
1. Experimental preparation
<ol>
	<li>Use a 70% ethanol solution to clean and sterilize the scissors and tweezers. Also, prepare disposable pipettes and a syringe.</li>
	<li>Prepare a wash medium by adding 3.595 g of NaCl to 495 mL of deionized water. Then, add 5 ml of Penicillin-Streptomycin (5 U/mL) to the medium. Fill a 100 mm Petri dish with the wash medium and warm it to 37 &#176;C.</li>
	<li>Prepare culture dishes according to Figure 1, which illustrates the overall configuration.
	<ol>
		<li>Prepare a piece of filter paper, cut it into a rectangular shape of approximately 17 mm &#215; 20 mm, and remove the four corners. Create a hollow center in the filter paper, approximately 7 mm &#215; 10 mm in size for securing the embryo (Figure 1, left). 
		NOTE: The collection of embryos is described in step 2.</li>
		<li>Attach a commercial available ring (&#934; = 1 inch, see Table of Materials) with a flexible film (i.e., paraffin film) layer, ensuring that the flexible film also has a hollow center. This ring with the film will be used to hold the filter paper with the embryo.</li>
		<li>Finally, place the prepared ring into a 35 mm Petri dish (Figure 1, right). 
		&#8203;NOTE: The filter paper is for securing and holding the extracted embryo by placing the filter paper on the membrane and leaving the embryo in the center hollow area (which will be detailed in step 2). The four corners were cut to fit the size of the outside ring (not necessary if it is already fitted). A 35 mm glass-bottom culture dish is used for better laser beam propagation. The dish has an inner well (&#934; = 23 mm), which can be filled with albumen for ex ovo culture. The diameter of the ring must be larger than the diameter of the inner well so that the well can be covered by the flexible film on the ring (Figure 1, inset). The size of the filter paper is not restricted and can be modified according to the size of the chosen ring and the culture dish. Alternatively, the bottom of the dish can be pre-coated with an agarose layer (thickness: ~1 mm). By removing agarose from the center of the layer using a blade, a well with a similar shape to the hollow center of the flexible film can be created for loading albumen. One critical point is that the four sides of the hollow filter paper need to have enough width (&#62; 5 mm) to ensure the attachment of the embryo.</li>
	</ol>
	</li>
</ol>
2. Extraction and ex ovo culture of the chicken embryo
NOTE: This step is modified from previously published reports40,41.
<ol>
	<li>After pre-culture, retrieve the egg from the incubator and place it on its long axis on the egg carton tray. Clean the eggshell with 70% ethanol, ensuring to cover the entire surface, and then allow it to rest for 15 min.</li>
	<li>Hold the egg on its short axis and crack it at the bottom. Open the egg over a clean 100 mm Petri dish to extract its contents, as illustrated in Figure 2. Ensure not to rotate the egg while holding and cracking it.</li>
	<li>Using a pipette, transfer and collect approximately 10 mL of the thin albumen (i.e., the liquid albumen42) into a 15 mL tube for ex ovo culture use. Fill the center well of the culture dish with the collected thin albumen (about 0.9 mL), as shown in Figure 3. The filling process should be slow, avoiding the formation of any bubbles inside the well. Ensure that the culture dish with albumen is warmed to 37 &#176;C. 
	NOTE: This dish will be used for culturing the embryo ex ovo. New culture dishes filled with wash medium will be used during Brillouin imaging (see step 3).</li>
	<li>Using tissue paper, gently remove the thick albumen (i.e., viscous albumen) attached to the embryo by carefully separating the albumen from the vitelline membrane, as demonstrated in Figure 4. Avoid direct contact with the vitelline membrane.</li>
	<li>After removing all the thick albumen, carefully affix the filter paper to the vitelline membrane. Ensure the body axis of the embryo aligns with the long axis of the center rectangle on the filter paper. Use scissors to cut the membrane surrounding the filter paper.</li>
	<li>Using tweezers, gently pull the isolated filter paper away from the yolk in an oblique direction. Flip the filter paper upside down to position the embryo with its dorsal side down (for inverted microscope configuration). Carefully immerse the entire filter paper from one side of the long axis into the 100 mm Petri dish with the wash medium in an oblique fashion.</li>
	<li>Wash away any remaining yolk by gently spraying the wash medium parallel to the filter paper using a clean pipette. Avoid directly spraying the wash medium onto the membrane.</li>
	<li>After clearing all the yolk, carefully remove the filter paper from the wash medium and use tissue paper to absorb any excess medium from the edges. Then, place the filter paper with the embryo onto the culture dish, ensuring that the dorsal side of the embryo is facing downward, as illustrated in Figure 5. To maintain humidity, place a moistened tissue paper in the dish.</li>
	<li>Transfer the culture dish to the on-stage incubator for ex ovo culture.</li>
</ol>
3. Brillouin measurement of the embryo
<ol>
	<li>Prepare another set of culture dishes and fill them with wash medium. Ensure the wash medium is warmed to 37 &#176;C.</li>
	<li>When the embryo reaches the desired developmental stage, transfer the filter paper with the embryo to the culture dish filled with wash medium. Place the culture dish into the on-stage incubator of the Brillouin microscope (see Table of Materials).
	<ol>
		<li>Before conducting the Brillouin measurement, save a bright-field image of the entire embryo for reference. The detailed configuration of the Brillouin microscope has been previously described30 and is summarized in the Results section (Figure 6).</li>
	</ol>
	</li>
	<li>Measure the Brillouin signal of water and methanol, which will be used in step 4 for the calibration process.</li>
	<li>Adjust the incident laser power and electron-multiplying charge-coupled device (EMCCD, see Table of Materials) camera exposure time to achieve at least 10,000 counts of Brillouin signal. Using the bright-field image as a guide, set up the scan range and step size. Acquire a Brillouin image of the region of interest by scanning the embryo using a 2D translational stage. 
	NOTE: The horizontal scanning range can be determined based on the bright-field image, and the vertical (i.e., depth) scanning range can be determined based on the signal strength obtained from a quick and coarse scan. To prevent any photodamage to the embryo, limit the incident power to 25 mW, and set the exposure time of the EMCCD camera to 50 ms. Choose a step size of 2 &#181;m in the horizontal direction and 1 &#181;m in the vertical direction to balance imaging quality and acquisition time.</li>
	<li>After completing the scan, carefully remove the filter paper with the embryo and use tissue paper to absorb any excess wash medium. Then, place the filter paper back into the culture dish filled with thin albumen for continuous culturing in the on-stage incubator.</li>
	<li>Repeat steps 3.2-3.5 at regular time intervals (e.g., 1.5 h) to capture time-lapse Brillouin images as the embryo develops.</li>
	<li>Reconstruct the 2D Brillouin image following step 4.</li>
</ol>
4. Brillouin image reconstruction
<ol>
	<li>Calibrate the Brillouin spectrometer using Brillouin signals of water and methanol to calculate the free spectral range (FSR) and the pixel-to-frequency conversion ratio (PR) of the spectrometer30. The calibrated values of FSR and PR will be used to calculate the Brillouin shift of the embryo at each pixel. 
	NOTE: Figure 7A displays a raw Brillouin spectrum captured by the EMCCD camera. By vertically summing the spectrum and then doing a Lorentzian fitting, the peak distance &#916;d of the two dots can be obtained (Figure 7B). By getting the peak distance of water &#916;dwater and methanol &#916;dmethanol, the FSR and PR can be computed based on the equations30:&#160;PR = 2&#183;(&#969;methanol-&#969;water)/(&#916;dwater- &#916;dmethanol), and FSR = 2&#183;&#969;water&#160;+ PR&#183;&#916;dwater, with the known Brillouin shift of water &#969;water = 6.01 GHz and methanol &#969;methanol = 4.49 GHz at 660 nm.</li>
	<li>Obtain the peak distance of the Brillouin signal at each pixel of the sample. Calculate the Brillouin shift based on the calibrated FSR and PR: &#969;sample = 0.5 (FSR - PR x &#916;dsample)30, where &#969;sample is the Brillouin shift of the sample, and &#916;dsample is the peak distance of the Brillouin signal.</li>
	<li>Reconstruct the 2D Brillouin image based on the Brillouin shifts of all pixels. 
	NOTE: To conduct local analysis, one can select a region of interest (e.g., the neural plate) from the acquired Brillouin image and quantify its mechanical properties. A common approach is to calculate the average Brillouin shift of the selected region.</li>
</ol>

Longitudinal Monitoring of Neural Plate Mechanics in Chick Embryo Development

The authors declare that they have no conflict of interest.

The early development of the embryo can be easily affected by external disturbances. Therefore, utmost caution is required during the sample extraction and transfer. One potential issue is the detachment of the embryo from the filter paper, which can lead to the shrinking of the vitelline membrane and result in a tilted artifact of the neural plate in Brillouin imaging. Furthermore, this shrinking may halt the development of the embryo. Attention should be paid to several critical steps to prevent detachment. First, in step 2.4, it is crucial to ensure thorough removal of albumen from the membrane surface. Any remaining albumen can impede the proper adhesion of the membrane to the filter paper46,47. Additionally, during the washing process, it is important to spray the washing medium in a direction parallel to the membrane. Directly hitting the membrane with the liquid flow may cause detachment or even tear the membrane. Furthermore, the entire washing process should be completed within a short time (e.g., &lt;3 min), as prolonged immersion can also lead to detachment of the embryo from the filter paper.
Before conducting Brillouin measurements, it is necessary to transfer the embryo to a dish with wash medium. This is because the Brillouin shift of the albumen is very close to the neural plate tissues, causing poor image contrast. Brillouin imaging is obtained by point scanning, which can be time-consuming, especially when dealing with many points. In this protocol, the scanning region is approximately 400 &#181;m horizontally and less than 100 &#181;m vertically. To avoid disturbance to the embryo's development, the total acquisition time was limited to within 30 min. This is achieved by utilizing a step size of 2 &#181;m horizontally and 1 &#181;m vertically. In case one is only interested in the average Brillouin shift of a specific region, a quick and coarse scan (e.g., with a step size of 4 &#181;m both horizontally and vertically) can be implemented, which will take less than 5 min and can mitigate the negative impact on embryo development.
Brillouin microscopy offers a real-time and non-invasive imaging approach for investigating the biomechanics of embryo development. One limitation of this method lies in its penetration depth, which is about 100-200 &#181;m depending on the embryo's development stage. While increasing the laser power and/or exposure time of the EMCCD can partially address this issue, it comes at the cost of potential phototoxicity and/or a long acquisition time. Alternatively, existing optical technologies, such as adaptive optics, could be potentially employed to improve the penetration depth48.
In conclusion, we have established a protocol for probing the mechanical properties of live chicken embryos using Brillouin microscopy. This non-contact method can fulfill current unmet needs and is complementary to other existing tools for studying biomechanics in embryonic 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Petri dish&#160;</td>
			<td>Fisherbrand</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>2D motorized stage&#160;</td>
			<td>Prior Scientific</td>
			<td>H117E2</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Brillouin Microscope with on-stage incubator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This is a custom-built Brillouin Microscope system based on Ref. 30</td>
		</tr>
		<tr>
			<td>Chicken eggs</td>
			<td>University of Connecticut</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Thorlabs</td>
			<td>CS2100M-USB</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>#2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1004-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator for in ovo culture</td>
			<td>GQF Manufacturing Company Inc.&#160;</td>
			<td>GQF 1502&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring</td>
			<td>Thorlabs</td>
			<td>SM1RR</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope body</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>On-stage incubator</td>
			<td>Oko labs</td>
			<td>OKO-H301-PRIOR-H117</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-711-6M</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Artman instruments</td>
			<td>N/A</td>
			<td>3pc Micro Scissors 5</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>305482</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue paper</td>
			<td>Kimwipes</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>DR Instruments</td>
			<td>N/A</td>
			<td>Microdissection Forceps Set&#160;</td>
		</tr>
	</tbody>
</table>

monitoring the mechanical evolution of tissue during neural tube closure of chick embryo

Neural tube closure (NTC) is a critical process during embryonic development. Failure in this process can lead to neural tube defects, causing congenital malformations or even mortality. NTC involves a series of mechanisms on genetic, molecular, and mechanical levels. While mechanical regulation has become an increasingly attractive topic in recent years, it remains largely unexplored due to the lack of suitable technology for conducting mechanical testing of 3D embryonic tissue in situ. In response, we have developed a protocol for quantifying the mechanical properties of chicken embryonic tissue in a non-contact and non-invasive manner. This is achieved by integrating a confocal Brillouin microscope with an on-stage incubation system. To probe tissue mechanics, a pre-cultured embryo is collected and transferred to an on-stage incubator for ex ovo culture. Simultaneously, the mechanical images of the neural plate tissue are acquired by the Brillouin microscope at different time points during development. This protocol includes detailed descriptions of sample preparation, the implementation of Brillouin microscopy experiments, and data post-processing and analysis. By following this protocol, researchers can study the mechanical evolution of embryonic tissue during development longitudinally.

Figure 6 shows the schematic of the Brillouin microscope. The system employs a 660 nm laser as the light source. An isolator is placed right after the laser head to reject any back-reflected light, and a neutral density (ND) filter is used to adjust the laser power. A pair of lenses, L1 and L2, with focal lengths of f1 = 16 mm and f2 = 100 mm, respectively, are used to expand the laser beam. A half-wave plate (HWP) and a linear polarizer (Polarizer 1) are employed to adjust the power of the beam shining on either the sample or the calibration materials (i.e., water and methanol) after the polarized beam splitter (PBS). To focus the laser into the sample and collect backward scattered light, an objective lens (OBJ2) with a numerical aperture (NA) of 0.6 and a magnification of 40 are used. The incident light and backward scattered light are separated by the same PBS after polarization adjustment using a quarter-wave plate (QWP2). Similarly, OBJ1 and QWP1 are used to guide the laser beam to the calibration materials. The Brillouin signal is delivered by a single-mode fiber to a Brillouin spectrometer43 for analysis. Inside the spectrometer, a cylindrical lens (C1) couples the Brillouin signal into the first VIPA (virtually imaged phased array) etalon. The output of the first VIPA etalon is reshaped by another cylindrical lens (C2) and focused onto Mask 1 to reject non-scattered laser light. Then, the Brillouin signal is coupled into a second VIPA etalon by a spherical lens (SL1), reshaped by another spherical lens (SL2), and projected onto Mask 2. The resulting spectral pattern then goes through a 4-f unit for noise rejection and is projected onto an EMCCD for recording. We used a commercial microscope body (see Table of Materials). Generally, any microscope body with different brands can be used as long as it has an available port for delivering the external light beam. The on-stage incubator is a metal chamber with temperature, gas, and air flow control. The position of the sample is adjusted via the 2D motorized stage. A complementary metal-oxide semiconductor (CMOS) camera is used to acquire the bright-field image.
Figure 8 shows the time-lapse bright-field and Brillouin images of a representative chicken embryo. The embryo was extracted after 29 h of in ovo culture, and the images were taken at 29 h, 30.5 h, and 32 h with ex ovo culture, which corresponds to somite numbers of 4, 5, and 8, respectively44. In general, the embryo can be cultured in the on-stage incubator for at least 24 h. The red line in the bright-field image indicates the location for Brillouin imaging. With the acquired Brillouin images, the neural plate region was selected (highlighted by the black dotted line in Figure 8) and calculated the average Brillouin shift of this region. As shown in Figure 9, the averaged Brillouin shift of the tissue increases during neural tube closure. The longitudinal modulus is also calculated based on equation (1), where the value of <img alt="Equation 3" src="/files/ftp_upload/66117/66117eq03.jpg" style="margin: auto;" /> = 1.3330 is estimated from literature using zebrafish embryos45, assuming the value is relatively constant for similar kinds of biological tissues28,33.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66117/66117fig01.jpg" /> 
Figure 1: Schematic of the whole culture dish configuration for embryonic development. This figure shows a filter paper for attaching the embryo on the left and a 35 mm culture dish with a ring attached with a flexible film layer on the right. The cross-section schematic is also provided. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66117/66117fig02.jpg" /> 
Figure 2: The process of opening the eggshell above a clean 100 mm Petri dish to extract the egg into the dish. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66117/66117fig03.jpg" /> 
Figure 3: Filling the center well of the culture dish with thin albumen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66117/66117fig04.jpg" /> 
Figure 4: Dragging away the thick albumen in the direction indicated by the red arrow, using a piece of tissue paper. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66117/66117fig05.jpg" /> 
Figure 5: The placement of the filter paper into the culture dish with the embryo on the upside of the filter paper. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66117/66117fig06.jpg" /> 
Figure 6: Schematic of the Brillouin microscope. The red arrow indicates the light path of the laser beam, and the orange arrow indicates the light path of the Brillouin signal. L1, L2, SL1-SL4: spherical lens; SF: spatial filter; C1, C2: cylindrical lens, HWP: half-wave plate; PBS: polarized beam splitter; QWP1, QWP2: quarter-wave plate; OBJ1, OBJ2: Objective lens; LP: lens pair. VIPA: virtually imaged phased array. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66117/66117fig07.jpg" /> 
Figure 7: Representative Brillouin signal. (A) A representative Brillouin spectrum captured by the EMCCD. (B) A vertically summed Brillouin spectrum and corresponding Lorentzian fitting result. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66117/66117fig08v2.jpg" /> 
Figure 8: Bright field images and the corresponding Brillouin cross-section images of the embryo at 29 h, 30.5 h and 32 h with 4, 5 and 8 somites. Red solid lines indicate the location for Brillouin imaging, and the black dotted line indicate the neural plate region. The grey area in 32 h (8 somites) image is an artifact of curve fitting due to the weak Brillouin signal and is excluded when calculating the average shift. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig08largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/66117/66117fig09.jpg" /> 
Figure 9: Brillouin shift and the estimated longitudinal modulus of the neural plate against somite number and incubation time. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66117/66117fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo at JoVE.com

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

This protocol was developed to longitudinally monitor the mechanical properties of neural plate tissue during chick embryo neurulation. It is based on the integration of a Brillouin microscope and an on-stage incubation system, enabling live mechanical imaging of neural plate tissue in ex ovo cultured chick embryos.

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

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

monitoring-mechanical-evolution-tissue-during-neural-tube-closure

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

616 Views.  Wayne State University. This protocol was developed to longitudinally monitor the mechanical properties of neural plate tissue during chick embryo neurulation. It is based on the integration of a Brillouin microscope and an on-stage incubation system, enabling live mechanical imaging of neural plate tissue in ex ovo cultured chick embryos.

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

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Biology

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

Neuroscience

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis is becoming increasingly recognized. In particular, the embryonic chick heart and brain tube, which undergo drastic morphological changes as they develop, are among the prime candidates to study the role of physical forces in morphogenesis. Progressive ventral bending&#160;and rightward torsion of the tubular embryonic chick brain happen at the earliest stage of organ-level left-right asymmetry in chick embryonic development. The vitelline membrane (VM) constrains the dorsal side of the embryo and has been implicated in providing the force necessary to induce torsion of the developing brain. Here we present a combination of new ex-ovo experiments and physical modeling to identify the mechanics of brain torsion. At Hamburger-Hamilton stage 11, embryos are harvested and cultured ex ovo (in media). The VM is subsequently removed using a pulled capillary tube. By controlling the level of the fluid and subjecting the embryo to a fluid-air interface, the fluid surface tension of the media can be used to replace the mechanical role of the VM. Microsurgery experiments were also performed to alter the position of the heart to find the resultant change in the chirality of brain torsion. Results from this protocol illustrate the fundamental roles of mechanics in driving morphogenesis.

Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis ...

Bioengineering

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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

Summary

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Chapters in this video

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Neural Tube Closure in Mouse Whole Embryo Culture

Assay for Neural Induction in the Chick Embryo

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Chicken Embryo Spinal Cord Slice Culture Protocol

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo