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

Published: November 10, 2023

doi:

Chenjun Shi, Chenchen Handler, Haden Florn, Jitao Zhang

¹Department of Biomedical Engineering, College of Engineering,Wayne State University, ²Fischell Department of Bioengineering,University of Maryland

Summary

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.

Abstract

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.

Introduction

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) 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 ω_R, following the equation³¹:

(1)

Here, is the refractive index of the material, λ is the wavelength of the incident light, M' is the longitudinal modulus, ρ is the mass density, and θ 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 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.

Protocol

The protocol has been approved by the Institutional Animal Care and Use Committee of Wayne State University. 1. Experimental preparation Use a 70% ethanol solution to clean and sterilize the scissors and tweezers. Also, prepare disposable pipettes and a syringe. 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 wa…

Representative Results

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 …

Discussion

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 s…

Declarações

The authors have nothing to disclose.

Acknowledgements

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

Materials

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

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Shi, C., Handler, C., Florn, H., Zhang, J. Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo. J. Vis. Exp. (201), e66117, doi:10.3791/66117 (2023).