此处包含的一些数字经版权许可中心许可已修改：斯普林格自然， 生物医学工程年鉴。用均匀的 z 轴菌株应变 3D 水凝胶，同时实现实时显微镜成像，A. Roitblat Riba，S. 纳坦，A. 科莱尔，H. 拉什金，O. 柴切扬，A. 莱斯曼，版权所有© （2019）。
https://doi.org/10.1007/s10439-019-02426-7

作者没有什么可透露的。

本文介绍的方法和协议主要基于我们之前由罗伊特布拉特·里巴等人进行的研究。 
与现有方法相比，该方法的主要优点包括有可能从其周长中拉紧非常柔软的 3D 水凝胶（弹性 Modulus +100 Pa），并在 实时 对焦成像下进行。其他方法通常限制其在 Z轴中应用应变场的能力，并且无法在拉伸时提供原位高放大显微镜图像，这主要是由于从样品?...

组织对机械力的反应是多种生物功能的组成部分，包括基因表达1、细胞分化2和组织改造3。此外，细胞外基质（ECM）的力诱发变化，如纤维对齐和密度化，可以影响细胞行为和组织形成4，5，6。ECM的纤维网状结构具有耐人寻味的机械特性，如非线性弹性、非擦合变形和塑料变形7、8、9、10、11、12。这些特性影响细胞及其周围微环境对外部机械力的反应13、14。了解 ECM 和组织对机械力的反应将推动组织工程领域以及更精确的计算和理论模型的开发取得进展。
最常见的机械拉伸样本的方法都集中在细胞载2D基材上，以探索对细胞行为的影响。例如，这些包括将菌株应用于多晶硅氧烷（PDMS）基板，以及分析与拉伸方向15、16、17、18、19相关的细胞重新定向角度。然而，研究3D细胞嵌入式水凝胶对外部拉伸的反应的方法更为有限，这种情况更密切地模仿组织微环境。与3D矩阵20相比，3D拉伸方法的进步尤为重要，因为细胞在2D基材上的行为不同。这些行为包括细胞重新调整，蛋白质表达水平，和迁移模式21，22，23。
允许3D样品拉伸的方法和设备包括市售的24、25、26、27、28和为实验室研究开发的方法和设备29。这些方法使用可拆解硅胶管30，多井室31，夹子26，32，生物反应器11，33，罐34，35，36，和磁铁37，38。有些技术会产生局部变形的3D水凝胶，例如从凝胶5中的两个单点拔针，而另一些技术允许凝胶16的大部分变形。此外，这些系统大多侧重于分析X-Y平面中的应变场，而Z方向的应变场信息有限。此外，这些设备中只有少数能够进行微观原位成像。原位高放大成像（例如共聚焦显微镜）的主要挑战是，从目标透镜到样品的几百微米工作距离有限。允许在拉伸过程中进行实时成像的设备牺牲了Z轴应变的均匀性，或者相对复杂且难以在其他实验室39，40中复制。
这种伸展3D水凝胶的方法允许在活体对焦显微镜期间进行静态或周期性单轴应变。拉伸设备（称为"智能环状单轴拉伸器 - SCyUS"）采用 3D 打印部件和低成本硬件构建，可在其他实验室中轻松复制。连接到设备的是一个市售的硅橡胶，其中心有几何切口。水凝胶组件聚合以填充切口。在聚合过程中，生物水凝胶，如纤维蛋白或胶原蛋白，自然粘附在切口的内壁上。使用SCyUS，硅胶条是非近似拉伸，转移控制菌株到嵌入式3D水凝胶41。
与其他现有方法相比，该系统允许将功能和优势的独特组合。首先，该系统允许从其外围单轴拉伸厚的 3D 软水凝胶（>100 微米厚，<1 kPa 刚度），在整个水凝胶中 Z- 同质变形。这些水凝胶太软，不能被传统的拉伸技术抓住和拉伸。其次，拉伸设备可以很容易地在其他实验室复制，因为 3D 打印可供研究人员使用，设计中使用的电子产品成本低廉。第三，也许也是最吸引人的特征，硅胶条中切口的几何形状和大小可以很容易地操纵，允许可调谐应变梯度和边界条件，以及使用各种样品体积，下至几微升。
所提出的协议包括将纤维蛋白凝胶成直径约2毫米的圆盘，在0.5毫米厚的硅橡胶条中，在活体共焦显微镜下进行单轴拉伸。以下详细讨论了测量和分析几何切口上的菌株、水凝胶中形成的内部菌株以及各种拉伸操作后产生的纤维对齐的实验程序。最后，讨论了将细胞嵌入水凝胶中并使其暴露在受控外部拉伸的可能性。

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 546 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A20002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Medium (DMEM High Glucose)</td>
			<td>Biological Industries</td>
			<td>01-052-1A</td>
			<td>Add 10% FBS, 1% PNS, 1% L-Glutamine, 1% Sodium Pyruvate</td>
		</tr>
		<tr>
			<td>Cover Slip #1.5</td>
			<td>Bar-Naor Ltd.</td>
			<td>BN72204-30</td>
			<td>22&#215;40 mm</td>
		</tr>
		<tr>
			<td>DIMETHYL SULPHOXIDE 99.5% GC DMSO</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>D-5879-500 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Biological Industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>EVICEL Fibrin Sealant (Human)</td>
			<td>Omrix Biopharmaceuticals</td>
			<td>3902</td>
			<td>Fibrinogen: 70 mg/mL, Thrombin: 800-1200 IU/mL</td>
		</tr>
		<tr>
			<td>Fibrinogen Buffer</td>
			<td>N/A</td>
			<td></td>
			<td>Recipe for 1L: 7g NaCl, 2.94g trisodium citrate dihydrate, 9g glycine, 20g arginine hydrochloride &#38; 0.15g calcium chloride dihydrate. Bring final volume to 1L with PuW (pH 7.0-7.2)</td>
		</tr>
		<tr>
			<td>Fluorescent micro-beads FluoSpheres (1 &#181;m)</td>
			<td>Invitrogen</td>
			<td>F8820</td>
			<td>Orange (540/560) 
			Provided as suspension (2% solids) in water plus 2 mM sodium azide</td>
		</tr>
		<tr>
			<td>High-Temperature Silicone Rubber</td>
			<td>McMaster-Carr</td>
			<td>3788T41</td>
			<td>580 &#181;m-thick 
			E = 1.5 Mpa 
			Poisson Ratio = 0.48 
			Tensile Strength = 4.8 MPa 
			Upper limit of stretch = +300% engineering strain</td>
		</tr>
		<tr>
			<td>HiTrap desalting column 5 mL (Sephadex G-25 packed)</td>
			<td>GE Healthcare</td>
			<td>17-1408-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HIVAC-G High Vacuum Sealing Compound</td>
			<td>Shin-Etsu Chemical Co., Ltd.</td>
			<td>HIVAC-G 100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ FIJI software39</td>
			<td>National Institute of Health, Bethesda, MD</td>
			<td>Version 1.8.0_112</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcontroller (Adruino Uno + Adafruit Motorshield v2.3)</td>
			<td>Arduino/Adafruit</td>
			<td>Arduino-DK001/Adafruit-1438</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroVL 21R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002470</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Primovert Light Microscope</td>
			<td>Carl Zeiss Suzhou Co., Ltd.</td>
			<td>491206-0011-000</td>
			<td></td>
		</tr>
		<tr>
			<td>SCyUS CAD (Solidworks)</td>
			<td>Dassault Syst&#232;mes</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>SCyUS Code37</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Servomotor - TowerPro SG-5010</td>
			<td>Adafruit</td>
			<td>155</td>
			<td></td>
		</tr>
		<tr>
			<td>SL 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004030</td>
			<td>For 50 mL tubes</td>
		</tr>
		<tr>
			<td>Sterile 10 cm non-culture plates</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin buffer</td>
			<td>N/A</td>
			<td></td>
			<td>Recipe for 1L: 20g mannitol, 8.77g NaCl, 2.72g sodium acetate trihydrate, 24 mL 25% Human Serum Albumin, 5.88g calcium chloride. Bring final volume to 1L with PuW (pH 7.0)</td>
		</tr>
		<tr>
			<td>Trypsin EDTA Solution B (0.25%), EDTA (0.05%)</td>
			<td>Biological Industries</td>
			<td>03-052-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>USB Cable (Type B Male to Type A Male)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 880 Confocal Microscope</td>
			<td>Carl Zeiss AG</td>
			<td>2811000417</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN 2.3 SP1 FP3 (black)</td>
			<td>Carl Zeiss AG</td>
			<td>Release Version 14.0.0.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

controlled strain of 3d hydrogels under live microscopy imaging

外力是组织形成、发育和维护的重要因素。这些力的作用通常使用专门的体外拉伸方法进行研究。各种可用系统使用基于 2D 基板的担架，而 3D 技术对软水凝胶应变的可及性则受到更多限制。在这里，我们描述了一种方法，允许软水凝胶从其周长外部拉伸，使用弹性硅胶条作为样品载体。本协议中使用的拉伸系统由 3D 打印部件和低成本电子产品构建，使其简单易于在其他实验室中复制。实验过程从聚合厚（>100微米）软纤维素水凝胶（弹性Modulus+100 Pa）开始，切口位于硅胶条的中心。然后，硅胶构造连接到打印拉伸装置上，并放置在共焦显微镜阶段。在实时显微镜下，拉伸装置被激活，凝胶以各种拉伸量度进行成像。然后，用于图像处理来量化由此产生的凝胶变形，在整个凝胶的 3D 厚度（Z-轴） 中显示相对均匀的菌株和纤维对齐。这种方法的优点包括能够在进行原位显微镜时以3D方式应变极其柔软的水凝胶，以及根据用户的需求自由地操纵样品的几何形状和大小。此外，通过适当的适应，该方法可用于拉伸其他类型的水凝胶（如胶原蛋白、聚丙烯酰胺或聚乙二醇），并允许在更逼真3D条件下分析细胞和组织对外部力量的反应。

图9显示了用于硅胶条的静态增量拉伸数据，该条带有3D纤维蛋白水凝胶，内嵌1μm荧光珠。分析表明硅胶拉伸对切口的几何变化以及凝胶中发达菌株的影响。整个凝胶的Z 堆栈图像用于评估原始圆形切口到椭圆几何形状的变形 （图 9A）。这些图像用于计算ε xx（孔） （方程 2）。在凝胶拉伸过程中放大并手动跟踪...

Watch this Scientific Journal Video about Controlled Strain of 3D Hydrogels under Live Microscopy Imaging at JoVE.com

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

提出的方法涉及嵌入硅橡胶的3D软水凝胶的单轴拉伸，同时允许活体共焦显微镜。演示了外部和内部水凝胶菌株的特征以及纤维对齐。开发的设备和协议可以评估细胞对各种菌株机制的反应。

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

controlled-strain-of-3d-hydrogels-under-live-microscopy-imaging

Research

JoVE Journal

Bioengineering

3.4K 次观看.  Tel-Aviv University. 提出的方法涉及嵌入硅橡胶的3D软水凝胶的单轴拉伸，同时允许活体共焦显微镜。演示了外部和内部水凝胶菌株的特征以及纤维对齐。开发的设备和协议可以评估细胞对各种菌株机制的反应。

视频: Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

磁力辅助的远程控制微导管尖端偏转根据磁共振成像

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

单轴拉伸应变下活细胞核动力学的高分辨率成像

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

用于使用受激拉曼散射显微镜进行延时活细胞成像的柔性腔室

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

具有长距离纤维排列的微工程3D胶原蛋白水凝胶

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...