Name: live-cell imaging of intact ex vivo globes using a novel 3d printed holder
Uploaded: 2022-10-06T14:15:00
Description: Watch this Scientific Journal Video about Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder at JoVE.com

我们要感谢NIH的以下赠款支持：RO1EY032079（VTR），R21EY029097-01（VTR），1F30EY033647-01（KS）和5T32GM008541-24（KS）。我们还要感谢马萨诸塞州狮子会眼科研究基金和新英格兰角膜移植基金。

涉及动物受试者的程序得到了视觉和眼科研究协会在眼科护理和视觉研究中使用动物以及波士顿大学IACUC协议（201800302）的批准。
1. 设计3D打印支架和盖杆
<ol><li>设计3D打印支架和盖杆，考虑鼠标地球仪的平均直径，并3D打印设计（图1A，B）。</li>
	<li>保持支架内壁的直径略大于地球仪的平均直径，以考虑个体小鼠的不同大小。?...

<ol>
	<li>Kneer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+fat+diet+induces+pre-type+2+diabetes+with+regional+changes+in+corneal+sensory+nerves+and+altered+P2X7+expression+and+localization.">High fat diet induces pre-type 2 diabetes with regional changes in corneal sensory nerves and altered P2X7 expression and localization.</a> Experimental Eye Research. 175, 44-55 (2018).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+Ca2++mobilizations:+A+quantitative+approach+to+predict+their+importance+in+cell-cell+communication+and+wound+healing.">Sustained Ca2+ mobilizations: A quantitative approach to predict their importance in cell-cell communication and wound healing.</a> PLoS One. 14 (4), 0213422 (2019).</li><li>Stepp, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wounding+the+cornea+to+learn+how+it+heals.">Wounding the cornea to learn how it heals.</a> Experimental Eye Research. 121, 178-193 (2014).</li><li>Klepeis, V. S., Cornell-Bell, A., Trinkaus-Randal, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+but+not+gap+junctions+play+a+role+in+injury-induced+Ca2++waves+in+epithelial+cells.">Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells.</a> Journal of Cell Science. 114 (23), 4185-4195 (2001).</li><li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-induced+changes+in+Ca(2+)+mobilization+and+protein+phosphorylation+implicated+in+impaired+wound+healing.">Hypoxia-induced changes in Ca(2+) mobilization and protein phosphorylation implicated in impaired wound healing.</a> American Journal of Physiology. Cell Physiology. 306 (10), 972-985 (2014).</li><li>Boucher, I., Rich, C., Lee, A., Marcincin, A., Trinkaus-Randall, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y2+receptor+mediates+the+epithelial+injury+response+and+cell+migration.">The P2Y2 receptor mediates the epithelial injury response and cell migration.</a> American Journal of Physiology. Cell Physiology. 299 (2), 411-421 (2010).</li><li>Awal, M. R., Wirak, G. S., Gabel, C. V., Connor, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapse+of+global+neuronal+states+in+Caenorhabditis+elegans+under+isoflurane+anesthesia.">Collapse of global neuronal states in Caenorhabditis elegans under isoflurane anesthesia.</a> Anesthesiology. 133 (1), 133-144 (2020).</li><li>Burnett, K., Edsinger, E., Albrecht, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+gentle+hydrogel+encapsulation+of+living+organisms+enables+long-term+microscopy+over+multiple+hours.">Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours.</a> Communications Biology. 1, 73 (2018).</li><li>Rhodes, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pannexin1:+Role+as+a+sensor+to+injury+is+attenuated+in+pretype+2+corneal+diabetic+epithelium.">Pannexin1: Role as a sensor to injury is attenuated in pretype 2 corneal diabetic epithelium.</a> Analytical Cellular Pathology. 2021, 4793338 (2021).</li><li>Segars, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+dependent+changes+in+corneal+epithelial+cell+signaling.">Age dependent changes in corneal epithelial cell signaling.</a> Frontiers in Cell and Developmental Biology. 10, 886721 (2022).</li><li>Xu, P., Londregan, A., Rich, C., Trinkaus-Randall, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+epithelial+and+stromal+corneal+stiffness+occur+with+age+and+obesity.">Changes in epithelial and stromal corneal stiffness occur with age and obesity.</a> Bioengineering. 7 (1), 14 (2020).</li><li>Minns, M. S., Teicher, G., Rich, C. B., Trinkaus-Randall, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinoreceptor+P2X7+regulation+of+Ca(2+)+mobilization+and+cytoskeletal+rearrangement+is+required+for+corneal+reepithelialization+after+injury.">Purinoreceptor P2X7 regulation of Ca(2+) mobilization and cytoskeletal rearrangement is required for corneal reepithelialization after injury.</a> The American Journal of Pathology. 186 (2), 285-296 (2016).</li><li>Tadvalkar, G., Pal-Ghosh, S., Pajoohesh-Ganji, A., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+euthanasia+and+enucleation+on+mouse+corneal+epithelial+axon+density+and+nerve+terminal+morphology.">The impact of euthanasia and enucleation on mouse corneal epithelial axon density and nerve terminal morphology.</a> The Ocular Surface. 18 (4), 821-828 (2020).</li></ol>

该协议描述了一种活细胞成像技术，该技术使用3D打印支架来稳定和固定完整的动物眼睛。它旨在规避先前离 体 角膜组织活细胞成像方案所识别的几个显着缺点。该协议为完整地球仪的活细胞成像提供了许多优势。它显着减少了不必要的组织损伤，这些损伤可能会干扰对实验诱导的划痕伤口的伤口愈合反应。这包括解剖和暴露于紫外线对神经和上皮的损害。此外，该协议通过固定组织的方...

角膜 角膜是一种透明的无血管结构，覆盖眼睛前表面，可折射光线以实现视力并保护眼睛内部免受损伤。由于角膜暴露在环境中，它容易受到机械原因（抓挠）和感染的损害。其他方面健康的患者的角膜损伤通常会在 1-3 天内愈合。然而，在患有包括角膜缘干细胞缺乏症和II型糖尿病在内的潜在疾病患者中，角膜伤口愈合过程可以大大延长1。由于角膜是高度神经支配的，这些不愈合的角膜溃疡和复发性角膜糜烂非常痛苦，并大大降低了经历它们的患者的生活质量1。
细胞信号传导 当原本健康的角膜受伤时，伤口附近细胞中的钙信号事件先于并促使细胞迁移到伤口床上，在那里它们闭合损伤而没有疤痕的风险2，3。这些信号传导事件已在使用活细胞成像的角膜上皮细胞培养模型中得到了很好的表征2。初步实验表明，与糖尿病细胞相比，非糖尿病细胞损伤后的钙信号传导明显更多。然而，在 离体 球中表征细胞信号事件已被证明是一项技术挑战。
活细胞成像 以前的研究已经成功地记录了角膜伤口愈合的体外细胞培养模型的钙信号传导事件4，5，6。开发一种方法在离体组织中产生这些信号事件的高质量图像是非常有趣的，因为它将允许在更复杂和逼真的系统中研究这些事件。以前的方法包括解剖角膜，然后固定在紫外线诱导的PEG凝胶7，8，9中。在处理活组织时，固定化是一个重要但具有挑战性的步骤，因为它必须在整个实验过程中保持活力和水合。此外，固定不得损坏组织。虽然PEG溶液固定了组织，但产生的图像的分辨率和质量并不一致。因此，开发了3D打印支架来固定完整的地球仪，以产生更高质量的图像，同时降低组织损伤的风险。
A. 方法 开发了一种独特的3D打印支架，用于固定完整的 离体 地球仪以进行活细胞成像。该支架可防止来自两个主要来源的损坏：它允许对去核眼球进行成像而无需解剖角膜，并且它消除了暴露于紫外线。没有这些损伤源，获得的图像更准确地代表了对实验造成的划痕伤害的反应。此外，3D打印支架被校准到小鼠眼睛的精确尺寸。这提供了比PEG溶液中固定更好的拟合，由于组织运动减少，在低倍物镜下可获得更高质量的图像。固定器顶部的盖杆可确保地球仪在整个实验过程中保持不动，并且在应用生长培养基以保持水合作用和活力时，地球仪不会移位。将支架打印到精确尺寸的能力也使我们能够为由于年龄或疾病状态而不同大小的鼠眼产生最佳贴合度。这项技术可以更广泛地应用于根据不同物种的尺寸开发支架。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.75 blue polylactic acid (PLA) plastic</td>
			<td>Creality (Shenzen, China)</td>
			<td>N/A</td>
			<td>Material for holder</td>
		</tr>
		<tr>
			<td>35 mm Dish, No. 1.5 Coverslip, 14 mm glass diameter, Poly-D-Lysine Coated</td>
			<td>MatTek Corporation (Ashland, MA)</td>
			<td>P35GC-1.5-14-C</td>
			<td>Well for imaging.&#160;</td>
		</tr>
		<tr>
			<td>Autodesk Fusion 360 software</td>
			<td>Autodesk (San Rafael, CA).</td>
			<td>N/A</td>
			<td>Software used for printing the holders.</td>
		</tr>
		<tr>
			<td>BD 25 G 7/8 sterile needles single use 100 needles/box</td>
			<td>Thermo Fisher Scientific (Waltham, MA)</td>
			<td>305124</td>
			<td>For experimentally-induced wounds to the globes</td>
		</tr>
		<tr>
			<td>CellMask DeepRed</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>C10046</td>
			<td>Cell membrane counterstain. Calcium indicator. 1:10,000 concentration with a final concentration of 1%(v/v) DMSO and 0.1% (w/v) pluronic acid</td>
		</tr>
		<tr>
			<td>Complete Home Super Glue</td>
			<td>Walgreens (Deerfield, IL)</td>
			<td>N/A</td>
			<td>For attaching the holder to the imaging well</td>
		</tr>
		<tr>
			<td>Ender 3 Pro 3D printer&#160;</td>
			<td>Creality (Shenzen, China)</td>
			<td>N/A</td>
			<td>For printing the holder</td>
		</tr>
		<tr>
			<td>FIJI/ImageJ</td>
			<td>ImageJ (Bethesda, MD)</td>
			<td>License Number: GPL2</td>
			<td>Softwareused for confirming consistency of wound depth and diameter between independent globes using Region of Interest analysis</td>
		</tr>
		<tr>
			<td>Fluo-4</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>F14201</td>
			<td>Calcium indicator. 1:100 concentration with a final concentration of 1%(v/v) DMSO and 0.1% (w/v) pluronic acid</td>
		</tr>
		<tr>
			<td>Keratinocyte Serum-Free Medium</td>
			<td>Gibco (Waltham, MA)</td>
			<td>17005042</td>
			<td>25 mg/mL bovine pituitary extract, 0.02 nM EGF, 0.3 mM CaCl2, and penicillin-streptomycin (100 units/mL, 100 &#181;g/mL, respectively) added to medium</td>
		</tr>
		<tr>
			<td>Phophate-Buffered Saline (PBS)</td>
			<td>Corning, Medlabtech (Manassas, VA)</td>
			<td>21-040-CV</td>
			<td>Used to wash excess stain off of corneas before imaging</td>
		</tr>
		<tr>
			<td>Zeiss Confocal 880 Microscope with AiryScan</td>
			<td>Zeiss (Thornwood, NY)</td>
			<td>N/A</td>
			<td>20x magnification objective was used</td>
		</tr>
	</tbody>
</table>

live-cell imaging of intact ex vivo globes using a novel 3d printed holder

角膜上皮伤口愈合是由上皮细胞上表达的嘌呤能受体的激活引发的迁移过程。这种激活导致钙动员事件在细胞之间传播，这对于启动细胞运动进入伤口床至关重要，促进有效的伤口愈合。Trinkaus-Randall实验室开发了一种在 离体 鼠球中实时成像角膜伤口愈合反应的方法。这种方法涉及从已按照既定方案安乐死的小鼠身上取出完整的球体，并立即用钙指示剂染料孵育球体。在此阶段可以应用染色细胞其他特征的复染剂，以帮助成像并显示细胞标志。该方案适用于用于复染的几种不同的活细胞染料，包括用于染色肌动蛋白的SiR肌动蛋白和用于染色细胞膜的深红色质膜染色。为了检查对伤口的反应，使用25G针损伤角膜上皮，并将地球仪放置在3D打印支架中。3D打印支架的尺寸经过校准，以确保在整个实验期间地球固定，并且可以修改以适应不同大小的眼睛。随着时间的推移，使用共聚焦显微镜在整个组织的不同深度连续进行伤口反应的活细胞成像。该协议使我们能够在共聚焦显微镜上使用20倍空气物镜生成高分辨率，出版质量的图像。其他目标也可用于此协议。它代表了 离体 鼠球活细胞成像质量的显着提高，并允许识别神经和上皮。

该协议已用于始终如一地生成出版质量的数据和图像10。与以前的方法相比，获得的图像具有显着的改进（图2）。使用3D打印支架，可以在整个角膜层中捕获图像，并且可以观察到不同z平面中的钙动员（图3）。该方法已被用于比较年轻和老年小鼠伤口处顶端和基底细胞层之间的细胞 - 细胞信号传导10。
<p class="jove...

Watch this Scientific Journal Video about Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder at JoVE.com

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

这项工作描述了一种新的实验方案，该方案利用3D打印支架来实现去核球的高分辨率活细胞成像。通过该协议，可以实时观察离 体 球受伤角膜上皮中的细胞钙信号活性。

使用新型3D打印支架对完整的 离体 地球仪进行活细胞成像

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

该协议中使用的3D打印支架使我们能够实时监测受伤角膜中的短期细胞信号传导和长期细胞迁移事件。通过保持眼球完整、可行和不动，该协议产生高质量的角膜细胞实时图像，包括上皮和神经。3D打印支架可以很容易地修改为各种尺寸和物种的地球仪。我们还可以以不同的方式定位眼睛以成像其他区域或进行纳米压痕实验。首先，在取出安乐死小鼠的头部后，立即将其放在冰上以保持组织的活力。然后用镊子通过道具打瞌睡来去除地球仪。并用解剖剪刀夹住视神经，就在镊子固定视神经的下方。在P-35细胞培养皿中将球体在两毫升培养基中孵育，包括钙指示剂和/或细胞膜染色剂在37摄氏度和5%二氧化碳的培养箱中在低光照条件下孵育一小时，确保将球浸没在染色培养基中以进行均匀染色。要将支架粘附在干净的玻璃底盖上，请在 70% 乙醇中清洗支架并将胶水涂在支架上。然后，将支架粘附在玻璃底盖上，确保支架内部区域内没有胶水，因为胶水会发出荧光，使成像复杂化。胶水凝固后，确认支架与盖玻片牢固。使用无菌滴管从染色溶液中取出地球仪，注意防止组织对感兴趣区域造成损害。在室温下洗涤地球仪五分钟，使用无菌磷酸盐缓冲盐水去除多余的污渍，并将地球仪放入培养基中以运输到显微镜。要在感兴趣的区域使用无菌 25 号针头缠绕地球仪，请使用无菌吸管从眼睛后部拿起并握住地球仪，以保持眼球稳定并防止其滚动。对于划痕伤口，轻轻移动无菌 25 号针穿过暴露的角膜，或将针头直接轻轻按入中央角膜以进行穿刺伤口，确保伤口不会刺穿角膜基底膜。将角膜或角膜缘区域放在支架内部区域的盖玻片上，同时确认地球仪位置正确且感兴趣部位与玻璃盖玻片接触。使用粘附在支架上的 3D 打印盖子，使用胶水稳定。并且不要试图取下地球仪，因为这可能会导致组织损伤。打开显微镜，将环境室设置为35摄氏度和5%二氧化碳，并验证加湿室。使用活细胞成像技术将盖玻片支架和稳定地球仪放在环境室内的显微镜载物台上并成像。通过在盖玻片上检查额外的生长培养基以防止脱水并保持组织活力，确保孔中有足够的培养基覆盖支架中的球体。使用活细胞成像技术开始实验，同时使用低功率激光设置来保存组织并防止组织损伤和适合长时间实验的目标。以CZI文件的首选文件格式记录和保存数据以进行数据记录。通过3D打印支架稳定，获得了X体内角膜多层结构的高质量成像数据，展示了角膜顶端，基底和基质层中的钙信号事件。获得角膜的Z-stack图像，固定在3D打印支架中，在划痕伤口处，以可视化整个角膜层的细胞膜和钙信号传导以及不同Z平面中的钙动员。进行了角膜的时间序列实验，以研究愈合反应期间细胞迁移到伤口床中的情况，揭示了眼球在X，Y或Z方向上的运动很小。据观察，通过将地球仪放在支架中，可以可视化角膜的不同区域，包括中央和角膜边缘区域。该协议使我们能够从表现出伤口愈合差异的疾病模型中对眼睛进行成像。我们可以表征活组织中细胞信号传导和运动中的畸变。

Research

JoVE Journal

Biochemistry

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

在质膜用新型荧光探针磷脂酰-4,5-二磷酸的单分子超分辨率成像

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

细胞外基质的Glycoproteomics：使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

用于大分子 X 射线晶体学的一体式样品支架,具有最小的背景散射

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

使用新型3D打印支架对完整的 离体 地球仪进行活细胞成像

使用新型3D打印支架对完整的离体地球仪进行活细胞成像