Name: continuous-wave thulium laser for heating cultured cells to investigate cellular thermal effects
Uploaded: 2017-06-30T13:00:00
Description: Watch this Scientific Journal Video about Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects at JoVE.com

这项工作得到了德国联邦教育研究部（BMBF）（授权号13GW0043C）和欧洲航空航天研究和开发办公室（EOARD，授权号FA9550-15-1-0443）的研究资助。

RPE细胞培养<ol><li> 从猪眼中分离RPE细胞 <ol><li>从当地屠宰场获取新鲜去核的猪眼。保持凉爽（4°C），并在黑暗的环境中。 </li><li>用剪刀去除细胞外组织，并将眼睛浸泡在消毒溶液中5分钟。 </li><li>将眼睛置于无钙和镁（PBS（ - ））的无菌磷酸盐缓冲盐水中直至使用。 </li><li>使用手术刀，在角膜缘后方约5毫米处穿透巩膜。用剪刀切割，平行于角膜缘，切除眼睛的整个前部。 </li><li>?...

<ol>
	<li>Inagaki, 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=Comparative+efficacy+of+pure+yellow+(577-nm)+and+810-nm+subthreshold+micropulse+laser+photocoagulation+combined+with+yellow+(561-577-nm)+direct+photocoagulation+for+diabetic+macular+edema.">Comparative efficacy of pure yellow (577-nm) and 810-nm subthreshold micropulse laser photocoagulation combined with yellow (561-577-nm) direct photocoagulation for diabetic macular edema.</a> Jpn J Ophthalmol. 59 (1), 21-28 (2015).</li><li>Roider, J., 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=Selective+retina+therapy+(SRT)+for+clinically+significant+diabetic+macular+edema.">Selective retina therapy (SRT) for clinically significant diabetic macular edema.</a> Graefes Arch Clin Exp Ophthalmol. 248 (9), 1263-1272 (2010).</li><li>Brinkmann, R., 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=Real-time+temperature+determination+during+retinal+photocoagulation+on+patients.">Real-time temperature determination during retinal photocoagulation on patients.</a> J Biomed Opt. 17 (6), 061219 (2012).</li><li>Yoshimura, N., 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=Photocoagulated+human+retinal+pigment+epithelial+cells+produce+an+inhibitor+of+vascular+endothelial+cell+proliferation.">Photocoagulated human retinal pigment epithelial cells produce an inhibitor of vascular endothelial cell proliferation.</a> Invest Ophthalmol Vis Sci. 36 (8), 1686-1691 (1995).</li><li>Denton, M. 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=Damage+Thresholds+for+Exposure+to+NIR+and+Blue+Lasers+in+an+In+Vitro+RPE+Cell+System.">Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System.</a> Invest Ophthalmol Vis Sci. 47 (7), 3065-3073 (2006).</li><li>Shrestha, R., Choi, T. Y., Chang, W., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-precision+micropipette+sensor+for+cellular-level+real-time+thermal+characterization.">A high-precision micropipette sensor for cellular-level real-time thermal characterization.</a> Sensors (Basel). 11 (9), 8826-8835 (2011).</li><li>Gao, F., Ye, Y., Zhang, Y., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+bath+hyperthermia+reduces+stemness+of+colon+cancer+cells.">Water bath hyperthermia reduces stemness of colon cancer cells.</a> Clin Biochem. 46 (16-17), 1747-1750 (2013).</li><li>Jansen, E. D., van Leeuwen, T. G., Motamedi, M., Borst, C., Welch, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+the+absorption+coefficient+of+water+for+midinfrared+laser+radiation.">Temperature dependence of the absorption coefficient of water for midinfrared laser radiation.</a> Lasers Surg Med. 14 (3), 258-268 (1994).</li><li>Iwami, H., Pruessner, J., Shiraki, K., Brinkmann, R., Miura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+a+laser-induced+sub-lethal+temperature+rise+on+RPE+cells+from+oxidative+stress.">Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress.</a> Exp Eye Res. 124, 37-47 (2014).</li><li>Denton, M. 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=Spatially+correlated+microthermography+maps+threshold+temperature+in+laser-induced+damage.">Spatially correlated microthermography maps threshold temperature in laser-induced damage.</a> J Biomed Optics. 16 (3), (2011).</li><li>Morgan, C. M., Schatz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrophic+creep+of+the+retinal+pigment+epithelium+after+focal+macular+photocoagulation.">Atrophic creep of the retinal pigment epithelium after focal macular photocoagulation.</a> Ophthalmology. 96 (1), 96-103 (1989).</li></ol>

在讨论温度相关的生物细胞反应时，不仅温度升高，而且温度升高的持续时间也是重要的，因为大多数生化过程都是时间依赖性的。特别是在激光诱导的眼科热疗领域，由于时间范围很短（从几毫秒到几秒），很难用精确的温度控制来研究细胞热效应。因此，期望适用于细胞培养模型的激光照射装置和能够进行严格的温度和时间控制的操作系统。热暴露后细胞反应（如蛋白质表达或分泌）的生物?...

了解温度依赖性细胞生物学反应对于成功的高热治疗非常重要。用于眼科的热激光视网膜激光凝固是医学中最成熟的激光治疗之一。视网膜激光治疗使用可见光，主要由绿色到黄色波长。光被视网膜色素上皮（RPE）细胞中的黑色素高度吸收，其形成视网膜的最外层细胞单层。医学界和研究人员近来对非常温和的热辐射（sub-visible光凝）感兴趣，作为不同种类的视网膜疾病1,2的新治疗策略。按照这种趋势，我们的兴趣在于在精确温度控制下，即温控光热疗法（TC-PTT）的技术在低温加热RPE细胞。 最近光电我们研究所的声学技术允许实时测量视网膜辐射部位的温度升高。这样可以控制照射期间的温度升高3 。然而，由于由于不可能测量和控制温度，以前没有考虑过由致死的RPE细胞引起的视网膜上的亚致死热疗，热激光照射后RPE细胞的温度依赖性细胞反应具有迄今为止研究很少。此外，不仅没有详细讨论温差，而且在致死和致死辐射后存活细胞的细胞行为的差异。因此，为了收集基于TC-PTT的治疗的科学证据，我们的目的是通过体外实验设置来阐明温度依赖性RPE细胞的生物反应及其机制。 对于t他的目的是建立满足以下条件的电池加热装置：1）快速升温的可能性，2）精确控制的时间和温度，3）生物实验检测细胞数量相对较多。关于加热方法，临床激光，例如倍频Nd.YAG激光（532nm），不幸的是不适合于细胞培养加热。这是因为培养的RPE细胞中黑素体数量大大减少。激光吸收可能是不均匀的，并且在实验之间，细胞水平的温度升高是可变的，即使用相同的辐射功率照射也是如此。以前的几项研究报道了在照射期间使用黑色纸底部的黑纸4或使用在实验5,6之前被培养细胞吞噬的其他黑素体。许多使用热板，水浴或温度为7 ℃的CO 2培养箱进行用于评估热疗诱导的细胞应答的体外生物学研究。这些方法需要较长的加热周期，因为它需要一些时间（ 即几分钟）达到所需的温度。此外，使用这些方法，难以在细胞水平上获得详细的热历史（ 即，温度乘以时间）。此外，一个培养皿中不同位置的细胞之间的温度可能由于可变的温度扩散而不同。在大多数情况下，即使生物细胞反应可能严重受温度升高和持续时间的影响，生物分析尚未考虑过热过程中的这种时间和空间温度信息。 为了克服这些问题，在这里使用湍流湍流激光来加热细胞。钍激光辐射（λ=1.94μm）被水8强烈吸收，培养皿底部的细胞仅通过热扩散热刺激。具有365μm直径的激光纤维设置在培养皿上方约12cm处，两者之间没有任何光学器件。激光束直径发散，几乎等于培养基表面培养皿（30mm）的内径。用一致量的培养基可以照射温度升高的细胞重复性高。可变功率设置可实现高达20 W的照射，细胞水平的介质温度可能会在10秒内增加到ΔT≈26°C。 通过改变照射条件，也可以改变激光束轮廓以改变温度分布在一个文化菜。例如，可以用当前研究中的高斯样温度分布或均匀的温度分布进行研究。后者可能有利于调查温度依赖性细胞反应对于亚致死温度升高的影响，而不是用于细胞死亡应激或伤口愈合反应。 总之，不同热暴露后，ium激光照射可以调查不同种类的生物因子，如基因/蛋白质表达，细胞死亡动力学，细胞增殖和细胞功能发育。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5796-500ML</td>
			<td>Add (2)-(4) before use. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955-100ML</td>
			<td>Containing 10000 units penicillin, 10 mg streptomycin and 25 &#956;g Amphotericin B in 1ml. Add 5.5 ml in 500 ml medium bottle (1) before use.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>Sigma-Aldrich</td>
			<td>S8636-100ML</td>
			<td>Add 5.5 ml in 500 ml medium bottle (1) before use (final concentration: 1 mM)</td>
		</tr>
		<tr>
			<td>Porcine serum</td>
			<td>Sigma-Aldrich</td>
			<td>12736C-500ML</td>
			<td>Add 50 ml in 500 ml medium bottole (1) before use (final: 10%)</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>T4799-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>ED-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF Quantikine ELISA Kit</td>
			<td>R&#38;D System</td>
			<td>DVE00</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxiselect Total Glutathione Assay Kit</td>
			<td>Cell Biolabs, Inc</td>
			<td>STA-312</td>
			<td></td>
		</tr>
		<tr>
			<td>Apoptotic/Necrotic/Healthy Cells Detection Kit</td>
			<td>PromoKine</td>
			<td>PK-CA707-30018</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thulium laser</td>
			<td>Starmedtec GmbH</td>
			<td>Prototype</td>
			<td>0-20 W</td>
		</tr>
		<tr>
			<td>365 mm core diameter fiber</td>
			<td>LASER COMPONENTS Germany</td>
			<td>CF01493-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouple</td>
			<td>Omega Engineering Inc</td>
			<td>HYP-0- 33-1-T-G-60-SMPW-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>MEDAX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader (spectrofluorometer)</td>
			<td>Molecular Device</td>
			<td>Spectramax M4</td>
			<td></td>
		</tr>
		<tr>
			<td>cell homogenizer</td>
			<td>QIAGEN</td>
			<td>TissueLyser LT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>mathematical software program</td>
			<td>The Mathworks. Inc</td>
			<td>MATLAB Release 2015b</td>
			<td></td>
		</tr>
		<tr>
			<td>system-design platform</td>
			<td>National Instrument</td>
			<td>Labview</td>
			<td>Laboratory Virtual Instrument Engineering Workbench</td>
		</tr>
	</tbody>
</table>

continuous-wave thulium laser for heating cultured cells to investigate cellular thermal effects

这里介绍一种使用1.94μm连续波ium激光加热培养细胞进行生物评估的原始方法。钍激光辐射被水强烈吸收，培养皿底部的细胞通过热扩散加热。直径为365μm的激光纤维设置在培养皿上方约12cm处，没有任何光学元件，使得激光束直径几乎等于培养皿的内径（30mm）。通过在每个实验中保持一定量的培养基，可以以高度可重现的温度升高来照射细胞。 为了校准每个功率设置的一个细胞培养皿中的温度升高及其分布，在不同位置和细胞水平的10秒照射期间测量温度。使用数学图形软件表示温度分布节目，其文化菜肴的格局是高斯形式。激光照射后，可以进行不同的生物实验来评估温度依赖性细胞反应。在本手稿中，介绍了活力染色（ 即区分活细胞，凋亡细胞和死细胞），以帮助确定不同时间点后细胞凋亡和死亡的阈值温度。 该方法的优点是温度和加热时间的精确度以及其在整个细胞培养皿中加热细胞的高效率。此外，它允许学习各种各样的温度和时间，这可以由计算机操作系统良好控制。

不同功率设置后的温度分布在温度校准中监测每个单次照射的所有温度发展。根据该数据，得到测定点的最高温度，定义为T max （℃）。 如图3A所示，在将培养皿放置在加热板上的时间点执行程序。在37℃下保存稳定的培养基温度需要140s的“预热时间1”之后，在8秒的“预热时间2”内除去培养皿的?...

Watch this Scientific Journal Video about Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects at JoVE.com

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

这里介绍了使用1.94μm连续波激光辐射在培养皿中加热细胞的原始实验装置。使用这种方法，可以研究不同热暴露后视网膜色素上皮（RPE）细胞的生物反应。

连续波ium激光加热培养细胞以研究细胞热效应

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

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