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>去除眼睛前部（ 即角膜和晶状体）和玻璃体。加入1 mL PBS（ - ），轻轻取出神经视网膜。 注意:这个"眼杯"，由巩膜，脉络膜和RPE组成，现在已经准备好了。 </li><li>在P中加入预热（37℃）0.25％胰蛋白酶BS（ - ）到眼杯。调节体积，使眼睛杯的大约80％充满这种胰蛋白酶溶液。 </li><li>使用胰蛋白酶溶液将眼杯在5％CO 2培养箱中于37℃孵育10分钟。 </li><li>从培养箱中取出眼罩，用含有0.05％胰蛋白酶+ 0.2％乙二胺四乙酸四钠盐（EDTA·4N）的PBS（ - ）溶液代替0.25％胰蛋白酶溶液。将孵育器中的眼杯孵育45分钟。 注意:45分钟后，RPE细胞将松弛地连接到Bruch膜上，或者已经分离并漂浮在胰蛋白酶-EDTA溶液中。 </li><li>通过温和吸液收集RPE细胞。将细胞和溶液收集在装有10mL培养基（含有L-谷氨酰胺的DMEM高葡萄糖）的锥形管中，包括10％猪血清，抗生素/抗真菌剂和丙酮酸钠（1mM）。 注意:血清可能会中和胰蛋白酶的作用。 </li><li>离心机室温400×g细胞悬液5分钟。 </li><li>取上清液加入10毫升新鲜培养基。在相同条件下再次离心5分钟。 </li><li>去除上清液并加入新培养基，使得细胞浓度达到5×10 5个细胞/ mL（通过使用血细胞计数器计数细胞测定）。通过温和移液混匀。 </li><li>在细胞培养皿中分配细胞悬浮液。每60毫米直径的培养皿使用3毫升。 注意:这种文化称为通过零（P0）。 </li><li>将细胞保持在37℃的5％CO 2培养箱中。将条件培养基的一半每隔一天更换成新鲜培养基。 </li><li>亚文化（步骤1.2）如果变得融合。 </li></ol></li><li> RPE细胞培养的亚文化 <ol><li>取出培养基，用PBS（ - ）冲洗细胞两次。 </li><li>用含有0.05％胰蛋白酶+ 0.2％EDTA的PBS（ - ）溶液孵育细胞na 5％CO 2培养箱中37℃5分钟。 </li><li>通过轻轻移液分离RPE细胞，并将细胞悬浮液收集在装有10mL培养基（包括10％猪血清）的锥形管中。 </li><li>在室温下将细胞悬浮液以400xg离心5分钟。 </li><li>去除上清液并添加新的培养基，使细胞浓度为5×10 5个细胞/ mL（通过用血细胞计数器计数细胞数确定）。将细胞分布在新的60毫米直径的培养皿中，如步骤1.1.13中所述。 注意:细胞培养现在是第1（P1）号。 </li><li>达到融合后，使用步骤1.2.1-1.2.5中所述的相同步骤将P1培养基传代培养至P2。从P2培养物中，将细胞种在更小的培养皿（30mm内径）上，而不是直径为60mm的培养皿。 </li><li>对于实验，使用P2或P3培养。 </li></ol></li></ol> Thul镭激光照射<ol><li> 辐照站的建设 <ol><li>将ium激光器件（1.94μm，功率范围:0-20W）连接到0.22-NA，365μm芯直径的光纤。 </li><li>将纤维头机械地固定在水平固定在照射台的垂直金属柱上的金属臂上。放置垂直柱，使得激光纤维的尖端位于照射期​​间放置细胞培养皿的热板上方。 </li><li>在热板上放置白纸，并将瞄准光束（λ= 635 nm，max = 1 mW，纸张直径≈30 mm）。将目标光束的圆周标记在白纸上，使得在照射期间放置培养皿的位置是已知的。 注意:纤维尖端的z平面可能是可变的。没有任何额外的成像光学元件，细胞培养平面上的激光光斑直径，放置在纤维尖端12厘米处，约30mm，几乎相当于细胞培养皿的内径。设置的示意图如图1所示。 </li></ol></li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/54326/54326fig1.jpg" /> 图1:ium激光照射站示意图。将培养皿放置在加热板上。将细胞放置在ium激光纤维尖端下方12cm处，使得束尺寸几乎与培养皿的内径相同（约30mm）。激光照射程序由定制系统设计平台的时间控制程序控制。功率设置必须在照射程序启动前确定。 <a href="http://ecsource.jove.com/files/ftp_upload/54326/54326fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol开始= "2"&gt; <li> 激光照射细胞培养 <ol><li>照射前1 h，用1.2 mL新鲜培养基完全取代培养基。 注意:这是一个关键步骤，必须严格遵循。 </li><li>将辐射站（ 即热板和固定激光光纤的柱）放在干净的工作台上。 </li><li>从培养箱中取出细胞培养皿，并将其放在热板上的标记位置（步骤2.1.3）。 </li><li>戴防护眼镜。打开ium激光。根据需要在激光设备上设置电源（可调0 - 20 W）。打开排放物。 </li><li>启动控制激光照射和定时协议（补充文件）的系统设计平台。 </li><li>将培养皿放置在热板上后，点击"预热时间"开始定时器140秒（"预热时间1"）;这将使培养基温度保持在3℃照射前7°C。 注意:140秒后，哔声会打开，下一个定时器（"预热时间2"）将自动开始计数8秒。在这8年的时间里，考官可以打开文化菜。在预热148秒后，将自动进行细胞培养10秒长的激光照射。在紧急情况下，为激光设备配备强制退出按钮，以立即停止激光。这是一个关键步骤，必须严格遵循。特别要注意的是，在8秒的预热时间开始之前，打开辐照前的盘盖。打开盖可能非常快地冷却介质表面。 </li><li>照射后立即将盖子放回培养皿中，将培养皿放置在热板上另外7秒，然后放回5％CO 2培养箱中37℃。 </li></ol></li><li> 测量温度分布细胞水平（温度校准） <ol><li>在30毫米直径的培养皿（无细胞）的四面（每90°）处接近底部的小孔（直径约300μm）;使用用本生灯加热的针头（20G）。用外部电隔离带密封孔，并用细针形成一个小孔，使得只有一个细微的热电偶（直径200μm）可以在水密条件下穿过该孔插入。 </li><li>在培养皿底部的外侧，绘制2个垂直直径，并将交叉点（ 即底侧的中心）设为坐标零点（0）。在沿着线的每个方向（ 图2 ，蓝点），每3mm径向向外标出盘（ 即， 0,3,6,9,12和15mm）;总分数应为21个。 </li><li>用1.2mL新培养基填充细胞培养皿。放置文化d在37℃的热板上，将一个细微的热电偶（直径200μm）插入侧孔，并将其敏感尖端放在要测量的标记位置。 </li><li>戴防护眼镜。打开ium激光器，并手动设置激光设备的功率（0到20 W，以0.1-W为增量）。 注意:对于温度校准，以3W为增量的功率测量应足够。 </li><li>打开系统设计平台，点击"开始温度采集"按钮（补充文件）开始温度测量。 </li><li>执行与步骤2.2.6相同的步骤。 注意:控制程序每100 ms测量插入的热电偶的温度，并显示在GUI中照射期间的温度进行。 </li><li>对所有21个测量点和不同的功率设置执行这些程序。对所有点和所有功率设置重复整个程序三次gs来实现可靠的数据。 </li><li>将温度数据导出为csv数据，最终可以将其转换为电子表格。在每一点对于一式三份的测量，平均照射结束时的最高温度。从同一个圆上的点平均值（总共4个点，中心点除外）。 </li><li>将获得的平均最大温度绘制在曲线图上，使得距离中心的距离（mm）为x轴，温度升高（ΔT，°C）为y轴。使用数学软件程序的拟合函数将高斯模型拟合到原始数据。创建高斯拟合温度分布。 </li></ol></li></ol><img alt="图2" class="xfigimg" src="/files/ftp_upload/54326/54326fig2.jpg" /> 图2:一个细胞培养皿中的温度校准点。温度数据在中心和5处测量4个不同角度的径向点（蓝点）。 <a href="http://ecsource.jove.com/files/ftp_upload/54326/54326fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 3.不同热辐射后细胞反应的生物学评估<ol><li> 在不同的功率设置和细胞死亡阈值的确定之后，评估细胞活力（ 即，生活，凋亡和死亡） <ol><li>在指定的时间点（ 即照射后3,24和48小时），用PBS（ - ）洗涤细胞，并使用市售试剂盒来评估细胞活力（ 即，重要的，凋亡的，死亡的），根据制造商的协议。 </li><li>通过加入5μL异硫氰酸荧光素（FITC）-annexin V，5μL乙锭均质二聚体III和5μLHoechst 33342至100μL的1x结合缓冲液（均为试剂盒）来制备染色溶液组件）。准备足够的染色溶液以覆盖细胞。孵育细胞15分钟。 </li><li>用结合缓冲液洗涤细胞培养物两次，用PBS（ - ）替换结合缓冲液，并将培养物置于荧光显微镜的阶段。 </li><li>将光路切换到眼镜，选择4&#39;，6-脒基-2-苯基吲哚（DAPI）过滤器，打开照明光，并用4x物镜找到聚焦平面。 </li><li>将光路更改为相机，在显微镜成像软件中在计算机屏幕上找到图像，并调整焦点。 </li><li>使用针迹功能（ 即，该功能在整个盘上记录多个图像，然后创建单个大图像）的显微镜专用软件，以获得整个细胞培养皿的荧光图像。使用3种不同的过滤器组--DAPI，FITC和四甲基罗丹明（TRITC）至图像Hoechst 33342阳性细胞（全细胞核），FITC-膜联蛋白V-定位ve细胞（凋亡）和同源二聚体III-阳性细胞（死亡）。 </li><li>测量染色细胞培养物中死亡（同源二聚体III-阳性）亚区域的半径（mm）和凋亡（膜联蛋白V阳性）带状区域的外/内半径。将这些半径应用于相应功率设置的温度分布的拟合高斯函数。计算死亡或凋亡区域边缘处的确切温度，以澄清细胞死亡和凋亡改变的阈值温度。 </li></ol></li></ol>

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作者没有什么可以披露的。

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><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium - high glucose</td><td>Sigma-Aldrich</td><td>D5796-500ML</td><td>Add (2)-(4) before use. Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Antibiotic Antimycotic Solution (100 &amp;times;)</td><td>Sigma-Aldrich</td><td>A5955-100ML</td><td>Containing 10,000 units penicillin, 10 mg streptomycin and 25 &amp;mu;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&amp;amp;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</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"内除去培养皿的盖子。...

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 ...

continuous-wave-thulium-laser-for-heating-cultured-cells-to

Research

JoVE Journal

Biology

7.7K 次观看.  University of Luebeck. 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。 

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

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

科研

癌症研究

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

遗传学

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient optical micro-imaging technique which can be used to map and quantify such behavior. Europium thenoyltrifluoroacetonate (EuTFC) has a 612 nm luminescence line whose activation efficiency drops strongly with increasing temperature, due to T-dependent interactions between the Eu3+ ion and the organic chelating compound. This material may be readily coated on to a sample surface by thermal sublimation in vacuum. When the coating is excited with ultraviolet light (337 nm) an optical micro-image of the 612 nm luminescent response can be converted directly into a map of the sample surface temperature. This technique offers spatial resolution limited only by the microscope optics (about 1 micron) and time resolution limited by the speed of the camera employed. It offers the additional advantages of only requiring comparatively simple and non-specialized equipment, and giving a quantitative probe of sample temperature.

Europium thenoyltrifluoroacetonate (EuTFC) has an optical luminescence line at 612 nm, whose activation efficiency decreases strongly with temperature. If a sample coated with a thin film of this material is micro-imaged, the 612 nm luminescent response intensity may be converted into a direct map of sample surface temperature.

高分辨率热显微成像采用铕螯合物的荧光涂料

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient ...

工程学

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

一分钟，小一瓦光热消融肿瘤使用Porphysomes，内在多功能纳米囊泡

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

生物工程

Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering thermal wave fields that are disturbed by the defect. This effect is measured and used to locate the defect. We form the destructively interfering wave fields by using a modified projector. The original light engine of the projector is replaced with a fiber-coupled high-power diode laser. Its beam is shaped and aligned to the projector&#39;s spatial light modulator and optimized for optimal optical throughput and homogeneous projection by first characterizing the beam profile, and, second, correcting it mechanically and numerically. A high-performance infrared (IR) camera is set up according to the tight geometrical situation (including corrections of the geometrical image distortions) and the requirement to detect weak temperature oscillations at the sample surface. Data acquisition can be performed once a synchronization between the individual thermal wave field sources, the scanning stage, and the IR camera is established by using a dedicated experimental setup which needs to be tuned to the specific material being investigated. During data post-processing, the relevant information on the presence of a defect below the surface of the sample is extracted. It is retrieved from the oscillating part of the acquired thermal radiation coming from the so-called depletion line of the sample surface. The exact location of the defect is deduced from the analysis of the spatial-temporal shape of these oscillations in a final step. The method is reference-free and very sensitive to changes within the thermal wave field. So far, the method has been tested with steel samples but is applicable to different materials as well, in particular to temperature sensitive materials.

This method aims at locating vertical subsurface defects. Here, we couple a laser with a spatial light modulator and trigger its video input to heat a sample surface deterministically with two anti-phased modulated lines while acquiring highly resolved thermal images. The defect position is retrieved from evaluating thermal wave interference minima.

使用激光投射光热法构造加热的地下缺陷定位

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

定制设计的基于激光的加热装置顺铂触发释放的热敏脂质体与磁共振成像制导

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

全细胞膜片钳红外神经刺激机制的调查

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

神经科学

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

磁性、声学和光学三响应性微泡，用于磁性高温和波托托热组合癌症治疗

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

生物学

水凝胶包埋干细胞的光生物调节生长增强 

Three&#45;Dimensional Cell Culture of Adipose-Derived Stem Cells in a Hydrogel with Photobiomodulation Augmentation

Adipose-derived stem cells (ADSCs), possessing multipotent mesenchymal characteristics akin to stem cells, are frequently employed in regenerative medicine due to their capacity for a diverse range of cell differentiation and their ability to enhance migration, proliferation, and mitigate inflammation. However, ADSCs often face challenges in survival and engraftment within wounds, primarily due to unfavorable inflammatory conditions. To address this issue, hydrogels have been developed to sustain ADSC viability in wounds and expedite the wound healing process. Here, we aimed to assess the synergistic impact of photobiomodulation (PBM) on ADSC proliferation and cytotoxicity within a 3D cell culture framework. Immortalized ADSCs were seeded into 10 &#181;L hydrogels at a density of 2.5 x 103 cells and subjected to irradiation using 525 nm and 825 nm diodes at fluencies of 5 J/cm2 and 10 J/cm2. Morphological changes, cytotoxicity, and proliferation were evaluated at 24 h and 10 days post-PBM exposure. The ADSCs exhibited a rounded morphology and were dispersed throughout the gel as individual cells or spheroid aggregates. Importantly, both PBM and 3D culture framework displayed no cytotoxic effects on the cells, while PBM significantly enhanced the proliferation rates of ADSCs. In conclusion, this study demonstrates the use of hydrogel as a suitable 3D environment for ADSC culture and introduces PBM as a significant augmentation strategy, particularly addressing the slow proliferation rates associated with 3D cell culture.

作者聚焦：通过光生物调节进行干细胞再生疗法的进展

Here, we present a protocol demonstrating the use of hydrogel as a three-dimensional (3D) cell culture framework for adipose-derived stem cell (ADSC) culture and introducing photobiomodulation (PBM) to enhance the proliferation of ADSCs within the 3D culture setting.