这项工作的一部分是由日本促进科学学会 (25350520、22500401、15K06105) 和美国陆军贸易中心-PAC 研究和开发项目 (FA5209-15-P-0175, FA5209-16-P-0132) 资助的一项科研补助金资助的。

1. 传统漫反射和总透射光谱分析系统的构造
注: 利用宽带白光光源、光导、消色差透镜、积分球、样品架、光纤和多通道光谱仪构造漫反射和总透射光谱测量系统。光阱的作用是从反射光谱中去除镜面反射分量。积分球的样品持有者由安装板和带有弹簧的夹紧夹具组件组成, 它将样品固定在该端口上。将燕尾和弹簧夹紧组件从样品架上取下, 将聚苯乙烯泡沫塑料的手工立方底座连接到安装板上。光学元件的布局, 如图 1a和1b所示, 可分别用于漫反射测量和总透射率测量的施工程序。
<ol><li>使用提供的通用串行总线 (USB) 电缆连接光谱仪器和个人计算机。</li>
	<li>将端口适配器连接到集成球体的探测器端口。使用光纤将积分球的光谱仪和端口适配器连接起来。连接 150 W 卤素灯光源和轻的指南。</li>
	<li>将样本持有者附加到积分球体的样本端口。当执行漫反射测量时, 将光阱连接到积分球体的适当端口。打开卤素灯光源,通过导光和消色差透镜照亮样品。</li>
	<li>打开光谱仪的操作软件。</li>
</ol>2. 丙烯酸模具的制备
注: 由两个矩形丙烯酸片和 U 形丙烯酸片组成的丙烯酸模, 用于创建单层凝胶幻影。图 2可以提到这个施工过程。
<ol><li>将两个矩形丙烯酸片从一个2毫米厚的丙烯酸板切割成可选尺寸。</li>
	<li>将丙烯酸片从1毫米厚的丙烯酸板中切出, 以选择尺寸。切割1毫米厚的丙烯酸片, 使其成为 U 形件, 用于模具制造1毫米厚的表皮幽灵。</li>
	<li>将丙烯酸片从5毫米厚的丙烯酸板中切出, 以选择尺寸。切割5毫米厚的丙烯酸片, 使其成为 U 形的一块被用作模具, 使5毫米厚的真皮幽灵。</li>
	<li>用金属锉除去每个丙烯酸片上的毛刺。</li>
	<li>通过持有1毫米厚的 U 形件与两个2毫米厚的丙烯酸片, 并用五保护夹固定, 使表皮幻影模具。</li>
	<li>使真皮幻影模具持有5毫米厚的 U 形件与两个2毫米厚的丙烯酸件, 并修复他们与五保护剪辑。</li>
</ol>3. 基础材料的制备
<ol><li>将500毫升标准生理盐水与 0.9% (瓦特/v) 氯化钠放在豆荚中。在搅拌混合物时, 慢慢加入5克琼脂糖粉, 避免结块。</li>
	<li>用 1000 W 功率设置为5分钟的电烹调加热器将琼脂糖粉和生理盐水的混合物加热。</li>
	<li>一旦混合物沸腾, 保持在低热量的混合物3分钟。</li>
	<li>将混合物冷却到大约70摄氏度的温度。然后将混合物倒入容器中, 并将其保存在恒温浴中, 在60摄氏度30分钟后再制造幻影。</li>
</ol>4. 仿皮光学幻影的制备
注: 咖啡溶液用于模拟黑色素的吸收谱。咖啡溶液中含有一种叫做豆豉的褐色颜料。据报道, 豆豉的吸收光谱与黑色素10相似。
<ol><li>
准备一个表皮幻影
	<ol>
<li>将100毫升纯净水倒入咖啡壶水库。在咖啡壶里放一个过滤器。将24克的地面咖啡添加到过滤器中。打开咖啡壶, 按下 brew 按钮开始酿造。</li>
		<li>在玻璃瓶中放入4毫升的煮咖啡和16毫升盐水, 制作咖啡溶液。</li>
		<li>将5毫升的脂质乳剂 (例如, 自制脂肪乳剂 10%) 和10毫升的咖啡溶液放入透明塑料杯中。在搅拌时, 将基材的35毫升缓慢地添加到这种混合物中。</li>
		<li>将混合物吸入注射器中, 慢慢注入表皮幻影霉菌, 避免任何气泡形成。将含有混合物的丙烯酸模具冷却5摄氏度, 20 分钟。</li>
		<li>从模具中取出保护剪辑。将其中一个丙烯酸片向外滑动, 从模具中取出。将1毫米厚的凝固凝胶幻影从模具中取出, 用手术手术刀将其切割成所需尺寸。</li>
		<li>放置并保持两个幻灯片玻璃之间的凝胶幻影。</li>
	</ol>
</li>
	<li>
制备含氧血的真皮幻影
	<ol>
<li>取5.0 毫升的脂质乳剂和0.4 毫升全马血 45%-红细胞压积, 放入透明塑料杯中。在搅拌混合物时, 慢慢加入44.6 毫升的基材。</li>
		<li>将混合物吸入注射器, 慢慢注入真皮幻影模具, 同时避免任何气泡的形成。将含有混合物的丙烯酸模具冷却5摄氏度, 20 分钟。</li>
		<li>从模具中取出保护剪辑。将其中一个丙烯酸片向外滑动, 从模具中取出。将5毫米厚的凝固凝胶幻影从模具中取出, 用手术手术刀将其切割成所需尺寸。</li>
		<li>放置并保持两个幻灯片玻璃之间的凝胶幻影。</li>
	</ol>
</li>
	<li>
准备一个含有脱氧血的真皮幻影
	<ol>
<li>将含有含氧血 (从台阶 4.2.3) 的真皮凝胶幻影放在玻璃盘子上。</li>
		<li>将1克硫酸钠钠 (Na2S2O4) 溶入玻璃瓶中的20毫升盐水中。</li>
		<li>添加0.05 克/毫升的 Na2S2O4解决方案上使用注射器 deoxygenate 的血液在幻影。</li>
		<li>放置和持有两个幻灯片玻璃之间的幻影, 以防止其干燥。</li>
	</ol>
</li>
	<li>
准备两层幻影
	<ol>
<li>将0.1 毫升生理盐水放到真皮幻影上, 以确保表皮和真皮层之间的光学耦合。将表皮幻影放在真皮幻影上。</li>
		<li>如果层间存在气泡, 请用指尖轻抚两层幻影的表面, 把它们推出来。</li>
		<li>在两个滑动玻璃之间保持两层的幻影, 以防止其干燥。</li>
	</ol>
</li>
</ol>5. 漫反射光谱的获取
<ol><li>
获取暗光谱 注: 光谱仪中的电荷耦合器件 (CCD) 传感器可以根据入射光产生的电信号估计光强。然而, 有暗噪声37 , 这是独立的信号产生的光子, 但依赖于器件的温度, 即使传感器没有检测到光。为了准确测量光的光谱强度, 应将暗电流信号测量为暗光谱, 然后从样本谱中减去。暗光谱是被阻挡的光路所采取的光谱。<ol>
<li>将积分球体定位于漫反射测量的最佳位置 (图 1a)。</li>
		<li>关闭卤素灯光源。使用端口插头或屏蔽板挡住光路到光谱仪。</li>
		<li>从 "文件" 菜单中选择 "存储深色" 命令以存储深色频谱。</li>
		<li>从 "文件" 菜单中选择 "减去深色频谱" 命令, 以从测量样本频谱中减去暗光谱 (见下文)。</li>
	</ol>
</li>
	<li>
参考谱的获取 注: 本实验所用元件的光学特性, 如光源、导光、消色差透镜、光纤、光谱仪等, 都有各自的波长依赖性。因此, 通过这些光学元件传递的光的光谱强度应作为参考谱来测量。对于漫反射光谱的测量, 参考光谱是一种光谱, 采用标准的白色扩散器, 光光源照射。<ol>
<li>按下电源按钮打开卤素灯光源。在获得参考谱之前, 先将灯预热至少10分钟。</li>
		<li>将标准的白色扩散器 (例如, Spectralon) 放置在集成球体的样本端口上。</li>
		<li>通过从光谱仪操作软件的下拉列表中选择合适的值来调整光谱仪的积分时间, 使峰值信号强度约为光谱仪强度最大值的75%。</li>
		<li>从 "文件" 菜单中选择 "存储引用" 命令以存储参考频谱。</li>
	</ol>
</li>
	<li>
样品谱的获取 注: 样品的漫反射光谱是在个人电脑的硬盘上获取并保存的, 使用相同的采集条件。<ol>
<li>将表皮幻影夹在样品口的两个滑动玻璃上。从 "文件" 菜单中选择 "保存" 命令, 以将漫反射光谱保存到文件中。</li>
		<li>对真皮和双层幽灵重复步骤5.3.1。</li>
	</ol>
</li>
</ol>6. 获得总透射率谱
<ol><li>
获取暗光谱 注: 光谱仪中的传感器可以根据入射光产生的电信号估计光强。然而, 有暗噪声是独立于光子产生的信号, 但依赖于器件的温度, 即使传感器没有探测到光。为了准确测量光的光谱强度, 应将暗电流信号测量为暗光谱, 然后从样本谱中减去。暗光谱是被阻挡的光路所采取的光谱。<ol>
<li>将积分球定位到总透射率测量的最佳位置 (图 1b)。</li>
		<li>从集成球体的端口上卸下光阱, 并将端口插头连接到端口。</li>
		<li>关闭卤素灯光源。使用端口插头或屏蔽板阻挡光路到积分球体。</li>
		<li>从 "文件" 菜单中选择 "存储深色" 命令以存储深色频谱。</li>
		<li>从 "文件" 菜单中选择 "减去深色频谱" 命令, 以从测量样本频谱中减去暗光谱 (见下文)。</li>
	</ol>
</li>
	<li>
参考谱的获取 注: 本实验所用元件的光学特性, 如光源、导光、消色差透镜、光纤、光谱仪等, 都有各自的波长依赖性。因此, 通过这些分量的光的光谱强度应测量为参考光谱。对于总透射率谱的测量, 参考谱是当光源的光通过样品端口直接进入积分球体时所采用的光谱。<ol>
<li>按下电源按钮打开卤素灯光源。在获得参考谱之前, 先将灯预热至少10分钟。</li>
		<li>通过从分光计运行软件中的积分时间下拉列表中选择合适的值来调节光谱仪的积分时间, 使最大光强显示的信号约为最大值的75%。值。</li>
		<li>从 "文件" 菜单中选择 "存储引用" 命令以存储参考频谱。</li>
	</ol>
</li>
	<li>
样品谱的获取 注: 样品的总透射率谱是在个人电脑的硬盘上获取并保存的, 使用相同的采集条件。<ol>
<li>将表皮幻影夹在样品口的两个滑动玻璃上。从 "文件" 菜单中选择 "保存" 命令, 以将总透射率频谱保存到文件中。</li>
		<li>对真皮和双层幽灵重复步骤6.3.1。</li>
	</ol>
</li>
</ol>7. 估计吸收和光散射特性
注: 将一组漫反射光谱和总透射率谱保存到个人电脑的硬盘驱动器上, 并进行离线分析。然后进行了8、38、39、40的反蒙特卡罗模拟, 以估计µ(λ) 的吸收系数谱和减小的散射系数。光谱µs'(λ)。在这一反蒙特卡罗模拟中, 在各向异性因子g为0的假设下, 估计散射系数µs, 被认为是减小的散射系数µs'.反射率和透射率数据都用于单个模拟运行。本协议中使用的详细算法已在以前的文献8、39中报道过。我们估计的吸收系数谱µa(λ) 和减少散射系数谱µs'(λ) 的表皮层从一组的漫反射从表皮层获得的光谱和总透射率谱。同样地, 我们估计从一组漫反射光谱和从真皮获得的总透射率谱µa(λ) 和µs'(λ) 的真皮层层。
<ol><li>为蒙特卡罗模拟打开输入文件。</li>
	<li>在输入数据文件中, 以 10 nm 间隔填充测量的漫反射率和在特定波长范围内的总透射率, 从400到 700 nm 的值。在输入数据文件中填充幻像粗细的值。</li>
	<li>将图层的折射率n设置为输入数据文件中的适当值 (例如, n = 1.33 在 550 nm)。将各向异性因子g的值设置为输入数据文件中的0。</li>
	<li>将吸收系数µa和散射系数µ的初始值设置为输入数据文件中适当的值 (例如, µa = 0.01, µs = 0。1).</li>
	<li>执行反蒙特卡罗模拟程序。</li>
	<li>键入输入文件名, 然后运行模拟。</li>
	<li>在迭代模拟终止后, 打开输出文件并检查µa和µ的最终值。</li>
	<li>对其他所需波长重复步骤 7.1-7.7。</li>
</ol>

作者没有什么可透露的。

该协议中最关键的一步是基材的温度控制。维持基材的温度从58到60摄氏度不等。如果温度超过70摄氏度, 则会发生脂质乳剂和全血的变性。因此, 幻影的光学特性将会恶化。如果温度小于40摄氏度, 基材将 ununiformly 胶体, 因此, 光散射和吸收剂将异种地分布在幻影。虽然基材保持在60摄氏度, 吸痰与注射器降低温度。当添加到血液溶液中时, 基材的温度降低到50摄氏度。
本文描述的光学幽灵受短可用寿命的影响, 通常只限于不超过一天。可以通过将幻像封装在密封容器中的基材或使用防腐剂来延长可用寿命。1毫米厚的表皮层幻影是比人表皮厚度大的数量级。然而, 在这个与丙烯酸模具的协议中, 很难创建一个小于0.5 毫米的层厚度。为了减少这种厚度对幽灵的漫反射光谱的预期影响, 对表皮幻影的散射和吸收系数进行了调节, 使漫反射光谱显示出相似的光谱人类的皮肤。一个自旋涂层方法42看起来有希望使一层薄比0.5 毫米。µ的价值 (λ) 和µs' (λ) 为人的皮肤被报道在文献43。
在琼脂幻层中, 黑色素或胆红素的均匀分布可能很难使用此处描述的协议, 因为这些染色体不完全溶于水。从烘焙咖啡豆和柠檬黄中提取的豆豉可以作为黑色素和胆红素的可比或替代材料使用。反蒙特卡罗模拟用于估计光学性质从测量的漫反射率和总透射率是相对耗时的, 由于其迭代的方式。另一种轻型运输计算模型, 如加加倍法44可用于缩短计算时间。减小散射系数µs'是一个集中光学性质, 包含散射系数µs和各向异性因子g。为了分别估计µs和g , 除了总透射率和漫反射率38、40外, 还必须测量幻影的准直透射率。在本研究中, 我们没有测量每一层的折射率。我们设置了在文献45中发表的水的折射率为反蒙特卡罗模拟的输入数据文件, 而不是因为琼脂糖凝胶主要由水组成。我们假设两层之间的折射率没有差别。我们还使用了玻璃的折射率 (例如, 1.524 在λ = 546.1 毫微米) 的名义价值的蒙特卡罗模拟。
这一协议具有成本效益, 这是有利的, 即一个集成球体而不是两个集成球体。另一方面, 使用单个积分球是很耗时的, 因为积分球的排列必须根据测量是否为总透射率或漫反射的变化而改变。本文所描述的协议可以通过改变模具设计来扩展以创建具有不同形状、大小和夹杂的单层或多层光学幽灵。幽灵层的表面在被取出模子后立即被润湿。因此, 将第二层紧密叠加在第一层上, 将表皮层和真皮层粘在一起。可以直接在第一个层上固化第二个图层, 而不是分别进行单独的制作, 然后再将其附加在一起。然而, 在这种情况下, 可能很难准确地使薄表皮层具有均匀的层厚度。我们把幻影夹在玻璃杯之间, 以防幻影的干燥。在蒙特卡罗模拟中, 我们考虑了玻璃的光学性质和厚度。因此, 对幽灵的估计光学特性没有影响。目前的技术对于现有方法的意义在于它能代表可见光到近红外波长区域内活体组织的漫反射光谱。该协议所制造的光学幽灵将可用于验证新开发的基于漫反射光谱和 spectrocolorimetry 的光学方法。

光学幻影是模仿生物组织光学特性的物体, 在生物医学光学领域得到了广泛的应用。它们的设计使光学特性, 如光散射和吸收系数, 与活体人体和动物组织相匹配。光学幽灵一般用于以下目的: 模拟生物组织中的光传输, 校准新开发的光学系统设计, 评估现有系统的质量和性能, 比较性能在系统之间, 并验证光学方法的能力, 以量化的光学性质1,2,3,4,5。因此, 制造光学幽灵需要易于获取的物质、简单的制造过程、高重现性和光学稳定性。
各种类型的光学幽灵用不同的基础材料, 如水悬浮6, 凝胶凝胶7, 琼脂糖凝胶8,9,10, 聚丙烯酰胺凝胶11, 树脂12, 13,14,15,16, 室温硫化硅胶17已在以前的文献报道。据报道, 凝胶和海藻酸盐凝胶是有用的光学幽灵与异构结构18。海藻酸盐幽灵有一个适当的机械和热稳定性, 以评估光热效应, 如激光消融研究和激光高温研究18。琼脂糖凝胶具有制造异质结构的能力, 其力学性能和物理性质在很长时间内稳定了18。高纯度琼脂糖凝胶具有极低的浊度和微弱的光吸收。因此, 用适当的光散射和吸收剂可以很容易地设计出基于琼脂糖的幽灵的光学特性。近年来, 苯乙烯-乙烯-丁烯-苯乙烯 (SEBS) 嵌段共聚物19和聚氯乙烯 (PVC) 凝胶20被报道为光学和光声技术的有趣的幻像材料。
高分子微球 7,12,21,22, 钛氧化物粉末1, 和脂乳剂23,24,25,26如牛奶和脂质乳剂用作光散射剂, 而黑色墨水27、28和分子染料29、30被用作光吸收器。大多数活体器官的漫反射光谱主要由红细胞中含氧和脱氧血红蛋白的吸收支配。因此, 血红蛋白溶液31,32和全血8,9,10,33,36经常被用作轻吸收剂在一种漫反射光谱和多光谱成像的幽灵。
本文所描述的方法用于创建模仿生物组织中的光传输的光学幻影, 并对其光学特性进行表征。以两层光学幻影为例, 对人体皮肤组织的光学特性进行了模拟。这种方法在替代技术上的优点是能够表示可见光到近红外波长区域内活体生物组织的漫反射光谱, 以及使用简便可得的简单性。材料和常规光学仪器。因此, 利用该方法制作的光学幽灵, 对于基于漫反射光谱和多光谱成像的光学方法的发展具有一定的参考价值。

<table>
	<tbody>
		<tr>
			<td>150-W halogen-lamp light source</td>
			<td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>LA-150SAE</td>
			<td></td>
		</tr>
		<tr>
			<td>Light guide</td>
			<td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>LGC1-5L1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrating Sphere</td>
			<td>Labsphere Incorporated, North Sutton, NH, USA</td>
			<td>RT-060-SF</td>
			<td></td>
		</tr>
		<tr>
			<td>Port adapter</td>
			<td>Labsphere Incorporated, North Sutton, NH, USA</td>
			<td>PA-050-SMA-SF</td>
			<td></td>
		</tr>
		<tr>
			<td>Light trap</td>
			<td>Labsphere Incorporated, North Sutton, NH, USA</td>
			<td>LTRP-100-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralon white standard with 99% diffuse reflectance</td>
			<td>Labsphere Incorporated, North Sutton, NH, USA</td>
			<td>SRS-99-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Ocean Optics Inc., Dunedin, Florida, USA</td>
			<td>P400-2-VIS-NIR</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature Fiber Optic Spectrometer</td>
			<td>Ocean Optics Inc., Dunedin, Florida, USA</td>
			<td>USB2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Achromatic lens</td>
			<td>Chuo Precision Industrial Co.,Ltd, Tokyo, Japan</td>
			<td>ACL-50-75M</td>
			<td></td>
		</tr>
		<tr>
			<td>Intralipid</td>
			<td>Fresenius Kabi AB, Uppsala, Sweden</td>
			<td>Intralipid 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>Coffee 
			(Blendy Mocha Blend Regular Coffee)</td>
			<td>Ajinomoto AGF, Inc. Tokyo, Japan</td>
			<td>Unavailable</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole blood</td>
			<td>Nippon Bio-Test Laboratories Inc. Saitama, Japan</td>
			<td>0103-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Nippon Genetics Co., Ltd, Tokyo, Japan</td>
			<td>NE-AG02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooking heater</td>
			<td>TOSHIBA CORPORATION Tokyo, Japan</td>
			<td>HP-103K</td>
			<td></td>
		</tr>
	</tbody>
</table>

agarose-based tissue mimicking optical phantoms for diffuse reflectance spectroscopy

本协议描述如何制作基于琼脂糖的组织模拟幽灵, 并演示如何使用一个集成球体的传统光学系统来确定它们的光学特性。利用宽带白光光源、光导、消色差透镜、积分球、样品架、光纤探针等方法, 建立了漫反射率和总透射率谱采集的测量系统, 以及多通道光谱仪。一种由两个矩形丙烯酸片和 U 形丙烯酸片组成的丙烯酸模, 用于制造表皮幻影和全血的真皮幻影。硫酸钠钠 (Na2S2O4) 溶液在真皮幻影中的应用使研究人员能够 deoxygenate 在真皮幻影中分布的红血球中的血红蛋白。采用积分球光谱仪测量的漫反射率和总透射率谱进行了反蒙特卡罗模拟, 确定了µ(λ) 的吸收系数谱和减小散射系数谱µ每层幻像的s' (λ)。通过在真皮幻影上堆积表皮幻影, 可以模拟人体皮肤组织漫反射的两层幻影。

图 3显示了表皮幻影和真皮幻影的减少散射系数和吸收系数的代表性估计谱。图 3所示的结果是十测量反射率和透射率谱的平均值。减小的散射系数µs'具有广泛的散射光谱, 在较短的波长上表现出更高的震级。光谱特征对应于软组织的典型散射光谱。随着波长的增加, 表皮幻影µ的吸收系数呈指数衰减, 与黑色素的吸收光谱相似。表皮幻层和黑色素41的吸收系数谱由指数函数拟合而成: <img alt="Equation" src="/files/ftp_upload/57578/57578eq1.jpg">
表皮层的B值被计算为 0.011, 而黑色素的价值估计为0.009。含氧血和脱氧血的真皮幽灵µ吸收系数的波长依赖性主要受氧血红蛋白和脱氧血红蛋白的光谱特性的支配,分别。
图 4显示了两层皮肤幽灵的典型数字彩色照片。图 4a显示了两层皮肤幻影的横断面图像。图 4b和4c显示了 3-3 个包含含氧血和脱氧血的幻像矩阵的最高视图。从上到下的行有咖啡溶液浓度cc 5%, 10% 和20%。从左到右的列有血浓度Cb的 0.2%, 0.4% 和0.6%。随着 c c 在表皮层中的值增加, 幻影的颜色变暗, 而随着cb值的增加, 它变成粉红色。含氧血的幽灵比脱氧的血有更红的颜色。这些变化代表皮肤颜色的变化, 由于生理条件, 如鞣制和低氧血症, 分别。
图 5显示了一个代表性测量的漫反射光谱的例子, 从两层皮肤组织幽灵获得不同的条件 (图 5a) 咖啡溶液的浓度cc, (图 5b)全血的浓度Cb, (图 5c) 血液的氧合状态。在图 5a中, 随着cc值的增大, 较长波长区域的漫反射率明显降低。这是由于咖啡溶液在较短波长区域中的强光吸收 (见图 3b)。图 5b显示了中间波长区域漫反射率的显著变化, 其值为Cb, 代表了血红蛋白在波长范围内的强光吸收量, 从500到 600 nm。图 5c所示的漫反射光谱清楚地观察了含氧血红蛋白与脱氧血红蛋白和血红蛋白等色点之间的光谱特征的差异。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57578/57578fig1.jpg"> 图 1: 实验装置的原理图.这些面板显示了测量 (a) 漫反射光谱和 (b) 总透射率谱的设置。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57578/57578fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57578/57578fig2.jpg"> 图 2: 制备基于琼脂糖的光学幽灵的步骤.这些面板显示 (a) 表皮层幻影的制作和 (b) 制作真皮层幻影。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57578/57578fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57578/57578fig3.jpg"> 图 3: 代表估计的幽灵的光学特性.(a) 本小组显示了表皮和真皮层的平均减少散射系数谱µs' (λ)。(b) 本小组显示了表皮层和真皮层的µ(λ) 的吸收系数谱。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57578/57578fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57578/57578fig4.jpg"> 图 4: 代表数码彩色照片的双层皮肤幽灵.(a) 本小组显示两层皮肤幻影的横断面视图。(b) 本小组展示了含有含氧血液的 3-3 幻像矩阵的最高视图。(c) 本小组展示了含有脱氧血的 3-3 幻像矩阵的最高视图。从上到下的行有咖啡溶液浓度cc 5%, 10% 和20%。从左到右的列有血浓度Cb的 0.2%, 0.4% 和0.6%。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57578/57578fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57578/57578fig5.jpg"> 图 5: 代表测量了从两层皮肤组织幽灵获得的漫反射光谱.这些面板显示了不同条件下的幽灵的漫反射光谱 (a) 咖啡溶液的浓度 c, (b) 全氧血浓度cb, 和 (c) 血液的氧合状态。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57578/57578fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy at JoVE.com

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

在这里, 我们演示了如何用琼脂糖为基础的组织模仿光学幽灵, 以及如何使用一个集成球体的传统光学系统来确定它们的光学特性。

基于琼脂糖组织模拟光学幽灵的漫反射光谱研究

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

agarose-based-tissue-mimicking-optical-phantoms-for-diffuse

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.3K 次观看.  Tokyo University of Agriculture & Technology. 在这里, 我们演示了如何用琼脂糖为基础的组织模仿光学幽灵, 以及如何使用一个集成球体的传统光学系统来确定它们的光学特性。

视频: Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

生物分子检测的干涉反射成像传感器（IRIS）

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

科研

生物工程

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

在体外合成的原住民，纤维长间距"和"分段长间距胶原蛋白

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

生物医学植入物的石墨涂料

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

幻影使用肿瘤尺寸测量的质量保证组织模仿材料的潜伏期多模工程

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

含有宏观结构的光学组织幻影的制备与表征

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the tissue motion and infer or quantify the underlying biomechanical characteristics. For abdominal aortic aneurysms (AAA), biomechanical properties such as changes in the tissue's elastic modulus and estimates of the tissue stress may be essential for assessing the need for the surgical intervention. Abdominal aortic aneurysms US elastography could be a useful tool to monitor AAA progression and identify changes in biomechanical properties characteristic of high-risk patients.
A preliminary goal in the development of an AAA US elastography technique is the validation of the method using a physically relevant model with known material properties. Here we present a process for the production of AAA tissue-mimicking phantoms with physically relevant geometries and spatially modulated material properties. These tissue phantoms aim to mimic the US properties, material modulus, and geometry of the abdominal aortic aneurysms. Tissue phantoms are made using a polyvinyl alcohol cryogel (PVA-c) and molded using 3D printed parts created using computer aided design (CAD) software. The modulus of the phantoms is controlled by altering the concentration of PVA-c and by changing the number of freeze-thaw cycles used to polymerize the cryogel. The AAA phantoms are connected to a hemodynamic pump, designed to deform the phantoms with the physiologic cyclic pressure and flows. Ultra sound image sequences of the deforming phantoms allowed for the spatial calculation of the pressure normalized strain and the identification of mechanical properties of the vessel wall. Representative results of the pressure normalized strain are presented.

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

超声弹性成像验证腹主动脉水凝胶组织模拟幻影的研制

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the ...