Three-dimensional (3D) cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models. The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness. While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D, they often have limitations, such as being too harsh for fragile 3D cell cultures, requiring complex handling protocols, or inducing tissue deformation with shrinkage or expansion. To address this issue, we proposed a modified optical clearing method for 3D cell cultures, called MACS-W, which is simple, highly efficient, and morphology-preserving. In our evaluation of MACS-W, we found that it exhibits excellent clearing capability in just 10min, with minimal deformation, and helps drug evaluation on tumor spheroids. In summary, MACS-W is a fast, minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

生物组织光透明技术针对生物组织具有的高散射及浑浊特性, 严重影响生物组织成像的对比度及分辨率等问题, 提供了较好的解决办法。把离体猪皮表面涂抹不同浓度的聚乙二醇、甘油、丙二醇这些光学透明剂(Optical Clearing Agents, OCA), 利用光学相干断层析(Optical Coherence Tomography, OCT)技术对其进行动态监测, 有效地提取测量信号在渗透过程中随测量深度的变化。结果表明, 利用光透明技术均可增加OCT的成像深度, 但对于不同OCA不同深度的作用效果不同。该研究为了解OCA浓度及作用时间对于不同深度最优选择, 提供有价值的数据参考, 进一步理解了OCA的作用机制, 对于OCA在医学方面的应用有一定的指导作用。

研究脉冲超声强度为0~0.8 W/cm²时的不同强度超声对在体的人皮肤中甘油溶液的渗透性和光穿透深度的影响。实验采用光学相干断层扫描（OCT）成像技术做定量连续的测量。研究结果表明，脉冲超声强度为0~0.8 W/cm²时，超声具有加速甘油溶液渗透、增强光穿透皮肤的能力，且皮肤的光透明效果随超声强度的增加而增大。以仅施加甘油溶液的样品为对照组，而施加超声作用及甘油溶液的样品为实验组。实验组和对照组的比较结果表明，在分别经超声强度为0.2，0.4，0.6，0.8 W/cm²的超声处理的人体皮肤中，15%甘油的平均渗透系数与仅施加15%甘油处理的人体皮肤相比分别增加了2.6%，9.5%，14.7%，19.8%。脉冲超声处理的30%甘油在人体皮肤中的渗透系数分别高于仅施加30%甘油处理的对照组3.7%，12%，16.7%，22.2%。结论表明，应用强度为0.2~0.8 W/cm²的超声，甘油的平均渗透系数随超声强度的增加而增加。

The current work is focused on the study of optical clearing of skeletal muscles under local compression. The experiments were performed on in vitro bovine skeletal muscle. The time dependence of optical clearing was studied by monitoring the luminescence intensity of NaYF4: Er,Yb upconverting particles located under tissue layers. This study shows the possibility to use upconverting nanoparticles (UCNPs) both for studying the dynamics of the optical clearing of biological tissue under compression and to detect moments of cell wall damage under excessive pressure. The advantage of using UCNPs is the presence of several bands in their luminescence spectra, located both at close wavelengths and far apart.

Confocal Raman microspectroscopy (CRM) with 633- and 785-nm excitation wavelengths combined with optical clearing (OC) technique was used for ex-vivo study of porcine skin in the Raman fingerprint region. The optical clearing has been performed on the skin samples by applying a mixture of glycerol and distilled water and a mixture of glycerol, distilled water and chemical penetration enhancer dimethyl sulfoxide (DMSO) during 30 min and 60 min of treatment. It was shown that the combined use of the optical clearing technique and CRM at 633nm allowed one to preserve the high probing depth, signal-to-noise ratio and spectral resolution simultaneously. Comparing the effect of different optical clearing agents on porcine skin showed that an optical clearing agent containing chemical penetration enhancer provides higher optical clearing efficiency. Also, an increase in treatment time allows to improve the optical clearing efficiency of both optical clearing agents. As a result of optical clearing, the detection of the amide-III spectral region indicating well-distinguishable structural differences between the type-I and type-IV collagens has been improved.

近年来组织光透明技术的飞速发展,为现代骨科临床基础研究带来了新的契机。组织光透明技术主要通过多种物理、化学手段,降低组织的光散射和光吸收,使光能在组织中更好地传播,从而增加光学成像的深度和对比度。结合多种荧光标记策略,实现更深层、更高分辨的骨组织及三维空间微结构信息,为突破骨这种高散射组织及骨疾病的分子影像学研究带来新的视角与方法。对组织光透明技术的原理及机制进行介绍,并重点从骨组织光透明成像技术的应用现状、新方法及透明机制三个方面加以详述,最后对该技术应用于骨及骨疾病的分子影像学研究前景进行展望。

现代光学成像技术与荧光标记技术不断发展,为高分辨地获取生物组织三维结构信息提供了重要的工具。然而,大多数生物组织具有不透明特性,限制了光在组织中的穿透深度,进而限制了光学成像技术在大组织或整体器官成像中的应用。近年兴起的组织光透明技术通过多种物理、化学手段降低组织对光的衰减,增加光穿透深度,从而提高光学成像的成像深度与成像质量,为整体组织器官的三维成像提供了全新的思路。本文从离体组织光透明方法、大组织器官标记方法、三维整体成像技术三个方面,对整体器官的光透明成像方法进行综述。

组织光透明技术结合双光子显微成像(TPM)技术能够有效提升生物样品的显微成像深度, 然而现有的光透明剂与常用显微物镜的浸润介质折射率并不匹配, 会引入球差从而降低深层组织成像的荧光强度和分辨率。针对该问题分析了球差对TPM荧光强度和分辨率的影响, 建立了由物镜特性(数值孔径和浸润介质)、聚焦深度和物体折射率等参数构成的球差补偿模型, 进而指导空间光调制器进行球差补偿。对荧光小球仿体样品和光透明脑组织样品的双光子成像结果显示, 该球差补偿方法能显著提升样品信号量和系统纵向分辨率。另外, 该方法在校正过程中无需多次成像, 操作简单且耗时短, 对光透明剂和显微物镜无特殊要求, 具有较强的通用性。

从定量的角度评价高渗制剂与组织光学特性参量改变的关系.利用FT-IR红外光谱仪与双积分球系统, 对离体猪皮组织在促渗剂二甲基亚砜与噻酮作用下的光谱进行测量, 并运用逆倍增方法计算得到组织的光学参量.结果表明: 离体猪皮组织的光学特性参量(吸收系数和散射系数)随着高渗制剂作用时间的增加明显减小, 光在组织中的穿透深度增加.由此可知猪皮组织的光学特性参量在两种促渗剂作用下随时间发生了动态改变, 且噻酮具有比二甲基亚砜更好的清透效果.