用单模光纤共焦显微镜定量光散射。

Quantifying light scattering with single-mode fiber -optic confocal microscopy.

机构信息

University of Missouri, Department of Biological Engineering, Columbia, MO 65211, USA.

出版信息

BMC Med Imaging. 2009 Nov 19;9:19. doi: 10.1186/1471-2342-9-19.

DOI:10.1186/1471-2342-9-19

PMID:19925674

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2793246/

Abstract

BACKGROUND

Confocal microscopy has become an important option for examining tissues in vivo as a diagnostic tool and a quality control tool for tissue-engineered constructs. Collagen is one of the primary determinants of biomechanical stability. Since collagen is also the primary scattering element in skin and other soft tissues, we hypothesized that laser-optical imaging methods, particularly confocal scattered-light scanning, would allow us to quantify scattering intensity and determine collagen content in biological layers.

METHODS

We built a fully automated confocal scattered-light scanner to examine how light scatters in Intralipid, a common tissue phantom, and three-dimensional collagen gels. Intralipid with 0.5%, 1.0%, 1.5%, and 2.0% concentration was filled between precisely spaced glass coverslips. Collagen gels at collagen concentrations from 0.30 mg/mL to 3.30 mg/mL were prepared, and all samples underwent A-mode scanning with multiple averaged scans. In Intralipid samples, light reflected from the upper fluid-glass interface was measured. In collagen gels, average scattering intensity inside the actual gel was measured. In both cases, intensity was correlated with concentration.

RESULTS

By measuring light attenuation at interface reflections of various thicknesses using our device, we were able to determine that the scattering coefficient at 660 nm of Intralipid at increasing concentrations in water to be 39 cm-1 for each percent increase of Intralipid. We were also able to measure the amount of scattering of various concentrations of collagen in gels directly using backscattered light. The results show a highly linear relationship with an increase of 8.2 arbitrary units in backscattering intensity for every 1 mg increase of collagen within a 1 mL gel volume.

CONCLUSION

The confocal scattered-light scanner allows to accurately quantify scattering in Intralipid and collagen gels. Furthermore, a linear relationship between collagen concentration and intensity was found. Confocal scattered-light scanning therefore promises to allow imaging of collagen content in soft tissue layers.

摘要

背景

共焦显微镜已成为一种重要的选择，用于检查组织在体内的诊断工具和组织工程构建的质量控制工具。胶原蛋白是生物力学稳定性的主要决定因素之一。由于胶原蛋白也是皮肤和其他软组织的主要散射元素，我们假设激光光学成像方法，特别是共焦散射光扫描，将使我们能够量化散射强度并确定生物层中的胶原蛋白含量。

方法

我们构建了一个全自动共焦散射光扫描仪，以检查光在 Intralipid（一种常见的组织仿体）和三维胶原蛋白凝胶中的散射方式。将浓度为 0.5%、1.0%、1.5%和 2.0%的 Intralipid 填充在精确间隔的玻璃盖玻片之间。制备浓度从 0.30 mg/mL 到 3.30 mg/mL 的胶原蛋白凝胶，所有样品均进行多次平均扫描的 A 模式扫描。在 Intralipid 样品中，测量来自上流体-玻璃界面的反射光。在胶原蛋白凝胶中，测量实际凝胶内部的平均散射强度。在这两种情况下，强度都与浓度相关。

结果

通过使用我们的设备测量不同厚度界面反射光的光衰减，我们能够确定在水中增加浓度的 Intralipid 的 660nm 处的散射系数为每增加 1%的 Intralipid 增加 39cm-1。我们还能够直接使用背散射光测量不同浓度的胶原蛋白在凝胶中的散射量。结果显示，在 1 毫升凝胶体积内，胶原蛋白增加 1 毫克，背散射强度增加 8.2 个任意单位，具有高度线性关系。