Leibniz Institute of Photonic Technology, 07745 Jena, Germany.
Justus Liebig University Giessen, Biochemistry and Molecular Biology, 35392 Giessen, Germany.
Anal Chim Acta. 2015 Sep 24;894:76-84. doi: 10.1016/j.aca.2015.08.025. Epub 2015 Aug 29.
A new setup for Raman spectroscopic wide-field imaging is presented. It combines the advantages of a fiber array based spectral translator with a tailor-made laser illumination system for high-quality Raman chemical imaging of sensitive biological samples. The Gaussian-like intensity distribution of the illuminating laser beam is shaped by a square-core optical multimode fiber to a top-hat profile with very homogeneous intensity distribution to fulfill the conditions of Koehler. The 30 m long optical fiber and an additional vibrator efficiently destroy the polarization and coherence of the illuminating light. This homogeneous, incoherent illumination is an essential prerequisite for stable quantitative imaging of complex biological samples. The fiber array translates the two-dimensional lateral information of the Raman stray light into separated spectral channels with very high contrast. The Raman image can be correlated with a corresponding white light microscopic image of the sample. The new setup enables simultaneous quantification of all Raman spectra across the whole spatial area with very good spectral resolution and thus outperforms other Raman imaging approaches based on scanning and tunable filters. The unique capabilities of the setup for fast, gentle, sensitive, and selective chemical imaging of biological samples were applied for automated hemozoin analysis. A special algorithm was developed to generate Raman images based on the hemozoin distribution in red blood cells without any influence from other Raman scattering. The new imaging setup in combination with the robust algorithm provides a novel, elegant way for chemical selective analysis of the malaria pigment hemozoin in early ring stages of Plasmodium falciparum infected erythrocytes.
提出了一种用于拉曼光谱宽场成像的新设置。它结合了基于光纤阵列的光谱转换器的优点和专门设计的激光照明系统，用于对敏感生物样品进行高质量的拉曼化学成像。照明激光束的高斯状强度分布通过方形芯光学多模光纤被成形为具有非常均匀强度分布的平顶轮廓，以满足科勒尔条件。30 米长的光纤和额外的振动器有效地破坏了照明光的偏振和相干性。这种均匀的、非相干的照明是稳定地对复杂生物样品进行定量成像的基本前提。光纤阵列将拉曼散射光的二维横向信息转换为具有非常高对比度的分离光谱通道。可以将拉曼图像与样品的相应白光显微镜图像相关联。新设置能够以非常高的光谱分辨率同时对整个空间区域的所有拉曼光谱进行定量，从而优于基于扫描和可调滤波器的其他拉曼成像方法。该设置用于快速、温和、敏感和选择性地对生物样品进行化学成像的独特功能应用于自动化血晶素分析。开发了一种特殊算法，用于根据红细胞中的血晶素分布生成拉曼图像，而不受其他拉曼散射的影响。新的成像设置与稳健的算法相结合，为恶性疟原虫感染红细胞的早期环阶段中疟色素血晶素的化学选择性分析提供了一种新颖、优雅的方法。
