Schönhals Arthur, Kröger-Lui Niels, Pucci Annemarie, Petrich Wolfgang
Kirchhoff Institute for Physics, Heidelberg University, Heidelberg, Germany.
IRM2 GmbH, Eppelheim, Germany.
J Biophotonics. 2018 Jul;11(7):e201800015. doi: 10.1002/jbio.201800015. Epub 2018 May 17.
A laser's high degree of coherence leads to interferences, which-in the absence of precautions-can cause severe image distortions such as fringes and speckles and which thereby strongly hamper a meaningful interpretation of hyperspectral images in laser-based widefield microspectroscopy. While images and spectra of homogenous samples may already suffer from interferences, any structured object such as a tissue thin section will add to these distortions due to wavelength- and, in particular, sample-dependent phase shifts (structure sizes, absorption coefficients, refractive indices). This effect is devastating for the universal applicability of laser-based microspectroscopy especially in the mid-infrared (MIR), where cell sizes are of the same dimension as the wavelength of the illumination source. Here, we show that the impact of interferences is strongly mitigated by reducing the time-averaged spatiotemporal coherence properties of the illumination using a moving plus a stationary scatterer. In this case, the illumination path provides a pseudothermal radiation source and spatially resolved spectra can be obtained at the quality of the reference method, that is, Fourier-transform infrared microspectroscopy, without compromising spectral or spatial resolution.
激光的高相干度会导致干涉现象，在没有预防措施的情况下，这种干涉会引起严重的图像失真，如条纹和散斑，从而极大地妨碍基于激光的宽场显微光谱中高光谱图像的有意义解读。虽然均匀样品的图像和光谱可能已经受到干涉影响，但任何结构化物体，如组织薄片，由于波长相关，特别是与样品相关的相移（结构尺寸、吸收系数、折射率），都会加剧这些失真。这种效应对于基于激光的显微光谱的普遍适用性是毁灭性的，尤其是在中红外（MIR）区域，那里细胞大小与照明源波长处于同一维度。在此，我们表明，通过使用一个移动散射体加一个固定散射体来降低照明的时间平均时空相干特性，可以显著减轻干涉的影响。在这种情况下，照明路径提供了一个伪热辐射源，并且可以在不影响光谱或空间分辨率的情况下，以参考方法（即傅里叶变换红外显微光谱）的质量获得空间分辨光谱。
