Hernández Candia Carmen Noemí, Gutiérrez-Medina Braulio
Program in Molecular Biology, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, Mexico.
Advanced Materials Division, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, Mexico.
PLoS One. 2014 Feb 18;9(2):e89106. doi: 10.1371/journal.pone.0089106. eCollection 2014.
In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive) objects if the corresponding phase (or absorptive) impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments) serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.
在透射光学显微镜中，样本的吸收结构和相位结构决定了图像的三维强度分布。因此，明场显微镜的基本脉冲响应由单独的吸收分量和相位分量组成，这使得线性的传统反卷积处理方法无法普遍应用于提高图像对比度和分辨率。然而，如果已知显微镜相应的相位（或吸收）脉冲响应，传统反卷积可应用于纯相位（或纯吸收）物体的情况。在这项工作中，我们展示了对在透射明场中操作的高孔径显微镜的相位点扩展函数和线扩展函数的直接测量。聚苯乙烯纳米颗粒和微管（生物聚合物细丝）分别用作纯相位点物体和线物体，使用标准显微镜加上数字图像处理以高对比度和低噪声对它们进行成像。我们的实验结果与所提出的响应函数模型一致，并证实了先前的理论预测。最后，我们使用测量的相位点扩展函数对活的、未染色细菌的明场图像应用传统反卷积，从而改善细胞边界和亚细胞特征的清晰度。这些进展证明了标准恢复方法在改善透射光显微镜中诸如细胞等相位物体成像方面的实际应用。
