Claveau Remy, Manescu Petru, Elmi Muna, Pawar Vijay, Shaw Michael, Fernandez-Reyes Delmiro
Department of Computer Science, Faculty of Engineering Sciences, University College London, London, WC1E 6BT, United Kingdom.
Biometrology Group, National Physical Laboratory, Teddington, TW11 OLW, United Kingdom.
Biomed Opt Express. 2019 Dec 11;11(1):215-226. doi: 10.1364/BOE.11.000215. eCollection 2020 Jan 1.
Fourier ptychography microscopy (FPM) is a recently developed microscopic imaging method that allows the recovery of a high-resolution complex image by combining a sequence of bright and darkfield images acquired under inclined illumination. The capacity of FPM for high resolution imaging at low magnification makes it particularly attractive for applications in digital pathology which require imaging of large specimens such as tissue sections and blood films. To date most applications of FPM have been limited to imaging thin samples, simplifying both image reconstruction and analysis. In this work we show that, for samples of intermediate thickness (defined here as less than the depth of field of a raw captured image), numerical propagation of the reconstructed complex field allows effective digital refocusing of FPM images. The results are validated by comparison against images obtained with an equivalent high numerical aperture objective lens. We find that post reconstruction refocusing (PRR) yields images comparable in quality to adding a defocus term to the pupil function within the reconstruction algorithm, while reducing computing time by several orders of magnitude. We apply PRR to visualize FPM images of Giemsa-stained peripheral blood films and present a novel image processing pipeline to construct an effective extended depth of field image which optimally displays the 3D sample structure in a 2D image. We also show how digital refocusing allows effective correction of the chromatic focus shifts inherent to the low magnification objective lenses used in FPM setups, improving the overall quality of color FPM images.
傅里叶叠层显微镜术（FPM）是一种最近开发的显微成像方法，它通过组合在倾斜照明下获取的一系列明场和暗场图像来恢复高分辨率的复数图像。FPM在低放大倍数下进行高分辨率成像的能力使其在数字病理学应用中特别具有吸引力，这些应用需要对大尺寸标本（如组织切片和血涂片）进行成像。到目前为止，FPM的大多数应用都局限于对薄样本成像，这简化了图像重建和分析。在这项工作中，我们表明，对于中等厚度的样本（在此定义为小于原始捕获图像的景深），重建复数场的数值传播允许对FPM图像进行有效的数字重聚焦。通过与使用等效高数值孔径物镜获得的图像进行比较，验证了结果。我们发现，重建后重聚焦（PRR）产生的图像质量与在重建算法中向光瞳函数添加散焦项相当，同时将计算时间减少了几个数量级。我们应用PRR来可视化吉姆萨染色外周血涂片的FPM图像，并提出了一种新颖的图像处理流程，以构建有效的扩展景深图像，该图像可在二维图像中最佳地显示三维样本结构。我们还展示了数字重聚焦如何有效地校正FPM设置中使用的低放大倍数物镜固有的色差焦点偏移，提高彩色FPM图像的整体质量。
