Department of Biomedical Engineering, University of Connecticut, Storrs, Connecticut, USA.
Department of Electrical and Computer Engineering, University of Connecticut, Storrs, Connecticut, USA.
J Biophotonics. 2020 Dec;13(12):e202000227. doi: 10.1002/jbio.202000227. Epub 2020 Sep 18.
Whole slide imaging (WSI) has moved digital pathology closer to diagnostic practice in recent years. Due to the inherent tissue topography variability, accurate autofocusing remains a critical challenge for WSI and automated microscopy systems. The traditional focus map surveying method is limited in its ability to acquire a high degree of focus points while still maintaining high throughput. Real-time approaches decouple image acquisition from focusing, thus allowing for rapid scanning while maintaining continuous accurate focus. This work reviews the traditional focus map approach and discusses the choice of focus measure for focal plane determination. It also discusses various real-time autofocusing approaches including reflective-based triangulation, confocal pinhole detection, low-coherence interferometry, tilted sensor approach, independent dual sensor scanning, beam splitter array, phase detection, dual-LED illumination and deep-learning approaches. The technical concepts, merits and limitations of these methods are explained and compared to those of a traditional WSI system. This review may provide new insights for the development of high-throughput automated microscopy imaging systems that can be made broadly available and utilizable without loss of capacity.
近年来，全玻片成像（WSI）技术使数字病理学更接近诊断实践。由于组织固有形貌的可变性，准确的自动聚焦仍然是 WSI 和自动化显微镜系统的一个关键挑战。传统的焦点图测量方法在获取高度聚焦点的能力方面存在局限性，同时仍保持高吞吐量。实时方法将图像采集与聚焦解耦，从而允许在保持连续准确聚焦的同时进行快速扫描。本文综述了传统的焦点图方法，并讨论了用于确定焦平面的焦点测量选择。还讨论了各种实时自动对焦方法，包括基于反射的三角测量、共焦针孔检测、低相干干涉测量、倾斜传感器方法、独立双传感器扫描、分光器阵列、相位检测、双 LED 照明和深度学习方法。解释了这些方法的技术概念、优点和局限性，并与传统的 WSI 系统进行了比较。本综述可能为开发高通量自动化显微镜成像系统提供新的思路，这些系统可以广泛应用，而不会降低其容量。
