LeSage Allan J, Kron Stephen J
Center for Molecular Oncology, The University of Chicago, Chicago, Illinois 60637, USA.
Cytometry. 2002 Dec 1;49(4):159-69. doi: 10.1002/cyto.10174.
Digital time-lapse microscopy using Nomarski-DIC requires that an autofocusing system adapt to changes in cell shape, size, and position while contending with drift, noise, and hysteresis in the microscope and imager. We have designed and implemented an autofocusing system that tracks subjects under dynamic conditions and maintains focus within a threshold of discriminability.
With the use of proven and novel algorithms for autofocusing in Nomarski, we performed "virtual" experiments on recorded image stacks to simulate drift and sudden displacements and test the search algorithm response.
We found that combining a simple [1, -1] contrast function with an adaptive "warmer-colder" focusing algorithm yields a reasonable compromise between focusing precision and noise tolerance. This method was implemented to record growth kinetics of yeast cells in single and multiple fields of view over several hours.
We have implemented a robust digital autofocus that maintains focus on optically complex samples imaged at high resolution. The tolerance of this system of drift and vibration suggests that it is a practical system for time-lapse imaging in many biological applications.
使用诺马斯基微分干涉衬度法（Nomarski-DIC）的数字延时显微镜要求自动聚焦系统在应对显微镜和成像仪中的漂移、噪声和滞后现象时，适应细胞形状、大小和位置的变化。我们设计并实现了一种自动聚焦系统，该系统可在动态条件下跟踪目标，并将焦点保持在可辨别阈值范围内。
利用已证实的和新颖的诺马斯基自动聚焦算法，我们对记录的图像堆栈进行了“虚拟”实验，以模拟漂移和突然位移，并测试搜索算法的响应。
我们发现，将简单的[1, -1]对比度函数与自适应“升温-降温”聚焦算法相结合，可在聚焦精度和噪声耐受性之间取得合理的平衡。该方法被用于记录酵母细胞在单个和多个视野中数小时的生长动力学。
我们实现了一种强大的数字自动聚焦功能，可在高分辨率下对光学复杂样本保持聚焦。该系统对漂移和振动的耐受性表明，它是许多生物应用中延时成像的实用系统。
