Chow S K, Hakozaki H, Price D L, MacLean N A B, Deerinck T J, Bouwer J C, Martone M E, Peltier S T, Ellisman M H
National Center for Microscopy and Imaging Research, University of California San Diego, 9500 Gilman Drive, Basic Science Building, Room 1000, La Jolla, CA 92093-0608, U.S.A.
J Microsc. 2006 May;222(Pt 2):76-84. doi: 10.1111/j.1365-2818.2006.01577.x.
An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide-field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast-scanning, multiphoton, confocal microscope fitted with a motorized, high-precision stage and custom-developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.
本文描述了一种用于多光子显微镜的自动拼接采集与处理系统，该系统用于在接近光学显微镜分辨率极限的条件下对大面积生物样本进行成像。在拼接图像中，通过在样本上系统地逐个采集一系列较小的图像来创建更大的图像。拼接技术允许在不牺牲分辨率的情况下获取生物样本的宽视野视图，从而在整体背景下提供生物样本的详细视图。该系统由一台快速扫描的多光子共聚焦显微镜组成，配备电动高精度载物台以及用于自动图像采集、图像归一化、图像对齐和拼接的定制软件开发程序。我们目前的能力使我们能够获取每个拼接图像由数千到数万张单独图像组成的数据集。创建单个拼接图像所涉及的大量单独图像需要开发软件来自动化拼接采集和处理步骤。在本报告中，我们描述了从用多种荧光探针标记的脑组织常规创建超大规模拼接图像所涉及的方法和挑战。
