King Michael A, Scotty Nicole, Klein Ronald L, Meyer Edwin M
Veterans Administration Medical Center, Research Services 151, University of Florida, Gainesville, FL 32602, USA.
Methods. 2002 Oct;28(2):293-9. doi: 10.1016/s1046-2023(02)00235-9.
Assessing the efficacy of in vivo gene transfer often requires a quantitative determination of the number, size, shape, or histological visualization characteristics of biological objects. The optical fractionator has become a choice stereological method for estimating the number of objects, such as neurons, in a structure, such as a brain subregion. Digital image processing and analytic methods can increase detection sensitivity and quantify structural and/or spectral features located in histological specimens. We describe a hardware and software system that we have developed for conducting the optical fractionator process. A microscope equipped with a video camera and motorized stage and focus controls is interfaced with a desktop computer. The computer contains a combination live video/computer graphics adapter with a video frame grabber and controls the stage, focus, and video via a commercial imaging software package. Specialized macro programs have been constructed with this software to execute command sequences requisite to the optical fractionator method: defining regions of interest, positioning specimens in a systematic uniform random manner, and stepping through known volumes of tissue for interactive object identification (optical dissectors). The system affords the flexibility to work with count regions that exceed the microscope image field size at low magnifications and to adjust the parameters of the fractionator sampling to best match the demands of particular specimens and object types. Digital image processing can be used to facilitate object detection and identification, and objects that meet criteria for counting can be analyzed for a variety of morphometric and optical properties.
评估体内基因转移的功效通常需要对生物对象的数量、大小、形状或组织学可视化特征进行定量测定。光学分割器已成为一种用于估计诸如脑亚区域等结构中神经元等对象数量的立体学方法。数字图像处理和分析方法可以提高检测灵敏度，并对组织学标本中的结构和/或光谱特征进行量化。我们描述了一个我们为进行光学分割器过程而开发的硬件和软件系统。一台配备摄像机、电动载物台和聚焦控制装置的显微镜与一台台式计算机相连。该计算机包含一个带有视频帧采集器的实时视频/计算机图形适配器组合，并通过一个商业成像软件包控制载物台、聚焦和视频。已经用该软件构建了专门的宏程序，以执行光学分割器方法所需的命令序列：定义感兴趣区域、以系统均匀随机的方式定位标本，以及逐步遍历已知体积的组织以进行交互式对象识别（光学解剖器）。该系统提供了灵活性，可在低放大倍数下处理超过显微镜图像视野大小的计数区域，并调整分割器采样的参数，以最佳匹配特定标本和对象类型的需求。数字图像处理可用于促进对象检测和识别，并且可以对符合计数标准的对象进行各种形态测量和光学特性分析。
