Goucher D R, Wincovitch S M, Garfield S H, Carbone K M, Malik T H
DVP/OVRR, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20892, USA.
Bioinformatics. 2005 Aug 1;21(15):3248-54. doi: 10.1093/bioinformatics/bti531. Epub 2005 Jun 9.
Recent advances in confocal microscopy have allowed scientists to assess the expression, and to some extent, the interaction/colocalization of multiple molecules within cells and tissues. In some instances, accurately quantifying the colocalization of two or more proteins may be critical. This can require the acquisition of multiple Z plane images (Z stacks) throughout a specimen and, as such, we report here the successful development of a freeware, open-source image analysis tool, IMAJIN_COLOC, developed in PERL (v. 5.8, build 806), using the PERLMagick libraries (ImageMagick). Using a pixel-by-pixel analysis algorithm, IMAJIN_COLOC can analyze images for antigen expression (any number of colors) and can measure all possible combinations of colocalization for up to three colors by analyzing a Z stack gallery acquired for each sample. The simultaneous (i.e. in a single pass) analysis of three-color colocalization, and batch analysis capabilities are distinctive features of this program.
A control image, containing known individual and colocalized pixel counts, was used to validate the accuracy of IMAJIN_COLOC. As further validation, pixel counts and colocalization values from the control image were compared to those obtained with the software packaged with the Zeiss laser-scanning microscope (LSM AIM, version 3.2). The values from both programs were found to be identical. To demonstrate the applicability of this program in addressing novel biological questions, we examined the role of neurons in eliciting an immune reaction in response to viral infection. Specifically, we successfully examined expression of the chemokine RANTES in measles virus (MV) infected hippocampal neurons and quantified changes in RANTES production throughout the disease period. The resultant quantitative data were also evaluated visually, using a gif image created during the analysis.
PERL (ActivePerl, version 5.8) is available at activestate.com; the PERLMagick libraries are available at imagemagick.org, and IMAJIN_COLOC, the source code and user documentation can be downloaded from http://www.fda.gov/cber/research/imaging/imageanalysis.htm.
共聚焦显微镜技术的最新进展使科学家能够评估细胞和组织内多种分子的表达情况，并在一定程度上评估它们的相互作用/共定位情况。在某些情况下，准确量化两种或更多种蛋白质的共定位可能至关重要。这可能需要在整个标本上采集多个Z平面图像（Z堆栈），因此，我们在此报告成功开发了一种免费的开源图像分析工具IMAJIN_COLOC，它是用PERL（v.5.8，构建806）开发的，使用了PERLMagick库（ImageMagick）。通过逐像素分析算法，IMAJIN_COLOC可以分析抗原表达的图像（任意数量的颜色），并通过分析为每个样本采集的Z堆栈图库来测量多达三种颜色的所有可能共定位组合。三色共定位的同时（即单次）分析和批量分析功能是该程序的独特之处。
使用包含已知单个像素计数和共定位像素计数的对照图像来验证IMAJIN_COLOC的准确性。作为进一步的验证，将对照图像的像素计数和共定位值与蔡司激光扫描显微镜（LSM AIM，版本3.2）附带软件获得的值进行比较。发现两个程序的值是相同的。为了证明该程序在解决新的生物学问题方面的适用性，我们研究了神经元在引发针对病毒感染的免疫反应中的作用。具体而言，我们成功检测了趋化因子RANTES在麻疹病毒（MV）感染的海马神经元中的表达，并量化了整个疾病期间RANTES产生的变化。还使用分析过程中创建的gif图像对所得的定量数据进行了可视化评估。
PERL（ActivePerl，版本5.8）可在activestate.com上获取；PERLMagick库可在imagemagick.org上获取，IMAJIN_COLOC的源代码和用户文档可从http://www.fda.gov/cber/research/imaging/imageanalysis.htm下载。
