Quantitative Imaging Laboratory, Department of Computer Science, University of Houston, 4800 Calhoun, 501 Philip G. Hoffman, Houston, TX 77204-3010, USA.
J Microsc. 2010 Sep 1;239(3):200-14. doi: 10.1111/j.1365-2818.2010.03366.x.
Over the past decade, there have been significant developments in the mechanisms for examination of biological and material samples. These developments exploit techniques in light microscopy to elucidate specific parts of cells and tissues, as well as inorganic particles. In recent years, spectral microscopy has become more prevalent for characterization of samples. Simultaneously, sensor technology has progressed as well and modern charge-coupled devices (CCD) cameras are now capable of achieving high spatial resolution and high sensitivity measurements of signals in the optical microscope. One major impediment in obtaining absolute quantitative information of imaged samples is the lack of automated photometric calibration mechanisms for spectral microscopes. In this paper, we present a methodology for achieving photometric calibration of an automated spectral imaging system targeted towards examination of biological samples. By acquiring spatial and spectral data simultaneously, spectral imaging allows one to exploit physical connections between a particle's morphology and its characteristic response to the optical spectrum. In composite biological material, the interpretation of the spectra is a complicated problem. This is because any light source and charge-coupled device camera used for data acquisition does not have a uniform illumination spectra and quantum efficiency, respectively, across the emitted light spectra. To balance the spectral response across individual wavelengths, our method modulates the exposure duration for the charge-coupled device camera during image acquisition. We present an image similarity based method to calibrate the system. Experiments to test the effectiveness of the calibration method under the various image similarity metrics are presented along with results to show the calibrated system's ability to accurately measure spectra based on the measured transmission profiles of optical filters.
在过去的十年中，生物和材料样本检测机制取得了重大进展。这些发展利用了显微镜技术来阐明细胞和组织的特定部分，以及无机颗粒。近年来，光谱显微镜在样品表征方面变得越来越流行。同时，传感器技术也取得了进展，现代电荷耦合器件（CCD）相机现在能够实现光学显微镜中信号的高空间分辨率和高灵敏度测量。获得成像样品绝对定量信息的主要障碍之一是缺乏用于光谱显微镜的自动光度校准机制。在本文中，我们提出了一种针对生物样品检查的自动化光谱成像系统的光度校准方法。通过同时获取空间和光谱数据，光谱成像可以利用颗粒形态与其对光谱特征响应之间的物理联系。在复合生物材料中，对光谱的解释是一个复杂的问题。这是因为用于数据采集的任何光源和电荷耦合器件相机在发射光谱上的光照度光谱和量子效率都不是均匀的。为了平衡各个波长的光谱响应，我们的方法在图像采集期间调制电荷耦合器件相机的曝光持续时间。我们提出了一种基于图像相似性的方法来校准系统。还提出了根据各种图像相似性度量测试校准方法有效性的实验，并展示了校准系统根据光学滤波器的测量透射谱准确测量光谱的能力。
