Methods for compensation of the light attenuation with depth of images captured by a confocal microscope
Methods for compensation of the light attenuation with depth of images captured by a confocal microscope
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DOI：
10.1002/jemt.20330
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发表时间：
2006-08-01
影响因子：
2.5
通讯作者：
Kubinova, Lucie
中科院分区：
文献类型：
--
作者：
Capek, Martin;Janacek, Jiri;Kubinova, Lucie
共聚焦激光扫描显微镜（CLSM）使我们能够从不同深度的生物标本中捕获图像，并获得一系列精确配准的荧光图像。然而，由于光损失失真，从样品的深层捕获的图像可能比来自最顶层的图像暗。这种效应给随后的生物对象分析造成困难。我们提出了一个解决方案，使用两种方法：一个在线的方法已经在图像采集或离线的方法，协助作为后处理步骤。在在线方法中，CLSM的光电倍增管的增益值是根据参考和当前采集的图像之间的平均图像强度的差异来控制的。离线方法包括两个阶段。在第一阶段中，从系列中的所有图像创建标准直方图，以保持灰度级的相对频率并提高亮度和对比度。在第二阶段中，根据该标准直方图来扭曲各个图像直方图。该方法进行了测试的真实的共聚焦图像数据从人类胎盘和大鼠骨骼肌标本。结果表明，这两种方法都减少了从样品深层捕获的图像中的光衰减。
A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.