Monck J R, Oberhauser A F, Keating T J, Fernandez J M
Department of Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota 55905.
J Cell Biol. 1992 Feb;116(3):745-59. doi: 10.1083/jcb.116.3.745.
The availability of the ratiometric Ca2+ indicator dyes, fura-2, and indo-1, and advances in digital imaging and computer technology have made it possible to detect Ca2+ changes in single cells with high temporal and spatial resolution. However, the optical properties of the conventional epifluorescence microscope do not produce a perfect image of the specimen. Instead, the observed image is a spatial low pass filtered version of the object and is contaminated with out of focus information. As a result, the image has reduced contrast and an increased depth of field. This problem is especially important for measurements of localized Ca2+ concentrations. One solution to this problem is to use a scanning confocal microscope which only detects in focus information, but this approach has several disadvantages for low light fluorescence measurements in living cells. An alternative approach is to use digital image processing and a deblurring algorithm to remove the out of focus information by using a knowledge of the point spread function of the microscope. All of these algorithms require a stack of two-dimensional images taken at different focal planes, although the "nearest neighbor deblurring" algorithm only requires one image above and below the image plane. We have used a modification of this scheme to construct a simple inverse filter, which extracts optical sections comparable to those of the nearest neighbors scheme, but without the need for adjacent image sections. We have used this "no neighbors" processing scheme to deblur images of fura-2-loaded mast cells from beige mice and generate high resolution ratiometric Ca2+ images of thin sections through the cell. The shallow depth of field of these images is demonstrated by taking pairs of images at different focal planes, 0.5-microns apart. The secretory granules, which exclude the fura-2, appear in focus in all sections and distinct changes in their size and shape can be seen in adjacent sections. In addition, we show, with the aid of model objects, how the combination of inverse filtering and ratiometric imaging corrects for some of the inherent limitations of using an inverse filter and can be used for quantitative measurements of localized Ca2+ gradients. With this technique, we can observe Ca2+ transients in narrow regions of cytosol between the secretory granules and plasma membrane that can be less than 0.5-microns wide. Moreover, these Ca2+ increases can be seen to coincide with the swelling of the secretory granules that follows exocytotic fusion.
比率型钙离子指示剂染料fura - 2和indo - 1的可得性，以及数字成像和计算机技术的进步，使得以高时空分辨率检测单个细胞内的钙离子变化成为可能。然而，传统落射荧光显微镜的光学特性并不能产生完美的样本图像。相反，所观察到的图像是物体经过空间低通滤波后的版本，并且被离焦信息所污染。结果，图像的对比度降低，景深增加。这个问题对于局部钙离子浓度的测量尤为重要。解决这个问题的一种方法是使用扫描共聚焦显微镜，它只检测焦平面上的信息，但这种方法对于活细胞中的低光荧光测量有几个缺点。另一种方法是使用数字图像处理和去模糊算法，通过了解显微镜的点扩散函数来去除离焦信息。所有这些算法都需要在不同焦平面拍摄的二维图像堆栈，尽管“最近邻去模糊”算法只需要图像平面上方和下方的一幅图像。我们对该方案进行了改进，构建了一个简单的逆滤波器，它能提取与最近邻方案相当的光学切片，但无需相邻图像切片。我们使用这种“无邻域”处理方案对来自米色小鼠的负载fura - 2的肥大细胞图像进行去模糊，并生成穿过细胞的薄切片的高分辨率比率型钙离子图像。通过在相隔0.5微米的不同焦平面拍摄成对图像，展示了这些图像较浅的景深。排除fura - 2的分泌颗粒在所有切片中都清晰可见，并且在相邻切片中可以看到它们的大小和形状有明显变化。此外，我们借助模型物体展示了逆滤波和比率成像的结合如何校正使用逆滤波器的一些固有局限性，并可用于局部钙离子梯度的定量测量。通过这项技术，我们可以观察到分泌颗粒和质膜之间胞质溶胶狭窄区域内宽度小于0.5微米的钙离子瞬变。此外，可以看到这些钙离子增加与胞吐融合后分泌颗粒的肿胀同时发生。
