Quantitative three‐dimensional confocal microscopy of synaptic structures in living brain tissue
Quantitative three‐dimensional confocal microscopy of synaptic structures in living brain tissue
复制标题
活体脑组织突触结构的定量三维共聚焦显微镜
DOI：
10.1002/jemt.1070290405
复制
发表时间：
1994
影响因子：
2.5
通讯作者：
A. Fine
中科院分区：
文献类型：
--
作者：
T. Hosokawa;T. Bliss;A. Fine
为了研究伴随学习和记忆而来的突触结构的变化，我们发展了一种光学方法，在体外维持的活的、电子监控的脑片上显示树突棘和突触前终末。荧光亲脂染料DII的焦点显微应用提供了少量细胞和过程的高尔基体染色，这些细胞和过程可以通过共聚焦显微镜清楚地分辨出来；染色细胞的活力是通过排除与DNA结合的荧光染料溴化乙锭来确定的。通过反卷积和其他图像处理方法对连续光学切片进行增强。由此产生的高分辨率图像在一个自动程序中被组合起来，以产生三维重建，其中亚微米突触结构可以被观察和测量。这些无偏见的方法允许在发育过程中或在对刺激、药物应用或其他干扰的反应中，在几小时或几天的时间内定量评估个体、活的突触结构的体积变化。©1994 Wiley-Liss，Inc.
In order to study changes in synaptic structure that accompany learning and memory, we have developed optical methods to visualize dendritic spines and presynaptic terminals in living, electrically monitored brain slices maintained in vitro. Focal microapplication of the fluorescent lipophilic dye DiI provides Golgi‐like staining of small numbers of cells and processes that can be resolved clearly using confocal microscoopy; viability of stained cells is established by exclusion of the fluorescent DNA‐binding dye ethidium bromide. Serial optical sections are enhanced by deconvolution and other image processing methods. The resulting high‐resolution images are combined in an automated procedure to generate three‐dimensional reconstructions, in which submicron synaptic structures can be viewed and measured. These unbiased methods allow volume changes in individual, living synaptic structures to be assessed quantitatively over periods of hours or days in development or in response to stimulation, drug application, or other perturbations. © 1994 Wiley‐Liss, Inc.