Von Wegner Frederic, Both Martin, Fink Rainer H A, Friedrich Oliver
Medical Biophysics Group, Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg BW 69120, Germany.
IEEE Trans Med Imaging. 2007 Jul;26(7):925-34. doi: 10.1109/TMI.2007.895471.
We used multifocal multiphoton microscopy to image fast, localized elevations of the cytosolic Ca2+ concentration in two spatial dimensions plus time (XYT). This technique extends the common spatially 1-D XT imaging and allows the acquisition of more than ten times longer time series (>500 images) and ten times larger areas of interest than for previously used confocal XYT imaging techniques due to lower phototoxicity and fast multifocal scanning. We recorded spontaneously occurring elementary Ca2+ release events in chemically permeabilized adult mammalian skeletal muscle fibers using two-photon excitation of the fluorescent dye Fluo-4. The resulting time series were analyzed with an automated denoising and detection algorithm based on the à trous implementation of the discrete wavelet transform. Wavelet coefficient hard-thresholding is used for denoising and event detection is performed across several wavelet scales. The spatiotemporal characteristics of the detected Ca2+ release events are followed throughout the XYT stack and are parametrized using a biophysically valid anisotropic Gaussian event model. The proposed method allows a detailed spatiotemporal analysis of elementary Ca2+ release events underlying the excitation-contraction coupling process in muscle.
我们使用多焦点多光子显微镜在二维空间加时间（XYT）中对胞质Ca2+浓度的快速、局部升高进行成像。该技术扩展了常见的一维空间XT成像，并且由于较低的光毒性和快速多焦点扫描，与先前使用的共聚焦XYT成像技术相比，能够获取时间序列长十倍以上（>500张图像）且感兴趣区域大十倍的图像。我们使用荧光染料Fluo-4的双光子激发，记录了化学通透的成年哺乳动物骨骼肌纤维中自发发生的基本Ca2+释放事件。使用基于离散小波变换的à trous实现的自动去噪和检测算法对所得时间序列进行分析。小波系数硬阈值化用于去噪，并在多个小波尺度上进行事件检测。在整个XYT堆栈中跟踪检测到的Ca2+释放事件的时空特征，并使用生物物理上有效的各向异性高斯事件模型对其进行参数化。所提出的方法允许对肌肉中兴奋-收缩偶联过程背后的基本Ca2+释放事件进行详细的时空分析。
