CIL:11924, Taricha granulosa, epithelial cell of lung. In Cell Image Library

Comparison of actin speckle microscopy with phalloidin staining. Primary cultures of newt lung epithelial cells were microinjected with X-rhodamine actin. Time-lapse FSM was performed on a spinning disk confocal microscope system. Light from a 50-mW Krypton-Argon ion laser (Melles Griot, OmniChrome) was delivered by a single-mode fiber optic (Point Source) to a Yokogawa spinning disk confocal scan-head (Ultra-View; PerkinElmer) on an inverted microscope (TE300 Quantum; Nikon). Excitation wavelength was selected by a filter-wheel apparatus (Sutter Instruments Co.) containing excitation filters for 488 and 568 nm (Chroma). Emission was selected by multiple bandpass dichromatic mirror and emission filter (Chroma). Images were collected by a 100x 1.4NA Plan-Apo DIC objective lens (Nikon) and captured with an Orca 2 camera (Hamamatsu). Images were collected in the ventral focal plane at 10s intervals. Following live cell imaging, cells were fixed with paraformaldehyde, permeabilized and stained with Alexa 488 phalloidin. Images were processed as follows: (1) background subtraction; (2) 3 x3 low pass filter; (3) unsharp mask filter. Video corresponds to Fig 1 and video 1 in J Cell Biol, 158:31-37, 2002.肌动蛋白斑显微镜与鬼笔环肽染色的对比研究。蝾螈肺上皮细胞原代培养物经显微注射导入X-罗丹明肌动蛋白（X-rhodamine actin），采用转盘式共聚焦显微镜系统开展延时荧光斑点显微镜（Fluorescence Speckle Microscopy, FSM）成像。实验光源为50 mW氪-氩离子激光器（Melles Griot，OmniChrome），光束经单模光纤（Point Source）传输至搭载于尼康TE300 Quantum倒置显微镜的横河电机Ultra-View转盘式共聚焦扫描头（PerkinElmer）。激发波长由搭载488 nm与568 nm激发滤光片（Chroma）的滤轮装置（Sutter Instruments Co.）进行选择，发射光路采用多波段双色分光镜与发射滤光片（Chroma）完成波段选择。图像通过100倍1.4NA Plan-Apo DIC物镜（Nikon）采集，并由滨松Orca 2相机（Hamamatsu）捕获，成像选取细胞腹侧焦平面，采集间隔为10秒。活细胞成像完成后，将细胞用多聚甲醛固定，透化处理后采用Alexa 488鬼笔环肽（Alexa 488 phalloidin）进行染色。图像经以下流程处理：(1) 背景扣除；(2) 3×3低通滤波；(3) 非锐化掩模滤波。本视频对应2002年《Journal of Cell Biology》（J Cell Biol）第158卷第31-37页的图1与视频1。