全内角反射荧光显微镜.pdf

METHODOLOGY Open Access Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana Yinglang Wan1, William M Ash III2, Lusheng Fan1,3, Huaiqin Hao1, Myung K Kim2 and Jinxing Lin1* Abstract Background: Total internal reflection fluorescence microscopy (TIRFM) is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM) was developed to circumvent this problem. However, the lack of a detailed analysis of the optical principles underlying VAEM has limited its applications in plant-cell biology. Results: Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein) and clathrin light chain (a vesicle coat protein) support our theoretical analysis. Conclusions: These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana. Keywords: Quantitative, VA-TIRFM, optical analysis, intact cell, cell wall Background Total internal reflection fluorescence microscopy (TIRFM), also known as evanescent wave microscopy (EWM), is a powerful tool for observing the distribution and movement of fluorescently labeled molecules in an aqueous environment. This technique can be used when the molecules of interest are very close to the boundary between the aqueous environment and ano