title: Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana.
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.
authors.affiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. linjx@ibcas.ac.cn.
authors.forename: Yinglang
authors.initials: Y
authors.lastname: Wan
authors.forename: William M
authors.initials: WM
authors.lastname: Ash
authors.forename: Lusheng
authors.initials: L
authors.lastname: Fan
authors.forename: Huaiqin
authors.initials: H
authors.lastname: Hao
authors.forename: Myung K
authors.initials: MK
authors.lastname: Kim
authors.forename: Jinxing
authors.initials: J
authors.lastname: Lin