Spatial Analysis of DHE on Plasma Membrane of Living Cells*

The objective of this study is to use image-processing techniques and statistical models to characterize the spatial properties of DHE (dehydroergosterol) on the PM (plasma membrane) of living cells. Our objective is to create a framework ( analysis algorithms, procedures and evaluation criteria) to assess spatial structures of DHE distribution on the PM. Multiple image processing algorithms and statistical measures are used to assert and cross check spatial properties of DHE distributions from the digital images of DHE acquired from MPLSM (multiphoton laser scanning microscopy) using multiple types of dyes, see figure 1.

(a)	(b)

Figure 1: (a) An Image of DHE in L-Cell Fibroblasts stained with Nile Red, and (b) the DHE intensity image extracted from it.

PM Extraction

The PM region is extracted by using a moments function to define a smooth geometric trajectory, on which an analysis confidence interval (the dash line envelope in figure 2) is defined to quantify the confidence of the assessment on the DHE analysis.

DHE Spatial Properties

Statistical inference techniques (Runs Test, Complete Spatial Randomness (CSR), and Poisson cluster process) were used to examine the spatial distributions of DHE on the PM. It was found that the (spatial) distributions of DHE at densely populated PM regions did not consititute a Poisson process. Through further analysis, it was found to exhibit stronger cluster than regular patterns. A parent-offspring clustering model with different parameter settings fit to the datasets showed that the DHE locations tended to be randomly distributed at sparsely populated area. The analysis for the first time demonstrated that sterol distribution was not random, but instead clustered into microdomains in plasma membranes of living cells. Similar analyses were applied to high intensity DHE pixels uing the CSR (complete spatial randomness) test on point process. The CSR test of S1 is used to illustrate the experiment outcomes, figure 3.

The peak DHE spot analysis showed that different PM segments exhibited clustered DHE distributions. For details, see [1]

References:

1. Weimin Zhang, Avery L. McIntosh, Hai Xu, Di Wu, Todd Gruninger, Barbara Atshaves, J. C. Steve Liu and Friedhelm Schroeder "Structural Analysis of Sterol Distributions in the Plasma Membrane of Living Cells,"Biochemistry, 44(8): pp. 2864-84, Mar. 2005.
2. F. Schroeder, B. Atshaves, A. Gallegos, A. McIntosh, J.C. Liu, A. Kier, H. Huang, J. Ball, “Caveolae organization and role in lipid cholesterol metabolism”, in Advances in molecular and cell biology, 36, (Caveolae and lipid rafts: roles in signal transduction and the pathogenesis of human diseases,) pp. 3-36, edited by M. Lisanti and P. Frank, Elsevier, 2005.

*Joint work with the Departments of Physiology and Pharmacology, Pathobiology, and Statistics, Texas A&M University.