AUTOMATIC SIZE DETERMINATION OF MEMBRANE-VESICLES BY FREEZE-FRACTURE ELECTRON-MICROSCOPY

In biological processes involving membrane vesicles, the vesicle sizes and the size distribution of the vesicle population are usually important process parameters. A previous method for determining these parameters by freeze-fracture electron microscopy exclusively makes use of concave profiles with half-cast shadow, the so-called equatorial profiles. This method suffers from two disadvantages: (i) the measurement is done manually and is therefore subjective, and (ii) only a small subpopulation of the profiles, those showing half-cast shadow, can be used. We describe an automatic, computerized method with which the vesicle size can be calculated from any concave profile with cast shadow. This method is based on the relationship between the angle of shadowing and the surface areas of the profile and the profile's uncoated region. Since the vesicles are processed automatically, the procedure is reproducible and not biased by subjective measurements. The method was tested both on synthetic, computer-generated profiles and on large unilamellar vesicles. It allows for the use of about four times as many profiles as the equator method in determining the vesicle size distribution. This means that the distribution is more reliable thanks to the larger population used for its determination. Furthermore, the reliability of our method is exemplified by its stability concerning errors in its parameters.