FIGURE 4.

Comparison of vesicle size distributions measured with cryoTEM and CLSM. Size-distribution histograms of extruded vesicles (SUV₁₀₀ extruded using a filter diameter of 100 nm and SUV₂₀₀ extruded using a filter diameter of 200 nm) were measured with cryoTEM and CLSM. Vesicles from SUV₁₀₀ (A) and SUV₂₀₀ (B) were trapped in an amorphous ice film and recorded with cryoTEM to construct histograms of vesicle sizes. Integrated fluorescence-intensity histograms of SUV₁₀₀ (C) and SUV₂₀₀ (D) immobilized on a glass surface and recorded with CLSM. To convert the integrated intensity histogram (C) to a physical size histogram, the mean diameter from the distribution in A was used to convert the abscissa of C to nanometers. Using SUV₁₀₀ as the reference sample, the physical size distribution of SUV₂₀₀ (D) could now be determined from the integrated intensity distribution. Comparing B and D, our model reproduces the mean peak of the physical size distribution obtained from cryoTEM within 7%. The shapes of the histograms obtained from cryoTEM and CLSM were found to be identical, thereby confirming that cryoTEM and CLSM reconstruct the vesicle populations in a similar way. Insets show representative micrographs. The brightness and contrast of the images has been optimized for visual purposes.