We propose a methodology to measure the mechanical properties of membranes from their fluctuations and apply this to optical microscopy measurements of giant unilamellar vesicles of lipids. We analyze the effect of the projection of thermal shape undulations across the focal depth of the microscope. We derive an analytical expression for the mode spectrum that varies with the focal depth and accounts for the projection of fluctuations onto the equatorial plane. A comparison of our model with existing approaches, that use only the apparent equatorial fluctuations without averaging out of this plane, reveals a significant and systematic reduction in the inferred value of the bending rigidity. Our results are in full agreement with the values measured through X-ray scattering and other micromechanical manipulation techniques, resolving a long standing discrepancy with these other experimental methods.
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