The mechanical properties of microtubules have been the subject of intense study during recent decades because of their importance to the many cell functions that they are involved in. Observations of microtubule thermal fluctuations have proven to be a reliable method to extract mechanical properties because they provide intrinsic calibration. While analysis of the entire microtubule shape is limited by spatial resolution to very long microtubules, we show that even for short microtubules, one can obtain high-precision fluctuation information from one point along the contour by the use of tracer particles attached to the microtubule. The information is sufficient to extract key mechanical parameters such as stiffness and first mode relaxation time. In this article, we discuss sample preparation as well as measurements and data analysis. Copyright 2010 Elsevier Inc. All rights reserved.