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Recovery of depth information plays a key role in the micromanipulation robot system for performing micron or sub-micron orientation and measurement tasks. Based on the model of defocused optical system and combined with characteristics of frequency response curves of defocus optical transfer function (OTF), a new method of estimating the depth information for micro-manipulation is proposed. The depth information can be calculated by using the depth equation derived from a sequence of calibration images when acquiring the energy parameter of the micromanipulator in the certain position. We experiment with the choice of the high-pass cut-off frequency in order to identify the optimum filter for the specific micromanipulation tool. Experimental results show that the method is efficient, robust, and useful in practical applications.