The invention provides methods of focusing an instrument for the acquisition of optical data from a tissue sample. Methods of the invention allow rapid focusing in the context of a diagnostic procedure in which rapid data acquisition is desirable. For example, inventive methods allow a user to focus an optical instrument quickly enough to obtain data within an optimal window of time following application of a chemical agent to the tissue. In one embodiment, a user focuses an optical instrument by aligning laser spots projected onto a tissue sample within rings that are superimposed at predetermined locations within the user's visual field. Preferred methods of the invention further comprise automatic validation to detect whether the spots are sufficiently well-aligned.