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Acquisition of quantitative information from microscopic biological samples is highly desirable in the context of the emerging field of systems biology. We derive a statistical approach to estimate the number of fluorescent molecules in the observation volume based on a confocal microscope for single-molecule detection. The method employs ps-pulsed laser sources for excitation and time-correlated single-photon counting with 4 avalanche photon diodes (APDs) for detection of individual photons. The feasibility for estimating the number of molecules is shown based on simultaneous emission and detection of multiple photons (photon-antibunching) under realistic experimental conditions. In theory, it should be possible to estimate the number of molecules with errors of less than 1% by using novel photo-stabilizing agents. The proposed method puts into perspective its application for high-resolution microscopy without the need for photo-switching or photo-activation of fluorescence dyes. [ABSTRACT FROM AUTHOR]