Method of calibration of a fluorescence microscope for quantitative studies

Confocal microscopy is based on measurement of intensity of fluorescence originating from a limited volume in the imaged specimen. The intensity is quantized in absolute (albeit arbitrary) units, producing a digital 3D micrograph. Thus, one may obtain quantitative information on local concentration of biomolecules in cells and tissues. This approach requires estimation of precision of light measurement (limited by noise) and conversion of the digital intensity units to absolute values of concentration (or number) of molecules of interest. To meet the first prerequisite we propose a technique for measurement of signal and noise. This method involves registration of a time series of images of any stationary microscope specimen. The analysis is a multistep process, which separates monotonic, periodic and random components of pixel intensity change. This approach permits simultaneous determination of dark and photonic components of noise. Consequently, confidence interval (total noise estimation) is obtained for every level of signal. The algorithm can also be applied to detect mechanical instability of a microscope and instability of illumination source. The presented technique is combined with a simple intensity standard to provide conversion of relative intensity units into their absolute counterparts (the second prerequisite of quantitative imaging). Moreover, photobleaching kinetics of the standard is used to estimate the power of light delivered to a microscope specimen. Thus, the proposed method provides in one step an absolute intensity calibration, estimate of precision and sensitivity of a microscope system.