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Optimizing laser and probe molecules for multi-photon microscopy

Multiple fluorescent probes (multi-dyes) and single or multi-laser configurations can significantly extend the applications and accuracy of microscopy. Multiple fluorescent probes enable the user to identify more than one target, but difficulties can arise due to overlapping spectral emissions of the different probes. In particular, spectral overlapping of fluorescent and/or phosphorescent emission signals can lead to incorrect analysis. We present a method to numerically calculate overlapping spectra. An accurate modeling tool would be valuable to predict the best laser-probes combinations for selection and screening stages. We use a numerical method that simulates both time and space so that we can calculate on a near-instantaneous basis the absorption of laser light and electron populations. We can then calculate the intensity of the emitted signal and determine the overlap of the spectra.