Optical Detection in Microscopic Domains. 2. Inner Filter Effects for Monitoring Nonfluorescent Molecules with Fluorescence

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Abstract
In this research, we test whether optical detection techniques show different characteristics in microscopic solution volumes (nano-, pico-, and femtoliter range) compared to the usual macroscopic samples. In part 1 (Lu, H.; et al. Anal. Chem. 2000, 72, 1569−1576.) absorption spectra of high quality were obtained, quantitatively obeying both Beer−Lambert's law and the law of superposition, despite the micrometer optical path lengths and the curvatures of the droplets studied. Addition and subtraction of absorbing molecules with diffusional microburets (DMBs), as well as more complex operations (simultaneous addition of one and subtraction of another molecule, and a consuming scheme), have been monitored with good spectral and temporal resolution. Despite the unexpectedly good performance of absorption microspectrometry, fluorescence-based detection schemes are considered more sensitive for microscopic studies (e.g., cell physiology). In this paper, we test whether fluorescence-based schemes can be used to indirectly measure nonfluorescent chemicals in microscopic domains. Absorption by such molecules will cause a corresponding decrease in overall fluorescence intensity of the added standard fluorescent dye. This phenomenon, the inner filter effect (IFE), was tested using Lucifer Yellow CH (LY) as the fluorescent standard dye. Its effective irradiation was absorbed by Orange G (primary IFE) or its emission by Bromophenol Blue (secondary IFE). By utilizing these phenomena, (1) we measured the concentration of absorbing molecules in microscopic samples by adding a standard amount of LY by a DMB, and (2) we monitored DMB delivery of nonfluorescent reagents into droplets preloaded with LY. The results prove that IFEs are sensitive indirect means of detection of absorbing molecules in microscopic domains. The techniques presented are expected to find applications in cellular studies where absorption spectrometry is usually not considered.