Optical detection in microscopic domains. 2. Inner filter effects for monitoring nonfluorescent molecules with fluorescence

In this research, we test whether optical detection techniques show differe nt characteristics in microscopic solution volumes (nano-, pico-, and femto liter range) compared to the usual macroscopic samples, In part 1 (Lu, H,; et at. 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 curv atures of the droplets studied, Addition and subtraction of absorbing molec ules with diffusional microburets (DMBs), as well as more complex operation s (simultaneous addition of one and subtraction of another molecule, and a consuming scheme), have been monitored with good spectral and temporal reso lution. Despite the unexpectedly good performance of absorption microspectr ometry, fluorescence-based detection schemes are considered more sensitive for microscopic studies (e,g., cell physiology). In this paper, we test whe ther fluorescence-based schemes can be used to indirectly measure nonfluore scent chemicals in microscopic domains, Absorption by such molecules will c ause a corresponding decrease in overall fluorescence intensity of the adde d standard fluorescent dye. This phenomenon, the inner filter effect (IFE), was tested using Lucifer Yellow CH (LY) as the fluorescent standard dye, I ts effective irradiation nas absorbed by Orange G (primary IFE) or its emis sion by Bromophenol Blue (secondary IFE). BY utilizing these phenomena, (1) we measured the concentration of absorbing molecules in microscopic sample s by adding a standard amount of LY by a DMB, and (2) we monitored DMB deli very of nonfluorescent reagents into droplets preloaded with LY. The result s prove that IFEs are sensitive indirect means of detection of absorbing mo lecules in microscopic domains. The techniques presented are expected to fi nd applications in cellular studies where absorption spectrometry is usuall y not considered.