Optical detection in microscopic domains. 1. Monitoring chemical manipulations with absorption microspectrometry

In this research, we test whether optical detection techniques, such as absorption microspectrometry, fluorescence detection, and inner filter effects, show different characteristics in microscopic domains (nano-, pico- and femtoliter range) with respect to usual solution volumes. In this part 1, characterization of absorption microspectrometry is facilitated by the use of a novel microscopic tool, the diffusional microburet (DMB), suitable for fine chemical manipulations of microscopic liquid samples. Since diffusional delivery of substances will not induce appreciable changes in volume and shape of the sample, a DMB makes it possible to obtain spectral recordings and a reference spectrum from the same microscopic droplet. Thus, good quantitative spectra of microscopic domains can be assessed. With this approach, despite the curvature of the sample boundaries, the large surface-to-volume ratios, and microscopic optical path lengths, both Beer-Lambert's law and the law of superposition were found to be directly applicable in microscopic domains without any corrections for absorption at sample boundaries. Use of a combination of microspectrometry and chemical manipulations by DMBs made it possible for the first time to record in real time, in truly microscopic domains, spectral evolution upon addition or subtraction of chemicals, as well as monitor the progress of chemical reactions. This approach is expected to contribute to the optical exploration of microchemistry and microanalysis.