Creating attoliter detection volume by microsphere photonic nanojet and fluorescence depletion

In this paper, a novel method is presented for creating a 3-dimensinal sub-diffraction effective observation volume based on microsphere photonic nanojet and fluorescence depletion effect. Using microsphere to achieve photonic nanojet focusing effect, the proposed method applies the radially polarized plane wave to excite fluorescence and the azimuthally polarized beam to obtain fluorescence depletion field. The effective detection volume of photonic nanojet can reach 0.002 μm3 (2aL). Compared with conventional confocal microscopy, this effective detection volume represents a reduction of almost 2 orders of magnitude. With simple configuration based on cost-effective microspheres, the proposed method is theoretically proved to be a potential tool for the fluorescence correlation spectroscopy (FCS) to have large analysis range and to investigate single molecule at high concentrations.