Chemical reactions in aqueous microdroplets often exhibit unusual kinetic and thermodynamic properties not observed in bulk solution. While an electric field has been implicated at the water interface, there has been no direct measurement in aqueous microdroplets, largely due to the lack of proper measurement tools. Herein, we employ newly developed stimulated Raman excited fluorescence microscopy to measure the electric field at the water−oil interface of microdroplets. As determined by the vibrational Stark effect of a nitrile-bearing fluorescent probe, the strength of the electric field is found to be on the order of 10 7 V/cm. This strong electric field aligns probe dipoles with respect to the interface. The formation of the electric field likely arises from charge separation caused by the adsorption of negative ions at the water−oil interface of microdroplets. We suggest that this strong electric field might account in part for the unique properties of chemical reactions reported in microdroplets.

Powerful optical tools have revolutionized science and technology. The prevalent fluorescence detection offers superb sensitivity down to single molecules but lacks sufficient chemical information [1][2][3] . In contrast, Raman-based vibrational spectroscopy provides exquisite chemical specificity about molecular structure, dynamics and coupling, but is notoriously insensitive [3][4][5] . Here we report a hybrid technique of Stimulated Raman Excited Fluorescence (SREF) that integrates superb detection sensitivity and fine chemical specificity. Through stimulated Raman pumping to an intermediate vibrational eigenstate followed by an upconversion to an electronic fluorescent state, SREF encodes vibrational resonance into the excitation spectrum of fluorescence emission. By harnessing narrow vibrational linewidth, we demonstrated multiplexed SREF imaging in cells, breaking the "color barrier" of fluorescence. By leveraging superb sensitivity of SREF, we achieved all-far-field single-molecule Raman spectroscopy and imaging without plasmonic enhancement, a long-sought-after goal in photonics. Thus, through merging Raman and fluorescence spectroscopy, SERF would be a valuable tool for chemistry and biology.

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding.

Water is arguably the most common and yet least understood material on Earth. The interface between water and a hydrophobic medium, such as air, oil, or lipids, plays a fundamental role in chemistry and biology. However, the behavior of molecules at interface of micron-sized water droplets (microdroplets) in such media is poorly characterized. Herein we employed two-photon fluorescence microscopy and Forster resonant energy transfer imaging to study the probe localization in water−oil microdroplets with high contrast and resolution. We found that there exists a general effect of surface enrichment and orientation alignment for watersoluble probes. Remarkably, probes are concentrated into a ∼10 nm thin layer at the microdroplet water−oil interface by up to 10 000-fold compared to the bulk counterpart. We suggest that the strong enrichment and alignment of water-soluble molecules, likely to be induced in part by a local electric field at the interface, could be a major factor accounting for orders of magnitude faster reaction rates observed in aqueous microdroplets compared to their bulk counterparts.

Imaging the spatial distribution of biomolecules is at the core of modern biology. The development of fluorescence techniques has enabled researchers to investigate subcellular structures with nanometer precision. However, multiplexed imaging, i.e. observing complex biological networks and interactions, is mainly limited by the fundamental ‘spectral crowding’ of fluorescent materials. Raman spectroscopy-based methods, on the other hand, have a much greater spectral resolution, but often lack the required sensitivity for practical imaging of biomarkers. Addressing the pressing need for new Raman probes, herein we present a series of Raman-active  nanoparticles (Rdots) that exhibit the combined advantages of ultra-brightness and compact sizes (~20 nm). When coupled with the emerging stimulated Raman scattering (SRS) microscopy, these Rdots are brighter than previously reported Raman-active organic probes by two to three orders of magnitude. We further obtain evidence supporting for SRS imaging of Rdots at single particle level. The compact size and ultra-brightness of Rdots allows immunostaining of specific protein targets (including cytoskeleton and low-abundant surface proteins) in mammalian cells and tissue slices with high imaging contrast. These Rdots thus offer a promising tool for a large range of studies on complex biological networks.