Intracellular diffusion underlies vital cellular processes. However, it remains difficult to elucidate how an unbound protein diffuses inside the cell with good spatial resolution and sensitivity. Here we introduce single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through local statistics of the instantaneous displacements of freely diffusing single molecules. We thus show that the diffusion of an average-sized protein in the mammalian cytoplasm and nucleus is spatially heterogeneous at the nanoscale, and that variations in local diffusivity correlate with the ultrastructure of the actin cytoskeleton and the organization of the genome, respectively. SMdM of differently charged proteins further unveils that the possession of positive, but not negative, net charges drastically impedes diffusion, and that the rate is determined by the specific subcellular environments. We thus unveil rich heterogeneities and charge effects in intracellular diffusion at the nanoscale.

The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter, up to six subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down to ~10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and Förster resonance energy transfer fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.

Under ambient conditions, the behavior of a solid surface is often dominated by a molecularly thin adsorbed layer (adlayer) of small molecules. Here we develop an optical approach to unveil the nanoscale structure and composition of small-molecule adlayers on glass surfaces through spectrally resolved super-resolution microscopy. By recording the images and emission spectra of millions of individual solvatochromic molecules that turn fluorescent in the adlayer phase, we obtain ~30 nm spatial resolution and achieve concurrent measurement of local polarity. This allows us to establish that the adlayer dimensionality gradually increases through a sequence of 0D (nanodroplets), 1D (nano-lines), and 2D (films) for liquids of increasing polarity. Moreover, we find that in adlayers, a solution of two miscible liquids spontaneously demixes into nanodroplets of different compositions that correlate strongly with droplet size and location. We thus reveal unexpectedly rich structural and compositional behaviors of surface adlayers at the nanoscale.

Water molecules can mediate charge transfer in biological and chemical reactions by forming electronic coupling pathways. Understanding the mechanism requires a molecular-level electrical characterization of water. Here, we describe the measurement of single water molecular conductance at room temperature, characterize the structure of water molecules using infrared spectroscopy, and perform theoretical studies to assist in the interpretation of the experimental data. The study reveals two distinct states of water, corresponding to a parallel and perpendicular orientation of the molecules. Water molecules switch from parallel to perpendicular orientations on applying an electric field, producing switching from high to low conductance states, thus enabling the determination of single water molecular dipole moments. The work further shows that water-water interactions affect the atomic scale configuration and conductance of water molecules. These findings demonstrate the importance of the discrete nature of water molecules in electron transfer and set limits on water-mediated electron transfer rates.