UC Berkeley

The advent of super-resolution microscopy over the past decade has excited many researchers. Single-molecule localization microscopy (SMLM; to include specific implementations including STORM, (f)PALM, and PAINT), a branch of super-resolution microscopy, offers outstanding spatial resolution of ~20 nm through single-molecule imaging. Another exciting but often overlooked feature of SMLM is that when properly designed, it may enable ultrahigh-throughput characterization of individual molecules for multiple physical parameters.

The first part of this dissertation describes the multidimensional characterization of single molecules through SMLM. We introduce spectrally-resolved SMLM, which captures the positions and spectra of single fluorescent molecules with ultra-high throughput. Combined with a solvatochromic fluorescent dye, we demonstrate spatial mapping of the local chemical environment for heterogeneous systems. We thus present polarity mapping of lipid membranes in live cells and discuss the origin of the heterogeneity. Difference between the plasma membrane and organelle membrane is visualized in live cells, along with their structures and dynamics. We also characterize the structure, polarity, and chemical compositions of adsorbed organic layers on a surface. Besides nanoscale structures of the surface adlayer, we reveal their spontaneous demixing on the surface. Finally, we develop a novel approach for the multidimensional characterization of single molecules through machine learning, in which the parameters of interest are directly extracted from the unmodified diffraction pattern of single fluorescent molecules. We thus demonstrate concurrent spectral separation and axial localization for two fluorescent dyes in fixed cells under SMLM settings.

In Part II, we discuss multiple strategies to improve the performance of optical microscopy, for which efforts we make unusual use of the exceptional mechanical, electrical, and optical properties of graphene, a single layer of bonded carbon atoms. We first take advantage of the superior conductivity of graphene to facilitate correlated SMLM and electron microscopy, in which graphene works as a protective and conductive layer to enable electron microscopy for wet cells. Both the molecular specificity from SMLM and the high spatial resolution from electron microscopy are thus conveniently achieved. We next demonstrate a novel electroporation-microscopy system based on graphene, which enables the spatially and temporally controlled delivery of fluorescent probes of various sizes into live cells with high efficiency. Remarkably, the superior optical and electrical properties of the graphene allow us to electroporate and image the cells in the same device with precise control and high resolution, thus broadening the palette of fluorescent probe for live cell SMLM. Lastly, we present new methodologies to achieve high-contrast and high-throughput microscopy for graphene itself on transparent substrates. Whereas traditional, transmission microscopy only provide ~3% optical contrast for each layer of graphene, we develop an approach based on interference reflection microscopy (IRM) to achieve >10-fold higher contrast on various inorganic and polymer transparent substrates, and demonstrate high-throughput fast imaging with diffraction-limited resolution.