IDBR: Development of cathodoluminescent near-field energy transfer microscopy for high frame-rate, nanoscale, non-invasive observation of aqueous biodynamics

University Of California-Berkeley, Berkeley CA

Investigators

Abstract

IDBR: Development of cathodoluminescent near-field energy transfer microscopy for high frame-rate, nanoscale, non-invasive observation of aqueous biodynamics The objective of this project is to develop a high-brightness, rapidly scannable, nanoscale light source appropriate for studying aqueous biological samples at the nanoscale under physiological conditions. Anticipated applications will investigate the dynamics of complexing soluble biomolecules and of heterogeneous lipid membranes. To realize these efforts requires a way to combine the most redeeming elements of electron and light optics: Inside a scanning electron microscope, a focused electron beam will be used to make a nano-optical spot in a thin film scintillator (a material that produces light when struck by electrons). This spot will in turn excite adjacent fluorophores in an encapsulated aqueous sample via highly localized near-field resonant energy transfer. By leveraging the nanoscale resolution and fast scanning of electron microscopy while enabling spectrally selective bio-compatible measurements of aqueous dynamics, this innovation will provide an entirely new way to perform near-field scanning optical microscopy, uninhibited by traditionally cited challenges such as low optical throughput, unwanted tip interactions, and active mechanical stabilization. Achieving tighter focal spots on the order of 10 nm both laterally and axially will revolutionize a host of biophysical fluorescence techniques. The ability to measure single-molecule fluorescence fluctuations at physiological concentrations up to a million times greater than traditional fluorescence correlation spectroscopy (FCS) will yield a much needed way to study binding and enzyme catalysis in the regime where complexes are stable. This has important implications in the understanding of diseases such as Alzheimer's and in the formation of molecular machinery such as the ribosome. The enabling of fluorescence recovery after photobleaching (FRAP) diffusion measurements on membranes that are themselves smaller than the diffraction limit, as is the case for example in the impressively crowded grana of plant chloroplasts, will reveal organizational heterogeneity and local variations in mobility that have yet to be uncovered, as other super-resolution microscopies are not compatible with FRAP. These spectrally selective studies at the nanoscale will literally provide a window into the inner workings of biomolecular interactions and will connect the high-level function of complex biomaterials with their nanoscopic origins. The developed platform will comprise optical detection in a standard scanning electron microscope (SEM), and a nanofabricated liquid cell that will both safely house the aqueous sample in vacuum and include a thin film scintillating window to convert an electron beam into a highly-localized optical field for sample illumination. The work will be performed at two different SEMs, both residing in shared facilities. In one case, the PI's group will train all new SEM users, and will offer assistance in teaching them to use the developed nano-optical capabilities. The PI's group will mentor the facility's volunteer undergraduates in the development of different nano-scintillators for use in the microscopy. The second SEM is in a national facility that has no associated user fees, and is positioned to provide a broad user base with exposure to the developed technology. Beyond these facilities, the technology will become easily reproducible as an accessible extension that others institutions will wish to incorporate into their pre-existing SEM facilities, broadening their user base to include more users in the life sciences. In undergraduate course material, the PI will use examples from this research project to highlight how physical principles are incorporated into real world biological research. The PI also promotes the participation of women and other minorities in science, as a frequent guest speaker for women's students groups in the natural sciences, and the PI's research group members participate in classroom visits to neighboring elementary schools through the Community Resources for Science Community in the Classroom program.