Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

SUMMARY The basis for region-specific neuronal toxicity in Huntington disease is unknown. Here, we show that region-specific neuronal vulnerability is a substrate-driven response in astrocytes. Glucose is low in HdhQ(150/150) animals, and astrocytes in each brain region adapt by metabolically reprogramming their mitochondria to use endogenous, non-glycolytic metabolites as an alternative fuel. Each region is characterized by distinct metabolic pools, and astrocytes adapt accordingly. The vulnerable striatum is enriched in fatty acids, and mitochondria reprogram by oxidizing them as an energy source but at the cost of escalating reactive oxygen species (ROS)-induced damage. The cerebellum is replete with amino acids, which are precursors for glucose regeneration through the pentose phosphate shunt or gluconeogenesis pathways. ROS is not elevated, and this region sustains little damage. While mhtt expression imposes disease stress throughout the brain, sensitivity or resistance arises from an adaptive stress response, which is inherently region specific. Metabolic reprogramming may have relevance to other diseases.

SUMMARY Through three-dimensional STORM super-resolution microscopy, we resolve the spectrin-actin-based membrane cytoskeleton of neural stem cells (NSCs) and NSC-derived neurons, astrocytes, and oligodendrocytes. We show that undifferentiated NSCs are capable of forming patches of locally periodic, one-dimensional (1D) membrane cytoskeleton with ~180 nm periodicity. Such periodic structures become increasingly ordered and long-ranging as the NSCs mature into terminally differentiated neuronal and glial cell types, and, during this process, distinct 1D periodic ‘‘strips’’ dominate the flat 2D membranes. Moreover, we report remarkable alignment of the periodic cytoskeletons between abutting cells at axon-axon and axon-oligodendrocyte contacts and identify two adhesion molecules, neurofascin and L1CAM, as candidates to drive this nanoscale alignment. We thus show that a conserved 1D periodic membrane cytoskeletal motif serves as a nanoscale scaffold and ruler to mediate the physical interactions between cell types of the NSC lineage.

The functional integration of atomically defined graphene nanoribbons (GNRs) into single-ribbon electronic device architectures has been limited by access to nondestructive high-resolution imaging techniques that are both compatible with common supports such as Si or Si/SiO wafers and capable of resolving individual ribbons in dilute samples. Conventional techniques such as scanning probe (AFM, STM) or electron microscopy (SEM, TEM) have been restricted by requisite sample preparation techniques that are incompatible with lithographic device fabrication. Here we report the design and synthesis of ultralong (∼10 μm) cove-type GNRs (cGNRs) featuring azide groups along the edges that can serve as a universal handle for late-stage functionalization with terminal alkynes. Copper-catalyzed click-chemistry with Cy5 fluorescent dyes gives rise to cGNRs decorated along the edges with fluorescent tags detectable by optical microscopy. The structures of individual dye-functionalized cGNRs spin-coated from a dilute solution onto transparent and opaque insulating substrates were resolved using diffraction-limited fluorescence microscopy and super-resolution microscopy (SRM) imaging techniques. Analysis of SRM images reveals an apparent width of cGNRs in the range 40-50 nm and lengths in excess of 10 μm, the longest GNRs imaged to date. Isolated cGNRs can even be distinguished from bundles and larger aggregates as long as the center-to-center distance is greater than the apparent width.

The incorporation of exogenous molecules into live cells is essential for both biological research and therapeutic applications. In particular, for the emerging field of super-resolution microscopy of live mammalian cells, reliable fluorescent labeling of intracellular targets remains a challenge. Here, utilizing the unique mechanical, electrical, and optical properties of graphene, a single layer of bonded carbon atoms, we report a facile approach that enables both high-throughput delivery of fluorescent probes into adherent live cells and in situ super-resolution microscopy on the same device. ~90% delivery efficiencies are achieved for free dyes and dye-tagged affinity probes, short peptides, and whole antibodies, thus enabling high-quality super-resolution microscopy. Moreover, we demonstrate excellent spatiotemporal controls, which, in combination with the ready patternablity of graphene, allow for the spatially selective delivery of two different probes for cells at different locations on the same substrate.We thus open up a new pathway to the microscopic manipulation and visualization of live cells.