Rapid adaptive optical recovery of optimal resolution over large volumes

Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain

Optical, magnetic, and electrical properties of single-walled carbon nanotubes

In this work we measure the optical, magnetic, and electrical properties of single-walled carbon nanotubes (SWNTs) and carbon nanotube materials. The bare polarized optical absorption cross sections of SWNTs are obtained for the first time, and a large anisotropy is found for light polarized parallel and perpendicular to the nanotube axes. This result validates predicted depolarization effects and also allows rapid measurement of the alignment of nanotube dispersions. Utilizing these calibrated cross sections, the mechanics of SWNTs in a magnetic field are investigated, and alignment of these molecules shows contributions from both the SWNT intrinsic diamagnetic response and external permanent moments. These magnetic alignment measurements are extended using resonant polarized photoluminescence, and we obtain the pure diamagnetic anisotropies for individual (n, m) SWNT species. Magnetic alignment spectroscopy is also used to detect the presence of SWNT bundles, and in all semiconducting SWNT bundles we discover new photoluminescence features providing direct evidence of energy transfer between SWNTs in a bundle. Micro-photoluminescence studies on individual tubes show SWNT emission properties depend strongly upon the SWNT environment. We find energy shifts in the photoluminescence emission can be controlled by varying the excitation power absorbed into the SWNT, and suggest these shifts originate from thermal outgassing of adsorbates on the SWNT sidewalls. Thermal properties of percolated nanotube networks embedded in SWNT-epoxy composites are obtained using a custom thermal conductivity measurement and show enhancements in excess of 50% over pure epoxy. The electrical resistance of novel conducting carbon nanotube aerogels are characterized, and a series of electrical pulses is found to increase the conductivity of polymer-reinforced varieties by several orders of magnitude. Transport and optical measurements on nanotube ensembles show unexplained effects at sub-Tesla magnetic fields with near identical field profiles. We investigate these low field effects as a function of temperature, surfactant, field direction, and discuss the results in the context of mechanisms established for effects in other materials

Optical, magnetic, and electrical properties of single-walled carbon nanotubes

In this work we measure the optical, magnetic, and electrical properties of single-walled carbon nanotubes (SWNTs) and carbon nanotube materials. The bare polarized optical absorption cross sections of SWNTs are obtained for the first time, and a large anisotropy is found for light polarized parallel and perpendicular to the nanotube axes. This result validates predicted depolarization effects and also allows rapid measurement of the alignment of nanotube dispersions. Utilizing these calibrated cross sections, the mechanics of SWNTs in a magnetic field are investigated, and alignment of these molecules shows contributions from both the SWNT intrinsic diamagnetic response and external permanent moments. These magnetic alignment measurements are extended using resonant polarized photoluminescence, and we obtain the pure diamagnetic anisotropies for individual (n, m) SWNT species. Magnetic alignment spectroscopy is also used to detect the presence of SWNT bundles, and in all semiconducting SWNT bundles we discover new photoluminescence features providing direct evidence of energy transfer between SWNTs in a bundle. Micro-photoluminescence studies on individual tubes show SWNT emission properties depend strongly upon the SWNT environment. We find energy shifts in the photoluminescence emission can be controlled by varying the excitation power absorbed into the SWNT, and suggest these shifts originate from thermal outgassing of adsorbates on the SWNT sidewalls. Thermal properties of percolated nanotube networks embedded in SWNT-epoxy composites are obtained using a custom thermal conductivity measurement and show enhancements in excess of 50% over pure epoxy. The electrical resistance of novel conducting carbon nanotube aerogels are characterized, and a series of electrical pulses is found to increase the conductivity of polymer-reinforced varieties by several orders of magnitude. Transport and optical measurements on nanotube ensembles show unexplained effects at sub-Tesla magnetic fields with near identical field profiles. We investigate these low field effects as a function of temperature, surfactant, field direction, and discuss the results in the context of mechanisms established for effects in other materials

Characterization, comparison, and optimization of lattice light sheets

Lattice light sheet microscopy excels at the noninvasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here, we undertake a theoretical and experimental analysis of various forms of light sheet microscopy, which demonstrates and explains why lattice light sheets provide substantial improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. We also introduce a new type of "harmonic balanced" lattice light sheet that improves performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view

An adaptive optics module for deep tissue multiphoton imaging in vivo

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo

Rapid reconstruction of neural circuits using tissue expansion and light sheet microscopy

Brain function is mediated by the physiological coordination of a vast, intricately connected network of molecular and cellular components. The physiological properties of neural network components can be quantified with high throughput. The ability to assess many animals per study has been critical in relating physiological properties to behavior. By contrast, the synaptic structure of neural circuits is presently quantifiable only with low throughput. This low throughput hampers efforts to understand how variations in network structure relate to variations in behavior. For neuroanatomical reconstruction, there is a methodological gulf between electron microscopic (EM) methods, which yield dense connectomes at considerable expense and low throughput, and light microscopic (LM) methods, which provide molecular and cell-type specificity at high throughput but without synaptic resolution. To bridge this gulf, we developed a high-throughput analysis pipeline and imaging protocol using tissue expansion and light sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many animals with single-synapse resolution and molecular contrast. Using Drosophila to validate this approach, we demonstrate that it yields synaptic counts similar to those obtained by EM, enables synaptic connectivity to be compared across sex and experience, and can be used to correlate structural connectivity, functional connectivity, and behavior. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.ISSN:2050-084

Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems