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Wavefront-shaping devices incorporated into optical microscopy systems are capable of correcting sample-induced aberrations and recovering diffraction-limited imaging performance. The widespread dissemination and application of adaptive optical techniques, however, requires easy integration of adaptive optical modules, both in terms of hardware and software, into existing microscopes. We built an adaptive optical module with reduced complexity and simplified integration by utilizing a novel segmented deformable mirror and a standalone control software program. We demonstrated its ability to improve image brightness and resolution at depth in the mouse, zebrafish, and fly brains in vivo.

Biological specimens are rife with optical inhomogeneities that seriously degrade imaging performance under all but the most ideal conditions. Measuring and then correcting for these inhomogeneities is the province of adaptive optics. Here we introduce an approach to adaptive optics in microscopy wherein the rear pupil of an objective lens is segmented into subregions, and light is directed individually to each subregion to measure, by image shift, the deflection faced by each group of rays as they emerge from the objective and travel through the specimen toward the focus. Applying our method to two-photon microscopy, we could recover near-diffraction-limited performance from a variety of biological and nonbiological samples exhibiting aberrations large or small and smoothly varying or abruptly changing. In particular, results from fixed mouse cortical slices illustrate our ability to improve signal and resolution to depths of 400 microm.

We describe a multiplexed aberration measurement method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine their phase gradients. Applicable to fluorescent-protein-labeled structures of arbitrary complexity, it allows us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improves structural and functional imaging of fine neuronal processes over a large imaging volume.

In traditional zonal wavefront sensing for adaptive optics, after local wavefront gradients are obtained, the entire wavefront can be calculated by assuming that the wavefront is a continuous surface. Such an approach will lead to sub-optimal performance in reconstructing wavefronts which are either discontinuous or undersampled by the zonal wavefront sensor. Here, we report a new method to reconstruct the wavefront by directly measuring local wavefront phases in parallel using multidither coherent optical adaptive technique. This method determines the relative phases of each pupil segment independently, and thus produces an accurate wavefront for even discontinuous wavefronts. We implemented this method in an adaptive optical two-photon fluorescence microscopy and demonstrated its superior performance in correcting large or discontinuous aberrations.

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 aberrations deteriorate the performance of microscopes. Adaptive optics can be used to improve imaging performance via wavefront shaping. Here, we demonstrate a pupil-segmentation based adaptive optical approach with full-pupil illumination. When implemented in a two-photon fluorescence microscope, it recovers diffraction-limited performance and improves imaging signal and resolution.

A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (

Inhomogeneous optical properties of biological samples make it difficult to obtain diffraction-limited resolution in depth. Correcting the sample-induced optical aberrations needs adaptive optics (AO). However, the direct wavefront-sensing approach commonly used in astronomy is not suitable for most biological samples due to their strong scattering of light. We developed an image-based AO approach that is insensitive to sample scattering. By comparing images of the sample taken with different segments of the pupil illuminated, local tilt in the wavefront is measured from image shift. The aberrated wavefront is then obtained either by measuring the local phase directly using interference or with phase reconstruction algorithms similar to those used in astronomical AO. We implemented this pupil-segmentation-based approach in a two-photon fluorescence microscope and demonstrated that diffraction-limited resolution can be recovered from nonbiological and biological samples.

We present scanning photoluminescence (PL) microscopy of freely suspended single-walled carbon nanotubes grown by chemically assisted vapor deposition (CVD) across micron-sized open apertures. Scans of the PL emission versus excitation position show unusual "holes"having subwavelength spatial features associated with abrupt blue shifts of the emission energy. By varying the excitation polarization, energy, intensity, and position, we demonstrate that optical switching in some nanotubes is controllable in a highly nonlinear manner by adjusting the nonequilibrium carrier density in the nanotube. Technologically important attributes include large spectral contrast between on/off states at room temperature, a dramatic response to small changes in light intensity near threshold, and the possibility that electrical charge injection could also be used to control emission energies.

We determine optical absorption cross sections of single-wall carbon nanotubes for visible light copolarized and cross polarized with respect to the nanotube axis. The need for perfectly aligned ensembles in absorbance measurements is eliminated by using Raman scattering to measure the nematic order parameter in magnetically aligned nanotube suspensions. The absorbance data allow the first quantitative, spectral comparisons with theories of local field depolarization, and provide benchmark spectra for simple, rapid, and quantitative measurements of alignment within nanotube dispersions.