An embedded adaptive optics real time controller

The design and realisation of a low cost, high speed control system for adaptive optics (AO) is presented. This control system is built around a field programmable gate array (FPGA). FPGA devices represent a fundamentally different approach to implementing control systems than conventional central processing units. The performance of the FPGA control system is demonstrated in a specifically constructed laboratory AO experiment where closed loop AO correction is shown. An alternative application of the control system is demonstrated in the field of optical tweezing, where it is used to study the motion dynamics of particles trapped within laser foci

Flicker-assisted localization microscopy reveals altered mitochondrial architecture in hypertension

Mitochondrial morphology is central to normal physiology and disease development. However, in many live cells and tissues, complex mitochondrial structures exist and morphology has been difficult to quantify. We have measured the shape of electrically-discrete mitochondria, imaging them individually to restore detail hidden in clusters and demarcate functional boundaries. Stochastic “flickers” of mitochondrial membrane potential were visualized with a rapidly-partitioning fluorophore and the pixel-by-pixel covariance of spatio-temporal fluorescence changes analyzed. This Flicker-assisted Localization Microscopy (FaLM) requires only an epifluorescence microscope and sensitive camera. In vascular myocytes, the apparent variation in mitochondrial size was partly explained by densely-packed small mitochondria. In normotensive animals, mitochondria were small spheres or rods. In hypertension, mitochondria were larger, occupied more of the cell volume and were more densely clustered. FaLM provides a convenient tool for increased discrimination of mitochondrial architecture and has revealed mitochondrial alterations that may contribute to hypertension

Compact, modular and in-plane AOSLO for high-resolution retinal imaging

The adaptive optics scanning laser ophthalmoscope (AOSLO) was first developed in 2002 and since then the technology has been adopted in several laboratories around the world, for both clinical and psychophysical research. There have been a few major design implementations of the AOSLO. The first used on-axis tilted spherical mirrors in a planar arrangement, and the second minimized the build up of astigmatism present in the first design by using a non-planar arrangement. Other designs have avoided astigmatism by using custom-made toroidal mirrors or by using lenses on-axis, rather than mirrors. We present a new design implementation for an AOSLO that maintains a planar optical alignment without the build up astigmatism using compact, reconfigurable modules based on an Offner relay system. We additionally use an off-the-shelf digital oscilloscope for data capture and custom-written Python code for generating and analyzing the retinal images. This design results in a compact system that is simple to align and, being composed of modular relays, has the potential for additional components to be added. We show that this system maintains diffraction-limited image quality across the field of view and that cones are clearly resolved in the central retina. The modular relay design is generally applicable to any system requiring one or more components in the pupil conjugate plane. This is likely to be useful for any point-scanned system, such as a standard scanning laser ophthalmoscope or non-ophthalmic confocal imaging system

Pressure-dependent regulation of Ca2+ signaling in the vascular endothelium

The endothelium is an interconnected network upon which hemodynamic mechanical forces act to control vascular tone and remodeling in disease. Ca2+ signaling is central to the endothelium's mechanotransduction and networked activity. However, challenges in imaging Ca2+ in large numbers of endothelial cells under conditions that preserve the intact physical configuration of pressurized arteries have limited progress in understanding how pressure-dependent mechanical forces alter networked Ca2+ signaling. We developed a miniature wide-field, gradient-index (GRIN) optical probe designed to fit inside an intact pressurized artery which permitted Ca2+ signals to be imaged with subcellular resolution in a large number (∼200) of naturally-connected endothelial cells at various pressures. Chemical (acetylcholine) activation triggered spatiotemporally-complex, propagating IP3-mediated Ca2+ waves that originated in clusters of cells and progressed from there across the endothelium. Mechanical stimulation of the artery, by increased intraluminal pressure, flattened the endothelial cells and suppressed IP3-mediated Ca2+ signals in all activated cells. By computationally modeling Ca2+ release, endothelial shape changes were shown to alter the geometry of the Ca2+ diffusive environment near IP3 receptor microdomains to limit IP3-mediated Ca2+ signals as pressure increased. Changes in cell shape produce a geometric, microdomain-regulation of IP3-mediated Ca2+ signaling to explain macroscopic pressure-dependent, endothelial-mechanosensing without the need for a conventional mechanoreceptor. The suppression of IP3-mediated Ca2+ signaling may explain the decrease in endothelial activity as pressure increases. GRIN imaging provides a convenient method that provides access to hundreds of endothelial cells in intact arteries in physiological configuration

Use of fiber optic technology to measure the effects of anesthesia on luciferase reaction kinetics

In vivo bioluminescent imaging (BLI) is a sensitive and reliable technique for studying gene expression, although experiments must be controlled tightly to obtain reproducible and quantitative measurements. The luciferase reaction depends on the availability of the reaction substrate, oxygen, and ATP, the distribution of which can vary markedly in different tissues. Here we used in vivo fiber optic technology, combined with stereotaxis-assisted surgery, to assess luciferase reaction kinetics in response to 2 anesthetic regimens, isoflurane and ketamine–xylazine. Transgenic rats that expressed luciferase under the control of the human prolactin promoter were used as a model organism. Anesthesia had a marked effect on luciferase reaction kinetics. The rise time to peak emission differed by 20 min between isoflurane and ketamine–xylazine. Optical imaging using a charge-coupled–device camera confirmed this delay. These results demonstrate that different anesthetics can have substantial effects on luciferase reaction kinetics and suggest that the timing of image acquisition after substrate injection should be optimized in regard to experimental conditions and the tissues of interest

Closed loop adaptive optics with a laser guide star for biological light microscopy

We report on the development of a widefield microscope that achieves adaptive optics correction through the use of a wavefront sensor observing an artificial laser guide star induced within the sample. By generating this guide star at arbitrary positions and depths within the sample we allow the delivery of high-resolution images. This approach delivers much faster AO correction than image optimization techniques, and allows the use of AO with fluorescent imaging modalities without generating excessive photo-toxic damage in the sample, or inducing significant photo-bleaching in the flurophore molecules

Realtime wavefront sensing in a SPIM microscope, and active aberration tracking

Adaptive optics (AO) can potentially allow high resolution imaging deep inside living tissue, mitigating against the loss of resolution due to aberrations caused by overlying tissue. Closed-loop AO correction is particularly attractive for moving tissue and spatially varying aberrations, but this requires direct wavefront sensing, which in turn requires suitable "guide stars" for use as wavefront references. We present a novel method for generating an orthogonally illuminated guide star suitable for direct wavefront sensing in a wide range of fluorescent biological structures, along with results demonstrating its use for measuring time-varying aberrations, in vivo

Spatially-structured cell populations process multiple sensory signals in parallel in intact vascular endothelium : stimulus-specific sensory cells in the intact vascular endothelium

Blood flow, blood clotting, angiogenesis, vascular permeability and vascular remodeling are each controlled by a large number of variable, noisy and interacting chemical inputs to the endothelium. The endothelium processes the entire chemical composition to which the cardiovascular system is exposed, carrying out sophisticated computations to determine physiological output. A major challenge faced by the endothelium is the requirement to process an enormous quantity of information held in the overall chemical environment to which the vascular system is exposed. We analyzed hundreds of endothelial cells and show that the endothelium segregates the chemical environment into small components of complementary information streams which are processed in parallel. Chemical stimuli arriving at the endothelium are mapped to different clusters of cells which each generate unique signal patterns. When there is more than one stimulus, cells communicate and combine inputs across information streams to generate new distinct signals. Our results establish the endothelium is a structured,collaborative, sensory network which simplifies the complex environment using separate cell clusters concerned with small distinct aspects of the overall information. These clusters interactively compute signals from the diverse but interrelated chemical inputs. These features permit the endothelium to selectively process separate signals and perform multiple computations in an environment that is noisy and variabl