Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo.

Recording of the concerted activity of neuronal assemblies and the dendritic and axonal signal integration of downstream neurons pose different challenges, preferably a single recording system should perform both operations. We present a three-dimensional (3D), high-resolution, fast, acousto-optic two-photon microscope with random-access and continuous trajectory scanning modes reaching a cubic millimeter scan range (now over 950 × 950 × 3000 μm3) which can be adapted to imaging different spatial scales. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (>290 × 290 × 200 μm3) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSFx < 1.9 μm, PSFz < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. The system contains new design concepts: it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors, and it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures. We demonstrate the use of the microscope at different spatial scales by first showing 3D optical recordings of action potential back propagation and dendritic Ca2+ spike forward propagation in long dendritic segments in vitro, at near-microsecond temporal resolution. Second, using the same microscope we show volumetric random-access Ca2+ imaging of spontaneous and visual stimulation-evoked activity from hundreds of cortical neurons in the visual cortex in vivo. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest

A Compact Multiphoton 3D Imaging System for Recording Fast Neuronal Activity

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at http://neurospy.org/ under an open source license

Technologies for imaging neural activity in large volumes

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Collecting data from individual planes, conventional microscopy cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here, we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for the processing and analysis of volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics, and help elucidate how brain regions work in concert to support behavior

High-Throughput Nonlinear Optical Microscopy

High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field.National Institutes of Health (U.S.) (9P41EB015871-26A1)National Institutes of Health (U.S.) (R01-EX017656)National Institutes of Health (U.S.) (5 R01 NS051320)National Institutes of Health (U.S.) (4R44EB012415-02)National Science Foundation (U.S.) (CBET-0939511)Singapore-MIT AllianceSkolkovo Institute of Science and TechnologySingapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology)Wellcome Trust (London, England) (Massachusetts Institute of Technology. Postdoctoral Fellowship 093831/Z/10/Z

Application of precision engineering for nanometre focussing of hard X-rays in synchrotron beam lines

This thesis was submitted for the degree of Master of Philosophy and awarded by Brunel University.Many modern synchrotron beamlines are able to focus X-rays to a few microns in size. Although the technology to achieve this is well established, performing routine experiments with such beams is still time consuming and requires careful set up. Furthermore there is a need to be able to carry out experiments using hard X-ray beams with even smaller beams of between 100nm and 10nm. There are focussing optics that are able to do this but integrating these optics into a stable and a usable experimental set up are challenging. Experiments can often take some hours and any change in position of the beam on the sample will adversely affect the quality of the results. Experiments will often require scanning of the beam across the sample and so mechanisms suitable for high resolution but stable scanning are required. Performing routine experiments with nanometre sized beams requires mechanical systems to be able to position the sample, focussing optics, detectors and diagnostics with significantly higher levels of stability and motion resolution than is required from so called micro focus beam lines. This dissertation critically reviews precision engineering and associated technologies that are relevant for building nano focus beamlines, and the following key issues are explored: • Long term position stability due to thermal effects • Short term position stability due to vibration • Position motion with nanometre incremental motion • Results of some tests are presented and recommendations given. Some test results are presented and guidance on designing nano focus beamlines presented.Diamond Light Sourc

Scanning thermal microscopy using nanofabricated probes

Novel atomic force microscope (AFM) probes with integrated thin film thermal sensors are presented. Silicon micromachining and high resolution electron beam lithography (EBL) have been used to make batch fabricated, functionalised AFM probes. The AFM tips, situated at the ends of Si3N4 cantilevers, are shaped either as truncated pyramids or sharp triangular asperites. The former gives good thermalisation of the sensor to the specimen for flat specimens whereas the latter gives improved access to highly topographic specimens. Tip radii for the different probes are 1 m and 50 nm respectively. A variety of metal structures have been deposited on the tips using EBL and lift-off to form Au/Pd thermocouples and Pd resistance thermometer/heaters. Sensor dimensions down to 35 nm have been demonstrated. In the case of the sharp triangular tips, holes were etched into parts of the cantilever in order to provide self alignment of the sensor to the tip. On the pyramidal tips it has been shown that multiple sensors can be made on a single tip with good definition and matching between sensors. A conventional AFM was constructed in order to test the micromachined thermal probes. During scans of a photothermal test specimen using improved access thermocouple probes, 80 nm period metal gratings were thermally resolved. This is equivalent to a thermal lateral resolution of 40 nm. Pyramidal tips with a resistance thermometer/heater, which were made for the microscopy and analysis of polymers, have been showed by others to produce high resolution thermal conductivity images. The probes have also been shown to be capable of locally heating a polymer specimen and thermomechanically measuring phase changes in small volumes of material. Also presented here is a study of scanning thermal microscopy of semiconductor structures using a commercial AFM. Included are scans of several specimens using both commercial andthe new micromachined probes. Subsurface images of voids buried under a SiO2 passivation layer were taken. It is shown that contrast caused by thermal conductivity differences in the specimen may be detected at a depth of over 200 nm

Surface analysis of xGnP/PEI nanocomposite

Tato Diplomová práce se zabývá povrchovou analýzou nanokompozitní folie polyetherimidu (PEI) vyztuženého exfoliovanými grafitickými nanodestičkami (xGnP). Analyzovány byly take vzorky nevyztužené PEI folie a samostatné nanodestičky. Vzorky nanokompozitu a PEI folie byly plazmaticky leptány s využitím argonového plazmatu po dobu 1, 3 a 10 hod. Skenovací elektronová mikroskopie (SEM) byla použita pro charakterizaci samostatných nanodestiček rozptýlených na křemíkovém substrátu, původních či leptaných vzorků PEI folie a nanokompozitu. Nanodestičky byly identifikovány při povrchu leptané nanokompozitní folie. Mikroskopie atomárních sil (AFM) byla použita pro zobrazení povrchové topografie separovaných nanodestiček a odkrytých destiček při povrchu leptaného kompozitu. Povrchová drsnost (střední kvadratická hodnota, vzdálenost nejnižšího a nejvyššího bodu) leptaného nanokompozitu narůstala s prodlužující se dobou leptání. Akustická mikroskopie atomárních sil (AFAM) byla použita pro charakterizaci elastické anizotropie leptaných kompozitních vzorků. Nanoindentační měření umožnila charakterizaci lokálních mechanických vlastností PEI a nanokompozitních folií.This Diploma thesis deals with surface analysis of nanocomposite foil – polyetherimide matrix (PEI) reinforced by exfoliated graphite nanoplatelets (xGnP). The PEI foil without reinforcement and separate xGnP particles were also analysed. Samples of the nanocomposite and the PEI foil were etched for various times by argon plasma. Scanning electron microscopy (SEM) was used to characterize xGnP agglomerates dispersed over silicon wafer and pristine/etched samples of PEI foil and nanocomposite xGnP/PEI foil. Graphite nanoplatelets were identified at surface of etched nanocomposite foil. Atomic force microscopy (AFM) was used for surface topography imaging of separate nanoplatelets and those uncovered at the surface of etched nanocomposite. Surface roughness (root mean square, peak to peak) of etched nanocomposite increased with prolonged etching time. Atomic force acoustic microscopy (AFAM) was used to characterize elastic anisotropy of etched nanocomposite. Nanoindentation measurements were employed to characterize the local mechanical properties of PEI and nanocomposite foils.

The development of a high speed 3D 2-photon microscope for neuroscience

The progress of neuroscience is limited by the instrumentation available to it for studying the brain. At present, there is a serious instrumentation gap between functional Magnetic Resonance Imaging (fMRI) of whole brains and the microscopic scale functional imaging possible with today’s optical microscopes and electrophysiology techniques, such as patch clamping of individual neurons. This thesis describes the development of a new extension to optical microscopy that enables refocusing within 25 microseconds rather than the large fraction of a second possible by moving the sample or objective. The system is capable of refocusing a laser beam that is monitoring activity in 3D samples of live brain tissue 300 times faster than previously possible. This will make practical a new type of optical functional imaging for studying small sub-networks of neurons containing up to about 30,000 neurons at up to 30,000 sub micrometre sized monitored points of interest per second. The thesis describes the development of a detailed design for a new type of 3D scanner that uses Acousto-Optic Deflectors (AODs) to diffractively deflect and focus an intense laser beam beneath a conventional microscope objective. The fluorescence of calcium sensitive dyes in live neurons is used to monitor action potentials conveying signals between neurons. The optical and systems engineering problems and design trade-offs involved are discussed in detail. The results of extensive computer modelling are described and innovative solutions to several key optical physics based engineering problems are explained. The practical problems found in building a prototype machine incorporating these innovations are described and the encouraging first operational results from the machine reported

THE DEVELOPMENT OF A NOVEL ELECTRO-MAGNETIC FORCE MICROSCOPE

This thesis describes the development of a new type of Magnetic Force Microscope (MFM) probe based on a unique electromagnetic design. In addition the design, construction and testing of a new MFM system, complete in both hardware and software, is also described. The MFM allowed initial tests on prototypes of the new probe, and is to provide a base for future new probe integration. The microscope uses standard MFM micro-cantilever probes in static modes of imaging. A new computer hosted DSP control system, software, and its various interfaces with the MFM have been integrated into the system. The system has been tested using standard probes with various specimens and satisfactory results have been produced. A novel probe has been designed to replace the standard MFM magnetic coated tip with a field generated about a sub-micron aperture in a conducting film. The field from the new probe is modelled and its imaging capability investigated, with iterative designs analysed in this way. The practical construction and potential problems therein, of the probe are also considered. Test apertures have been manufactured, and an image of the field produced when operating is provided as support to the theoretical designs. Future methods of using the new probe are also discussed, including the examination of the probe as a magnetic write mechanism. This probe, integrated into the MFM, can provide a new method of microscopic magnetic imaging, and in addition opens a new potential method of magnetic storage that will require further research