High speed multiphoton imaging

Intravital multiphoton microscopy has emerged as a powerful technique to visualize cellular processes in-vivo. Real time processes revealed through live imaging provided many opportunities to capture cellular activities in living animals. The typical parameters that determine the performance of multiphoton microscopy are speed, field of view, 3D imaging and imaging depth; many of these are important to achieving data from in-vivo. Here, we provide a full exposition of the flexible polygon mirror based high speed laser scanning multiphoton imaging system, PCI-6110 card (National Instruments) and high speed analog frame grabber card (Matrox Solios eA/XA), which allows for rapid adjustments between frame rates i.e. 5 Hz to 50 Hz with 512 x 512 pixels. Furthermore, a motion correction algorithm is also used to mitigate motion artifacts. A customized control software called Pscan 1.0 is developed for the system. This is then followed by calibration of the imaging performance of the system and a series of quantitative in-vitro and in-vivo imaging in neuronal tissues and mice

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2‐fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub‐diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro‐vascular dynamics after laser‐induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological‐imaging capacity of any polygon scanning microscope system.Australian Research Council, Grant/Award Number: DE16010084

Raster adaptive optics for video rate aberration correction and large FOV multiphoton imaging

Removal of complex aberrations at millisecond time scales over millimeters in distance in multiphoton laser scanning microscopy limits the total spatiotemporal imaging throughput for deep tissue imaging. Using a single low resolution deformable mirror and time multiplexing (TM) adaptive optics, we demonstrate video rate aberration correction (5 ms update rate for a single wavefront mask) for a complex heterogeneous distribution of refractive index differences through a depth of up to 1.1 mm and an extended imaging FOV of up to 0.8 mm, with up to 167% recovery of fluorescence intensity 335 µm from the center of the FOV. The proposed approach, termed raster adaptive optics (RAO), integrates image-based aberration retrieval and video rate removal of arbitrarily defined regions of dominant, spatially varied wavefronts. The extended FOV was achieved by demonstrating rapid recovery of up to 50 distinct wavefront masks at 500 ms update rates that increased imaging throughput by 2.3-fold. Because RAO only requires a single deformable mirror with image-based aberration retrieval, it can be directly implemented on a standard laser scanning multiphoton microscope.Australian Research Council (CE140100011, DE160100843, DP190100039)

Volumetric adaptive optical multiphoton microscope for in vivo imaging and photomanipulation

Modern biological studies are mainly built upon observational sciences and thus relies heavily on imaging and manipulation tools. Light microscopy is one of the most widely recognized fundamental instrument in life science that can decipher biological processes over several length scales. Modern optical microscopes are combinations of laser engineering, optical design, software development and biochemical labelling. A well-orchestrated microscope should accumulate both high throughput information and also manipulate biological events of living cells within an animal in its native microenvironment. Multiphoton microscopy is widely used to study cellular and sub-cellular events ranging from immunological diseases model to neurological responses in living mice. A key success in the multiphoton microscope is the unprecedented imaging depth and imaging resolution from the nonlinear optical absorption. It is because of this nonlinear effect, fluorophores and specific proteins can be imaged and tracked over time. However, the inefficiencies of multiphoton microscopy during the delivering of a highly concentrated amount of photons over space and time within highly scattering tissue. In this thesis, I shall elaborate upon the design, development and application of a rapid volumetric scanning multiphoton microscope platform with adaptive optics capability for the intravital imaging and photomanipulation. The development of both the hardware and software was shown to a range of in vivo imaging experiments that especially focus on micro-vasculature imaging. A major milestone achievement is the experimental demonstration of video-rate adaptive optics capable of achieving 20 fps through raster aberration correction with time multiplexing. Overall, the final system achieves volumetric imaging (1~10 VPS) with an extendable imaging field of view (FOV) from 0.3 x 0.45 x 0.08 mm to 0.8 x 0.6 x 0.08 mm and a space bandwidth product (SBP- digital pixel density from 512 x 512 pixels ~ 10240 x 10240 pixels) in a single acquisition. These devices and functions are fully integrated into a customized control software named PScan, that provides not only an accessible operation interface for high throughput imaging but also offers photomanipulation capabilities. In the first chapter, I shall provide a general introduction and discussion of the development of fluorescence microscopy with the focus on multiphoton effect for deep tissue imaging and implementation of multiphoton microscopy. In the next three chapters, I shall describe our approach in designing and constructing the next generation of polygon based multiphoton system along with the control software PScan. Chapter 3 then illustrates our approach to achieve video-rate AO correction, named raster-scanning AO (RAO) that shows the capability of compensating for field-dependent aberration using time multiplexing with 10 ms update rate. Then, chapter 4 demonstrates the application of a piezo-deformable mirror to achieve volumetric imaging rates and a slit-based approach to achieve localized photomanipulation for fluorescence recovery after photobleaching (FRAP) and laser induced injury for in vivo thrombus studies. Finally, in chapter 5, I shall summaries the main features of the setup and also proposes several plans to further expanding the many functions of the microscope system for in vivo longitudinal photoconversion imaging. In summary, this thesis presents the detail of designing and building a state-of-art volumetric microscope that achieves multi-functionality imaging performance (speed, FOV, contrast, aberration-free) for in vivo studies of mice models. I anticipate that this volumetric microscope will be beneficial for a broader field of laser scanning microscopy for biologists and I believe that this thesis will impact various areas of biological sciences such as neurobiology, immunology and cancer research

PScan 1.0: flexible software framework for polygon based multiphoton microscopy

Multiphoton laser scanning microscopes exhibit highly localized nonlinear optical excitation and are powerful instruments for in-vivo deep tissue imaging. Customized multiphoton microscopy has a significantly superior performance for in-vivo imaging because of precise control over the scanning and detection system. To date, there have been several flexible software platforms catered to custom built microscopy systems i.e. ScanImage, HelioScan, MicroManager, that perform at imaging speeds of 30-100fps. In this paper, we describe a flexible software framework for high speed imaging systems capable of operating from 5 fps to 1600 fps. The software is based on the MATLAB image processing toolbox. It has the capability to communicate directly with a high performing imaging card (Matrox Solios eA/XA), thus retaining high speed acquisition. The program is also designed to communicate with LabVIEW and Fiji for instrument control and image processing. Pscan 1.0 can handle high imaging rates and contains sufficient flexibility for users to adapt to their high speed imaging systems. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only

The pro-competitive gains from trade with variable markup and multiple factors

The paper investigates the welfare gains from trade liberalization in the presence of multiple factors, multiple industries and variable markup. We extend Bernard, Redding and Schott (2007) to incorporate variable markup, using additively separable utility. We then characterize the equilibrium under costly trade and discuss the welfare implications of Heckscher-Ohlin mechanism, as well as the variable markup. Our main nding is that gains from trade liberalization are higher in models with variable markup than those with constant markup when the Heckscher-Ohlin force is strong enough. The larger the trade cost decreases, the stronger the Heckscher-Ohlin force is. We conclude that the pro-competitive e ect of trade liberalization does exist in the presence of Heckscher-Ohlin comparative advantage and variable markup.Bachelor of Art

Optical toolkits for in vivo deep tissue laser scanning microscopy: a primer

Life at the microscale is animated and multifaceted. The impact of dynamic in vivo microscopy in small animals has opened up opportunities to peer into a multitude of biological processes at the cellular scale in their native microenvironments. Laser scanning microscopy (LSM) coupled with targeted fluorescent proteins has become an indispensable tool to enable dynamic imaging in vivo at high temporal and spatial resolutions. In the last few decades, the technique has been translated from imaging cells in thin samples to mapping cells in the thick biological tissue of living organisms. Here, we sought to provide a concise overview of the design considerations of a LSM that enables cellular and subcellular imaging in deep tissue. Individual components under review include: long working distance microscope objectives, laser scanning technologies, adaptive optics devices, beam shaping technologies and photon detectors, with an emphasis on more recent advances. The review will conclude with the latest innovations in automated optical microscopy, which would impact tracking and quantification of heterogeneous populations of cells in vivo

Divergent Responses of Summer Precipitation in China to 1.5°C Global Warming in Transient and Stabilized Scenarios

International audienceResponses of summer precipitation in China to global 1.5°C warming with transient and stabilized pathways are investigated through the analysis of the Common Earth System Model climate simulations. Precipitation increases more significantly in southeast China in the stabilized scenario compared with the transient one. The difference is primarily attributable to dynamic factors related to circulation changes and driven by differences of land-ocean thermal contrast between the two scenarios. The stabilized warming favors conditions of larger ocean warming and smaller land warming, leads to enhanced meridional temperature gradient and ultimately speeds up the jet stream over East Asia. Ageostrophic wind at the entrance of the accelerated jet strengthens ascending motion and precipitation along the coast of Asia from southeast China to India. The enhanced heating over India further induces a Kelvin-wave response with an anomalous anticyclone that contributes to the enhanced westward shifted of the western North Pacific Subtropical High. Southerly winds at the west flank of the anticyclone transport moisture northward from tropical oceans, and strengthen precipitation in southeast China

Geological characteristics of the southern segment of the Late Sinian—Early Cambrian Deyang—Anyue rift trough in Sichuan Basin, SW China

Based on the latest drilling, seismic and field outcrop data, the geological characteristics (e.g. strata, development and sedimentary evolution) of the southern segment of the Late Sinian—Early Cambrian Deyang—Anyue rift trough in the Sichuan Basin are analyzed. First, the strata in the southern segment are complete. The first to second members of Dengying Formation (Deng 1 + Deng 2) are found with relatively stable thickness (400–550 m), and the third to fourth members (Deng 3 + Deng 4) show great thickness difference between the marginal trough and the inner trough, which is up to 250 m. The Cambrian Maidiping Formation and Qiongzhusi Formation in southern Sichuan Basin are relatively thin, with the thickness changing greatly and frequently. Second, the Deyang—Anyue rift trough extended southward during the Deng 4 period, affecting southern Sichuan Basin. Compared to the middle and northern segments of the rift trough, the southern segment is generally wide, gentle and shallow, with multiple steps, and alternating uplifts and sags, which are distributed in finger shape. Third, the Deng 1 + Deng 2 in southern Sichuan Basin records the dominance of carbonate platform and unobvious sedimentary differentiation, and the Deng 4 exhibits obvious sedimentary differentiation, namely, basin—slope—secondary slope—slope—secondary slope—platform margin—restricted platform, from the inner trough to the marginal trough. Fourth, the rift trough in southern Sichuan Basin has evolved in four stages: stabilization of Deng 1—Deng 2, initialization of Deng 3—Deng 4, filling of Maidiping—Qiongzhusi, and extinction of Canglangpu Formation