Development of 3D super-resolution tomographic STED microscopy and its application to studies on bone resorption

In this doctoral thesis, a tomographic STED microscopy technique for 3D super-resolution imaging was developed and utilized to observebone remodeling processes. To improve upon existing methods, wehave used a tomographic approach using a commercially available stimulated emission depletion (STED) microscope. A certain region of interest (ROI) was observed at two oblique angles: one at a standard inverted configuration from below (bottom view) and another from the side (side view) via a micro-mirror positioned close to the ROI. The two viewing angles were reconstructed into a final tomogram. The technique, named as tomographic STED microscopy, was able to achieve an axial resolution of approximately 70 nm on microtubule structures in a fixed biological specimen. High resolution imaging of osteoclasts (OCs) that are actively resorbing bone was achieved by creating an optically transparent coating on a microscope coverglass that imitates a fractured bone surface. 2D super-resolution STED microscopy on the bone layer showed approximately 60 nm of lateral resolution on a resorption associated organelle allowing these structures to be imaged with super-resolution microscopy for the first time. The developed tomographic STED microscopy technique was further applied to study resorption mechanisms of OCs cultured on the bone coating. The technique revealed actin cytoskeleton with specific structures, comet-tails, some of which were facing upwards and some others were facing downwards. This, in our opinion, indicated that during bone resorption, an involvement of the actin cytoskeleton in vesicular exocytosis and endocytosis is present. The application of tomographic STED microscopy in bone biology demonstrated that 3D super-resolution techniques can provide new insights into biological 3D nano-structures that are beyond the diffraction-limit when the optical constraints of super-resolution imaging are carefully taken into account.Tomografisen STED superresoluutio mikroskooppitekniikan kehittäminen, sekä sen sovellus luubiologian tutkimuksessa Väitöskirja käsittelee STED super-resoluutio kerroskuvausmikroskooppitekniikan kehittämistä, sekä sen sovellusta luuston uusiutumisprosessin havainnoinnissa. Tekniikka kehitettiin kaupallista STED mikroskooppia hyödyntäen. Kerroskuvausmenetelmä perustuu näytteen kuvaamiseen kahdesta eri suunnasta, alta sekä sivusta; sivunäkymä muodostettiin mikroskooppinäytteen päälle, kuvattavan kohteen yläpuolelle vinosti asetetun pienen peilin kautta, jonka jälkeen projektiot yhdistetään laskennallisesti yhdeksi kerroskuvaksi. STED kerroskuvaustekniikka mahdollisti solun sisäisten mikroputkirakenteiden havainnoinnin 70nm pitkittäisresoluutiolla. Pystyäksemme kuvaamaan luuta aktiivisesti resorpoivia osteoklastisoluja korkealla optisella erottelukyvyllä, kehitimme mikroskooppilasille muodostettavan, optisesti läpinäkyvän pinnoitteen, joka imitoi murtunutta luupintaa. Pinnoite mahdollisti osteoklastisolujen luun resorptioon liittyvien soluelinten kuvantamisen 2D STED mikroskooppia käyttäen 60 nm lateraaliresoluutiolla; tämä on ensimmäinen kerta kun kyseisiä solurakenteita on pystytty kuvaamaan super-resoluutio mikroskoopilla. Käytimme kehitettyä tomografista STED tekniikkaa osteoklastisolujen resorptiomekanismin kuvantamisessa, uutta läpinäkyvää luupinnoitetta hyväksikäyttäen. Tomografiatekniikalla muodostetuista kuvista voidaan nähdä aktiini tukirakenteiden spesifisiä rakenteita, jotka meidän mielestämme viittaavat niiden osallistumiseen vesikkeleiden endo- ja eksosytoosiin luun resorptioprosessin aikana. Tomografinen STED mikroskopia, yhdistettynä uuteen luupinnoitteeseen avaa uusia mahdollisuuksia kolmiulotteisten nanorakenteiden havainnointiin, tutkittaessa luuston uusiutumisprosessia.Siirretty Doriast

Observations of SiO Maser Sources within a Few Parsec from the Galactic Center

Mapping and monitoring observations of SiO maser sources near the Galactic center were made with the Nobeyama 45-m telescope at 43 GHz. Rectangular mapping an area of approximately 200'' x 100''$ in a 30'' grid, and triangular mapping in a 20'' grid toward the Galactic center, resulted in 15 detections of SiO sources; the positions of the sources were obtained with errors of 5--10'', except for a few weak sources. Three-year monitoring observations found that the component at V_lsr=-27 km/s of IRS 10 EE flared to about 1.5 Jy during 2000 March--May, which was a factor of more than 5 brighter than its normal intensity. Using the radial velocities and positions of the SiO sources, we identified 5 which are counterparts of previously observed OH 1612 MHz sources. The other 10 SiO sources have no OH counterparts, but two were previously detected with VLA, and four are located close to the positions of large-amplitude variables observed at near-infrared wavelengths. A least-squares fit to a plot of velocities versus Galactic longitudes gives a rather high speed for the rotation of the star cluster around the Galactic center. The observed radial-velocity dispersion is roughly consistent with a value obtained before. It was found that all of the SiO sources with OH 1612 MHz counterparts have periods of light variation longer than 450 days, while SiO sources without OH masers often have periods shorter than 450 days. This fact suggests that lower-mass AGB stars are more often detected in SiO masers than in the OH 1612 MHz line.Comment: 9 pages, 7 figures. PASJ 54, No. 1 (2002) in pres

Industrial Applications of Tunable Diode Laser Absorption Spectroscopy

Tunable diode laser absorption spectroscopy (TDLAS) utilizes the absorption phenomena to measure the temperature and species concentration. The main features of the TDLAS technique are its fast response and high sensitivity. Extensive research has been performed on the utilization of diode laser absorption spectroscopy for the system monitoring and its control. The TDLAS technique gives self-calibrations to reduce the noise such as particles and dusts because the laser wavelength is rapidly modulated at kHz rates. In addition, two dimensional (2D) temperature and concentration distributions can be obtained by combining computed tomography (CT) with TDLAS. The TDLAS applications have been extensively studied with great progress. This chapter largely focuses on the engineering fields, especially the practical industrial applications

Better than a lens -- Increasing the signal-to-noise ratio through pupil splitting

Lenses are designed to fulfill Fermats principle such that all light interferes constructively in its focus, guaranteeing its maximum concentration. It can be shown that imaging via an unmodified full pupil yields the maximum transfer strength for all spatial frequencies transferable by the system. Seemingly also the signal-to-noise ratio (SNR) is optimal. The achievable SNR at a given photon budget is critical especially if that budget is strictly limited as in the case of fluorescence microscopy. In this work we propose a general method which achieves a better SNR for high spatial frequency information of an optical imaging system, without the need to capture more photons. This is achieved by splitting the pupil of an incoherent imaging system such that two (or more) sub-images are simultaneously acquired and computationally recombined. We compare the theoretical performance of split pupil imaging to the non-split scenario and implement the splitting using a tilted elliptical mirror placed at the back-focal-plane (BFP) of a fluorescence widefield microscope

Simultaneous two cross-sectional measurements of NH3 concentration in bent pipe flow using CT-tunable diode laser absorption spectroscopy

Urea Selective Catalytic Reduction (urea SCR) system is widely used for diesel engine to reduce the emission of NOx by NH3 which is provided by a hydrolysis of urea water. Concentration distribution of NH3 in an exhaust pipe is an important factor for improvement of the SCR efficiency and prevention of NH3 slip and urea deposit. Therefore, it is necessary to measure two-dimensional (2D) concentration of NH3 in detail. The purpose of this study is to develop the real-time two cross-sectional measurements technology of NH3 concentration using the computed tomography-tunable diode laser absorption spectroscopy (CT-TDLAS). Theoretical NH3 concentration distribution which was reconstructed by CT agreed to CFD results and quadruple pipe’s results showed good resolution by 14th order reconstruction. Therefore, this method has enough resolution and accuracy for measuring the concentration distribution of NH3. And this method was employed in a bent pipe model demonstrated a urea SCR system. The experimental results of two cross-sectional 2D concentration of NH3 show differences of the concentration distribution of NH3 each cross-section and flow pattern like swirl flow. It was found that CT-TDLAS was an effective method to measure concentration distribution of NH3 and observe characteristics of flow. In addition, observing flow pattern enable to validate CFD results, and it helps to improve efficiency of after treatment system

MONITORING OF THE CHANGE IN COASTAL ENVIRONMENT IN SOUTHERN PART OF RED-RIVER DELTA FROM SATELLITE IMAGES AND THE MECHANISM OF BEACH EROSION

Joint Research on Environmental Science and Technology for the Eart

Feasibility of Controlling Gas Concentration and Temperature Distributions in a Semiconductor Chamber with CT-TDLAS

The feasibility to control the gas concentration and temperature distributions in a semiconductor process chamber by measuring them was investigated. Gas concentration and temperature distributions for various flow rates were measured with the computed tomography-tunable diode laser absorption spectroscopy (CT-TDLAS). The infrared absorption spectra of multiple laser paths passing through the measured area were collected and the distributions of methane concentration and temperature in the chamber were reconstructed with the computed tomography (CT) calculations. The measured results indicated that the distributions can be independently controlled by measuring with the CT-TDLAS and adjusting the flow rates and the susceptor temperature

Validation of real-time 2D temperature measurement method using CT tunable diode laser absorption spectroscopy

Two dimensional (2D) temperature and concentration distribution plays an important role for the combustion structure and the combustor efficiency in engines, burners, gas turbines and so on. Recently, as a multi-species measurement technique with high sensitivity and high response, tunable diode laser spectroscopy (TDLAS) has been developed and applied to the actual engine combustions. With these engineering developments, transient phenomena such as start-ups and load changes in engines have been gradually elucidated in various conditions. In this study, the theoretical and experimental research has been conducted in order to develop the non-contact and fast response 2D temperature and concentration distribution measurement method. The method is based on a computed tomography (CT) using absorption spectra of water vapor at 1388 nm. The computed tomography tunable diode laser spectroscopy (CT-TDLAS) method was employed in engine exhausts to measure 2D temperature distribution. The measured 2D temperature shows a good agreement with the temperature measured by a thermocouple. The temporal and spatial resolutions of this method have also been discussed to demonstrate its applicability to various types of combustor

Pulverized coal combustion application of laser-based temperature sensing system using computed tomography : Tunable diode laser absorption spectroscopy (CT-TDLAS)

The investigation of combustion phenomena in pulverized coal flames is significant for combustion optimization related to energy conservation and emission reduction. Real-time two dimensional (2D) temperature and concentration distributions play an important role for combustion analysis. The non-contact and fast response 2D temperature and concentration distribution measurement method was developed in this study. The method is based on a combination of computed tomography (CT) and tunable diode laser absorption spectroscopy (TDLAS). The accuracy evaluation of developed 32-path CT-TDLAS demonstrated its feasibility of 2D temperature measurement. 32-path CT-TDLAS was applied to CH4 and 5 kg/h coal combustion fields for 2D temperature measurement. The time-series 2D temperature distribution in coal combustion furnace was measured using 32-path CT-TDLAS measurement cell with kHz time resolution. The transient temperature field of combustion flame directly reflects the combustion mode and combustion stability. The measurement results demonstrate its applicability of CT-TDLAS to various types of combustor, especially the combustion fields with coal and ash particles. CT-TDLAS method with kHz response time enables the real-time 2D temperature measurement to be applicable for combustion analysis