Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

Density functional theory embedding for correlated wavefunctions

Methods that exploit the intrinsic locality of molecular interactions show significant promise in making tractable the electronic structure calculation of large-scale systems. In particular, embedded density functional theory (e-DFT) offers a formally exact approach to electronic structure calculations in which the interactions between subsystems are evaluated in terms of their electronic density. In the following dissertation, methodological advances of embedded density functional theory are described, numerically tested, and applied to real chemical systems.

First, we describe an e-DFT protocol in which the non-additive kinetic energy component of the embedding potential is treated exactly. Then, we present a general implementation of the exact calculation of the non-additive kinetic potential (NAKP) and apply it to molecular systems. We demonstrate that the implementation using the exact NAKP is in excellent agreement with reference Kohn-Sham calculations, whereas the approximate functionals lead to qualitative failures in the calculated energies and equilibrium structures.

Next, we introduce density-embedding techniques to enable the accurate and stable calculation of correlated wavefunction (CW) in complex environments. Embedding potentials calculated using e-DFT introduce the effect of the environment on a subsystem for CW calculations (WFT-in-DFT). We demonstrate that WFT-in-DFT calculations are in good agreement with CW calculations performed on the full complex.

We significantly improve the numerics of the algorithm by enforcing orthogonality between subsystems by introduction of a projection operator. Utilizing the projection-based embedding scheme, we rigorously analyze the sources of error in quantum embedding calculations in which an active subsystem is treated using CWs, and the remainder using density functional theory. We show that the embedding potential felt by the electrons in the active subsystem makes only a small contribution to the error of the method, whereas the error in the nonadditive exchange-correlation energy dominates. We develop an algorithm which corrects this term and demonstrate the accuracy of this corrected embedding scheme.

Ghost imaging with thermal light by third-order correlation

Ghost imaging with classical incoherent light by third-order correlation is investigated. We discuss the similarities and the differences between ghost imaging by third-order correlation and by second-order correlation, and analyze the effect from each correlation part of the third-order correlation function on the imaging process. It is shown that the third-order correlated imaging includes richer correlated imaging effects than the second-order correlated one, while the imaging information originates mainly from the correlation of the intensity fluctuations between the test detector and each reference detector, as does ghost imaging by second-order correlation.

Spontaneous and stimulated light emission due to radiative recombination in forward biased lead telluride P-N junctions

Since the discovery in 1962 of laser action in semiconductor diodes made from GaAs, the study of spontaneous and stimulated light emission from semiconductors has become an exciting new field of semiconductor physics and quantum electronics combined. Included in the limited number of direct-gap semiconductor materials suitable for laser action are the members of the lead salt family, i.e . PbS, PbSe and PbTe. The material used for the experiments described herein is PbTe . The semiconductor PbTe is a narrow band- gap material (E_g = 0.19 electron volt at a temperature of 4.2°K). Therefore, the radiative recombination of electron-hole pairs between the conduction and valence bands produces photons whose wavelength is in the infrared (λ ≈ 6.5 microns in air).

The p-n junction diode is a convenient device in which the spontaneous and stimulated emission of light can be achieved via current flow in the forward-bias direction. Consequently, the experimental devices consist of a group of PbTe p-n junction diodes made from p –type single crystal bulk material. The p - n junctions were formed by an n-type vapor- phase diffusion perpendicular to the (100) plane, with a junction depth of approximately 75 microns. Opposite ends of the diode structure were cleaved to give parallel reflectors, thereby forming the Fabry-Perot cavity needed for a laser oscillator. Since the emission of light originates from the recombination of injected current carriers, the nature of the radiation depends on the injection mechanism.

The total intensity of the light emitted from the PbTe diodes was observed over a current range of three to four orders of magnitude. At the low current levels, the light intensity data were correlated with data obtained on the electrical characteristics of the diodes. In the low current region (region A), the light intensity, current-voltage and capacitance-voltage data are consistent with the model for photon-assisted tunneling. As the current is increased, the light intensity data indicate the occurrence of a change in the current injection mechanism from photon-assisted tunneling (region A) to thermionic emission (region B). With the further increase of the injection level, the photon-field due to light emission in the diode builds up to the point where stimulated emission (oscillation) occurs. The threshold current at which oscillation begins marks the beginning of a region (region C) where the total light intensity increases very rapidly with the increase in current. This rapid increase in intensity is accompanied by an increase in the number of narrow-band oscillating modes. As the photon density in the cavity continues to increase with the injection level, the intensity gradually enters a region of linear dependence on current (region D), i.e. a region of constant (differential) quantum efficiency.

Data obtained from measurements of the stimulated-mode light-intensity profile and the far-field diffraction pattern (both in the direction perpendicular to the junction-plane) indicate that the active region of high gain (i.e. the region where a population inversion exists) extends to approximately a diffusion length on both sides of the junction. The data also indicate that the confinement of the oscillating modes within the diode cavity is due to a variation in the real part of the dielectric constant, caused by the gain in the medium. A value of τ ≈ 10^-9 second for the minority- carrier recombination lifetime (at a diode temperature of 20.4°K) is obtained from the above measurements. This value for τ is consistent with other data obtained independently for PbTe crystals.

Data on the threshold current for stimulated emission (for a diode temperature of 20. 4°K) as a function of the reciprocal cavity length were obtained. These data yield a value of J’_th = (400 ± 80) amp/cm² for the threshold current in the limit of an infinitely long diode-cavity. A value of α = (30 ± 15) cm^-1 is obtained for the total (bulk) cavity loss constant, in general agreement with independent measurements of free- carrier absorption in PbTe. In addition, the data provide a value of n_s ≈ 10% for the internal spontaneous quantum efficiency. The above value for n_s yields values of t_b ≈ τ ≈ 10^-9 second and t_s ≈ 10^-8 second for the nonradiative and the spontaneous (radiative) lifetimes, respectively.

The external quantum efficiency (n_d) for stimulated emission from diode J-2 (at 20.4° K) was calculated by using the total light intensity vs. diode current data, plus accepted values for the material parameters of the mercury- doped germanium detector used for the measurements. The resulting value is n_d ≈ 10%-20% for emission from both ends of the cavity. The corresponding radiative power output (at λ = 6.5 micron) is 120-240 milliwatts for a diode current of 6 amps.

20-W average power, high repetition rate, nanosecond pulse with diffraction limit from an all-fiber MOPA system

In this article, we report an all-fiber master oscillator power amplifier (MOPA) system, which can provide high repetition rate and nanosecond pulse with diffraction-limit. The system was constructed using a (2 + 1) X 1 multimode combiner. The Q-Switched, LD pumped Nd:YVO4 solid-state laser wets used (is master oscillator. The 976-nm fiber-coupled module is used as pump source. A 10-m long China-made Yb3+-doped D-shape double-clad large-mode-area fiber was used as amplifier fiber. The MOPA produced as much as 20-W average power with nanosecond pulse and near diffraction limited. The pulse duration is maintained at about 15 its during 50-175 kHz. The system employs a simple and compact architecture and is therefore suitable for the use in practical applications such as scientific and military airborne LIDAR and imaging. Based oil this system. the amplification performances of. the all fiber amplifier is investigated. (C) 2008 Wiley Periodicals, Inc.

Quantum ricochets: surface capture, release and energy loss of fast ions hitting a polar surface at grazing incidence

A diffraction mechanism is proposed for the capture, multiple bouncing and final escape of a fast ion (keV) impinging on the surface of a polarizable material at grazing incidence. Capture and escape are effected by elastic quantum diffraction consisting of the exchange of a parallel surface wave vector G= 2p/ a between the ion parallel momentum and the surface periodic potential of period a. Diffraction- assisted capture becomes possible for glancing angles F smaller than a critical value given by Fc 2- 2./ a-| Vim|/ E, where E is the kinetic energy of the ion,. = h/ Mv its de Broglie wavelength and Vim its average electronic image potential at the distance from the surface where diffraction takes place. For F< Fc, the ion can fall into a selected capture state in the quasi- continuous spectrum of its image potential and execute one or several ricochets before being released by the time reversed diffraction process. The capture, ricochet and escape are accompanied by a large, periodic energy loss of several tens of eV in the forward motion caused by the coherent emission of a giant number of quanta h. of Fuchs- Kliewer surface phonons characteristic of the polar material. An analytical calculation of the energy loss spectrum, based on the proposed diffraction process and using a model ion-phonon coupling developed earlier (Lucas et al 2013 J. Phys.: Condens. Matter 25 355009), is presented, which fully explains the experimental spectrum of Villette et al (2000 Phys. Rev. Lett. 85 3137) for Ne+ ions ricocheting on a LiF(001) surface.

Flame imaging of gas-turbine relight

High-altitude relight inside a lean-direct-injection gas-turbine combustor is investigated experimentally by highspeed imaging. Realistic operating conditions are simulated in a ground-based test facility, with two conditions being studied: one inside and one outside the combustor ignition loop. The motion of hot gases during the early stages of relight is recorded using a high-speed camera. An algorithm is developed to track the flame movement and breakup, revealing important characteristics of the flame development process, including stabilization timescales, spatial trajectories, and typical velocities of hot gas motion. Although the observed patterns of ignition failure are in broad agreement with results from laboratory-scale studies, other aspects of relight behavior are not reproduced in laboratory experiments employing simplified flow geometries and operating conditions. For example, when the spark discharge occurs, the air velocity below the igniter in a real combustor is much less strongly correlated to ignition outcome than laboratory studies would suggest. Nevertheless, later flame development and stabilization are largely controlled by the cold flowfield, implying that the location of the igniter may, in the first instance, be selected based on the combustor cold flow. Copyright © 2010.

Fast, sensitive and low-noise nanowire and graphene field effect transistors for room-temperature detection of Terahertz quantum cascade laser emission

Antenna-coupled field effect transistors have been developed as plasma-wave THz detectors in both InAs nanowire and graphene channel materials. Room temperature operation has been achieved up to 3 THz, with noise equivalent power levels < 10-10 W/Hz1/2, and high-speed response already suitable for large area THz imaging applications. © 2013 IEEE.

An experimental study about the influence of well thickness on the electroluminescence of InGaN/GaN multiple quantum wells

The influence of well thickness on the electroluminescence (EL) of InGaN/GaN multiple quantum wells (MQWs) grown by metalorganic chemical vapor deposition is investigated. It is found that the peak wavelength of EL increases with the increase of well thickness when the latter is located in the range of 3.0-5.1 nm. The redshift is mainly attributed to the quantum confined Stark effect (QCSE). As a contrast, it is found that the EL intensity of InGaN/GaN MQWs increases with the increase of well thickness in spite of QCSE. The result of X-ray diffraction demonstrates that the interface become smoother with the increase of well thickness and suggests that the reduced interface roughness can be an important factor leading to the increase of EL intensity of InGaN/GaN MQWs. (C) 2009 Elsevier B.V. All rights reserved.

Antibunching and blinking from a single colloidal CdSe quantum dot

We have investigated the optical properties of single CdSe/ZnS nanocrystals by conducting combinations of experiments on antibunching and photoluminescence intermittence under different experimental conditions. Based on photoluminescence in an antibunching experiment, we analyzed the emission lifetime of QDs by using stretched exponentials. The difference between the parameters obtained from average lifetimes and stretched exponents were analyzed by considering the effect of nonradiative emission. An Auger-assisted tunneling model was used to explain the power law exponents of off time distribution. The power law exponent under high excitation power was correlated with a higher Auger ionization rate. Using the parameters obtained from stretched exponential function and power law, the antibunching phenomena at different time and under different excitation intensity were analyzed.

Effect of Interface Roughness and Dislocation Density on Electroluminescence Intensity of InGaN Multiple Quantum Wells

Effects of interface roughness and dislocation density on the electroluminescence (EL) intensity of InGaN multiple quantum wells (MQWs) are investigated. It is found that the EL intensity increases with the number of satellite peaks in the x-ray diffraction experiments of InGaN MQW samples. It is indicated that the rough interface will lead the reduction of EL intensity of InGaN MQW samples. It is also found that the EL intensity increases with the decrease of dislocation density which is characterized by the x-ray diffraction measurements. It is suggested that the EL intensity of InGaN MQWs can be improved by decreasing the interface roughness and dislocation density.

Fabrication and excitation-power-density-dependent micro-photoluminescence of hexagonal nanopillars with a single InGaAs/GaAs quantum well

Hexagonal nanopillars with a single InGaAs/GaAs quantum well (QW) were fabricated on a GaAs (111) B substrate by selective-area metal-organic vapor phase epitaxy. The standard deviations in diameter and height of the nanopillars are about 2% and 5%, respectively. Zincblende structure and rotation twins were identified in both the GaAs and the InGaAs layers by electron diffraction. The excitation-power-density-dependent micro-photoluminescence (mu-PL) of the nanopillars was measured at 4.2, 50, 100 and 150 K. It was shown that, with increasing excitation power density, the mu-PL peak's positions shift to a higher energy, and their intensity and width increase, which were rationalized using a model that includes the effects of piezoelectricity, photon-screening and band-filling. It was also revealed that the rotation twins significantly reduce the diffusion length of the carriers in the nanopillars, compared to that in the regular semiconductors.

Solid source MBE growth of quantum cascade lasers

High material quality is the basis of quantum cascade lasers (QCLs). Here we report the solid source molecular beam epitaxy (MBE) growth details of realizing high quality of InGaAs/InAlAs QCL structures. Accurate control of material compositions, layer thickness, doping profile, and interface smoothness can be realized by optimizing the growth conditions. Double crystal x-ray diffraction discloses that our grown QCL structures possess excellent periodicity and sharp interfaces. High quality laser wafers are grown in a single epitaxial run. Room temperature continuous-wave (cw) operation of QCLs is demonstrated.

Generation and behavior of pure-edge threading misfit dislocations in InxGa1-xN/GaN multiple quantum wells

The relaxation of the misfit strain by the formation of misfit dislocations in InxGa1-xN/GaN multiple quantum wells grown by metal-organic chemical-vapor deposition was investigated by the cross-sectional transmission electron microscopy, double crystal x-ray diffraction, and temperature-dependent photoluminescence. It is found that the misfit dislocations generated from strain relaxation are all pure-edge threading dislocations with burgers vectors of b=1/3<11 (2) over bar0>. The misfit dislocations arise from the strain relaxation due to the thickness of strained layer greater than the critical thickness. The relaxation of strained layer was mainly achieved by the formation of dislocations and localization of In, while the dislocations changed their slip planes from {0001} to {10 (1) over bar0}. With the increasing temperature, the efficiency of photoluminescence decrease sharply. It indicates that the relaxation of the misfit strain has a strong effect on optical efficiency of film. (C) 2004 American Institute of Physics.

Characterization of InAs quantum dots on lattice-matched InAlGaAs/InP superlattice structures

Self-assembled InAs quantum dots (QDs) in an InAlGaAs matrix, lattice-matched to InP substrate, have been grown by molecular beam epitaxy (MBE). Transmission electron microscopy (TEM), double-crystal X-ray diffraction (DCXRD) and photoluminescence (PL) are used to study their structural and optical properties. In InAs/InAlGaAs/ InP system, we propose that when the thickness of InAs layer deposited is small, the random strain distribution of the matrix layer results in the formation of tadpole-shaped QDs with tails towards random directions, while the QDs begin to turn into dome-shaped and then coalesce to form islands with larger size and lower density to release the increasing misfit strain with the continuous deposition of InAs. XRD rocking curves showing the reduced strain with increasing thickness of InAs layer may also support our notion. The results of PL measurements are in well agreement with that of TEM images. (C) 2004 Elsevier B.V. All rights reserved.