Transfection and imaging of diamond nanocrystals as scattering optical labels

We report on the first demonstration of nanodiamond (ND) as a scattering optical label in a biological environment. NDs were efficiently transfected into cells using cationic liposomes, and imaged using differential interference and Hoffman modulation ‘space’ contrast microscopy techniques. We have shown that 55 nm NDs are biologically inert and produce a bright signal compared to the cell background. ND as a scattering label presents the possibility for extended biological imaging with relatively little thermal or biochemical perturbations due to the optical transparency and biologically inert nature of diamond.     

Spatially resolved Raman spectroscopy evaluation of residual stresses in 3C-SiC layer deposited on Si substrates with different crystallographic orientations

Raman scattering studies were performed on hot-wall chemical vapor deposited (heteroepitaxial) silicon carbide (SiC) films grown on Si substrates with orientations of (1 0 0), (1 1 1), (1 1 0) and (2 1 1), respectively. Raman spectra suggested that good quality cubic SiC single crystals could be obtained on the Si substrate, independent of its crystallographic orientation. Average residual stresses in the epitaxially grown 3C-SiC films were measured with the laser waist focused on the epilayer surface. Tensile and compressive residual stresses were found to be stored within the SiC film and in the Si substrate, respectively. The residual stress exhibited a marked dependence on the orientation of the substrate. The measured stresses were comparable to the thermal stress deduced from elastic deformation theory, which demonstrates that the large lattice mismatch between cubic SiC and Si is effectively relieved by initial carbonization. The confocal configuration of the optical probe enabled a stress evaluation along the cross-section of the sample, which showed maximum tensile stress magnitude at the SiC/Si interface from the SiC side, decreasing away from the interface in varied rate for different crystallographic orientations. Defocusing experiments were used to precisely characterize the geometry of the laser probe in 3C-SiC single crystal. Based on this knowledge, a theoretical convolution of the in-depth stress distribution could be obtained, which showed a satisfactory agreement with stress values obtained by experiments performed on the 3C-SiC surface.     

Age-induced phase transitions on mechanically alloyed amorphous GaSe

After aging it for four years at room temperature, a mechanically alloyed amorphous GaSe powder was transformed to a multi-phase crystalline alloy, where major phase is the trigonal Se one. The structural, thermal and optical properties of this aged amorphous GaSe were investigated through systematic X-ray diffraction, differential scanning calorimetry and Raman scattering measurements. The X-ray diffraction results on the aged GaSe powder suggest the presence of oxides, and X-ray absorption spectroscopy was employed to further investigate it.     

Porous silicon as efficient surface enhanced Raman scattering (SERS) substrate

Silver nanocrystallites are obtained through immersion of porous silicon samples in AgNO₃ solutions and a successive thermal annealing. The efficiency of nanostructures as surface enhanced Raman scattering (SERS) substrates is checked on cyanine-based dyes and horseradish peroxidase, evidencing detectable concentrations as low as 10⁻⁷ to 10⁻⁸ M. The substrate efficiency is strictly related to the Ag particle morphology, which could yield to either local surface plasmons (LSP) coupled to individual particles or to inter-particle short-range interaction.     

The interpretation of the LII signal in optically dense combusting sprays

A numerical investigation was made of the generation and behaviour of the LII signal in optically dense combusting sprays at conditions similar to those in the combustion chamber of compression ignition engines and gas turbines. The influence of particle size, particle morphology and size distribution on the behaviour of the LII signal, and the scattering and absorption of light, and the consequences that different calibration procedures have on the accuracy of the results were studied. Results show that, as the particle size or aggregation increases, light extinction is not caused only by absorption but also by scattering, which contributes more than 10% to the total extinction of light. Particle shape effects are important, irrespective of particle size. The form, soot concentration gradients and optical thickness of the flame cause an uneven laser fluence across the measuring volume that affects the generation of the LII signal. In addition, the quotient between the transmitted and incoming laser pulses across the flame borders can be as small as a percentage of unity. The interpretation of the induced signal is further challenged by the loss of signal between the measuring volume and the detection arrangement, thus causing the detection of spectrally distorted and weaker signals with an erroneous profile of the local amount of carbonaceous particles. An appropriate calibration procedure must be followed to obtain results that are quantitatively representative. External calibration was found to be inappropriate for these systems since it can lead one to underestimate the local volume fraction for almost two orders of magnitude. Implementing an in situ calibration along a line can lead to underestimate or overestimate the local mean volume fraction by a factor of two. However, the use of an in situ calibration procedure using a laser sheet that propagates through the complete measuring volume can reduce the error in estimating the mean soot volume fraction to a 30%. The latter was found to be the most adequate among the studied calibration routines.     

Optical properties of fullerene and non-fullerene peapods

Single-wall carbon nanotubes (SWNTs) encapsulating fullerenes, so-called fullerene peapods, were synthesized in high yield by using diameter-selected nanotubes as pods. Transmission electron microscopy revealed high-density fullerene chains inside the nanotubes. X-ray-diffraction measurements indicate 85% filling for C₆₀ and 72% filling for C₇₀ molecules as a total yield. Interestingly, C₆₀ peas do not show any thermal expansion while C₇₀ peas show normal behavior. Room-temperature Raman spectra show one-dimensional photopolymerization of C₆₀ inside nanotubes by blue-laser irradiation, suggesting molecular rotation inside them. In C₇₀ peapods, no photopolymerization was observed but the relative Raman intensity of each peak is different from the C₇₀ 3D crystal. This is probably caused by mixing of two different crystal structures in C₇₀ peas. Furthermore, we synthesized Zn-diphenylporphyrin peapods. Optical absorption and Raman spectra suggest that the encapsulated molecules are deformed by interaction with the SWNT. Received: 12 November 2001 / Accepted: 3 December 2001 / Published online: 4 March 2002     

Optical tweezers for confocal microscopy

In confocal laser scanning microscopes (CLSMs), lasers can be used for image formation as well as tools for the manipulation of microscopic objects. In the latter case, in addition to the imaging lasers, the light of an extra laser has to be focused into the object plane of the CLSM, for example as optical tweezers. Imaging as well as trapping by optical tweezers can be done using the same objective lens. In this case, z-sectioning for 3D imaging shifts the optical tweezers with the focal plane of the objective along the optical axis, so that a trapped object remains positioned in the focal plane. Consequently, 3D imaging of trapped objects is impossible without further measures. We present an experimental set-up keeping the axial trapping position of the optical tweezers at its intended position whilst the focal plane can be axially shifted over a distance of about 15 μm. It is based on fast-moving correctional optics synchronized with the objective movement. First examples of application are the 3D imaging of chloroplasts of Elodea densa (Canadian waterweed) in a vigorous cytoplasmic streaming and the displacement of zymogen granules in pancreatic cancer cells (AR42 J). Received: 24 March 2000 / Revised version: 23 June 2000 / Published online: 11 October 2000     

Sensitivity enhancement in thermal lens laser spectrometry

A novel experimental configuration for thermal lens detection is described. The method makes use of optical filtering of the probe beam by means of a circular aperture. This considerably reduces noise associated with intensity fluctuations of the probe beam. The technique provides an enhancement of the signal-to-noise ratio by almost one order of magnitude as compared to other thermal lens laser spectrometers. A theoretical calculation of the signal enhancement associated with optical filtering of the probe beam is presented. Furthermore, experiments on methyl blue dissolved in ethylalcohol are described which verify the theoretical predictions.     

Time-resolved fluorescence intensity issued from a heterogeneous slab: Sensitivity characterization

Optical imaging using fluorescent contrast agents has become an interesting tool to differentiate diseased lesions from normal tissue. However, several sensitivity characterizations may strongly influence the time-dependent fluorescence measurements. Herein, we present a numerical model based on the finite element method that allows the simulation of time-resolved reflectance and transmittance signals from heterogeneous media mimicking breast tissues with an embedded fluorescent object (tumor). The influence, on the computed signals, of several tumor depths, as well as various fluorophore concentrations and several fluorescent markers targeting are analyzed. The results show the possibility of uncoupling location depth from the shape of the target. Therefore, the analysis of the time to reach half the maximum intensity is validated as a good localization scheme. Then, the transmitted data show that the maximal detected intensity at the bottom of the medium is very sensitive to the dye concentration but not to the tumor shape. Moreover, the strong competition between concentration determination and fluorophore distribution is presented. These results will lead to a better detection and localization of tumors.     

Nanoscale characterization of single Au nanorods by confocal microscopy

We analyzed the scattering patterns of individual Au nanorods detected by means of confocal interference scattering microscopy in combination with a higher order laser mode. We placed the Au nanorods at the interface between two dielectric media and examined the influence of different interfaces on the signal amplitude, the signal-to-noise ratio as well as on the precision of topological measurements. Approaching the index matching regime allows for topological measurements with high accuracy minimizing the acquisition time. We were also able to track the position and the orientation of particles embedded in water even when they were not thoroughly sticking to the glass surface. These results underscore the potential of the presented technique for applications in life sciences.     

Modeling tip performance for combined STM-luminescence and aperture-SNOM scanning probe: Spatial resolution and collection efficiency

Finite-element simulations of the performance of the tip intended for use in combined aperture-SNOM and the scanning tunneling microscope (STM)-luminescence microscopy are presented. Tip geometry and the role of the opening in the protective metal coating were addressed. It is shown that the tip shape can affect transmittance for the excitation SNOM mode by nearly two orders of magnitude and the metal coating can enhance collection efficiency for the STM-luminescence mode. Desired tip configuration can be chosen based on the interplay between the improving collection efficiency and the deteriorating spatial resolution with increasing opening size.     

TEM studies on the formation of nano crystallites of Si by metal induced crystallization

In the present investigations, we have grown the nano-crystallites of Si by metal induced crystallization process. Layers of two different metals (Al and Au) were deposited on either side of Si using thermal evaporation technique to study metal induced crystallization. Annealing of such samples was carried out in the hot stage of TEM. We have found that the crystallization of amorphous silicon starts at 150 °C through the formation of metal silicides. Formation of metal silicides was observed through selected area diffraction. Nearly complete formation of nano crystallites of Si throughout the sample was observed at 200 °C. High-resolution TEM studies confirm the formation of nano-crystallites of Si all along the film.     

Confocal fluorescence microscopy for minimal-invasive tumor diagnosis

The goal of the project “stereotactic laser-neurosurgery” is the development of a system for careful and minimal-invasive resection of brain tumors with ultrashort laser pulses through a thin probe. A confocal laser-scanning-microscope is integrated in the probe. In this paper, the simulation of its optical properties by a laboratory setup and the expansion by the ability for fluorescence microscopy are reported. For a valuation of the imaging properties, the point-spread-function in three dimensions and the axial depth-transfer-function were measured and thus, among other things, the resolving power and the capacity for depth discrimination were analysed. The microscope will enable intra-operative detection of tumor cells by the method of immunofluorescence. As a first model of the application in the brain, cell cultures, that fluorescein-labelled antibodies were bound to specifically, were used in this work. Due to the fluorescence signal, it was possible to detect and identify clearly the areas that had been marked in this manner, proving the suitability of the setup for minimal-invasive tumor diagnosis. Received: 11 January 1999 / Revised version: 9 August 1999 / Published online: 21 January 2000     

Enhancement of the thermal transport in a culture medium with Au nanoparticles

In this work, it is reported the gold nanoparticles synthesis, their characterization, and their application to the enhancement of the thermal transport in a cellular culture medium. The Au nanoparticles (NPs), with average size of 10 nm, contained into a culture medium (DMEM (1)/F12(1)) (CM) increased considerably the heat transfer in the medium. Thermal lens spectrometry (TLS) was used to measure the thermal diffusivity of the nanofluids. The characteristic time constant of the transient thermal lens was obtained by fitting the theoretical expression, for transient thermal lens, to the experimental data. Our results show that the thermal diffusivity of the culture medium is highly sensitive to the Au nanoparticle concentration and size. The ability to modify the thermal properties to nanometer scale becomes very important in medical applications as in the case of cancer treatment by using photodynamic therapy (PDT). A complementary study with UV-vis and TEM techniques was performed to characterize the Au nanoparticles.     

Room temperature ferromagnetism in nanocrystalline Ni-doped ZnO synthesized by co-precipitation

Ni-doped ZnO powder was synthesized by thermal co-decomposition of a mixture of bis(acetylacetonato) zinc(II)hydrate and bis(dimethylglyoximato)nickel(II) complexes. The samples were characterised by X-ray diffraction (XRD), Energy dispersion X-ray fluorescence (EDXRF), and FT-IR spectroscopy. The atomic ratio Ni/Zn of the samples was determined by the EDXRF method to be 1%, 4.3%, 7.4% and 22.5 wt%. The XRD studies show the formation of nanocrystalline (14-18 nm) of Ni-doped ZnO along with nanoparticles of NiO. By magnetic measurements, it was observed that powder contains 1%Ni, 4.3%Ni, 7.4%Ni exhibits superparamagnetic behaviour while the sample of 22.5%Ni prepared in closed atmospheric environment shows clear ferromagnetic (FM) loop at room temperature due to the formation of solid solution Zn_1−xNi_xO.     

Measurement of thermal lensing in a power amplifier of a terawatt Ti:sapphire laser

We have measured detailed thermal lensing in a power amplifier of a terawatt Ti:sapphire laser operating at 50 Hz. The thermal lensing in the amplifier was evaluated by measuring the optical path difference (OPD) using a Shack–Hartmann-type wavefront sensor. It was found that the radial dependence of the OPD was almost quadratic in the pumping region, despite inhomogeneous pumping. Therefore, a simple spherical lens or convex mirror effectively compensates for the thermal lens in our amplifier. We found that the thermal lens profile was temporally stable, and did not degrade the pointing stability of the amplified laser pulses. We also found that the time constant of the thermal distortion in our power amplifier was approximately 0.5 s. Received: 3 September 2001 / Revised version: 23 January 2002 / Published online: 14 March 2002     

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10^-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns). Received: 23 February 2003 / Published online: 22 May 2003 RID="*" ID="*"Corresponding author. Fax: +1-734/9361-905, E-mail: mycek@umich.edu     

基于光热效应的显微光谱技术在单粒子检测中应用和发展

高灵敏度的单粒子检测技术是纳米粒子在生物医学、化学、光电子等领域应用的前提条件。常见的单粒子检测技术主要包括基于粒子的荧光、拉曼、散射和吸收等信号而发展起来的光学显微成像及光谱技术。其中,拉曼光谱和荧光光谱技术主要适用于一些具有拉曼活性的分子/粒子或可发光的荧光分子或粒子,然而即使对于荧光效率高的有机染料分子和半导体纳米粒子,固有的光漂白和blinking现象也对单粒子探测形成了挑战。散射光谱测量是应用于单粒子检测的另外一种方法,从理论上讲,由于瑞利散射随着尺寸的减小而呈六次方减弱的趋势,在细胞或生物组织内,小尺寸粒子的散射信号很难从背景散射噪声中分离出来。众所周知,介质吸收激发光后会引起介质内的折射率变化,进而在光加热区附近出现折射率的梯度分布,称为光热效应(photothermal effect)。基于粒子光热效应的光学显微成像和光谱测量技术具有信号灵敏度高、无背景散射、原位和免标记等优点,在单粒子检测领域展现了良好的应用潜力。综述了近年来基于光热效应的显微光谱技术在单粒子检测中应用和研究发展,首先介绍了光热效应的测量原理;接着分别讨论了光热透镜测量技术、微分干涉相差测量技术和光热外差测量技术的实验装置,比较了各种测量技术的信噪比、灵敏度、分辨率等特点,并且介绍这些测量技术在单粒子检测中的应用研究进展;接着,论述了近年来研究人员在提高光热显微测量的信噪比、改善动态测量性能以及在红外波段拓展等方面的最新研究成果;最后,简单总结了光热测量技术在单粒子检测领域所面临的挑战。     

Influence of the cross section and the permittivity on the plasmon-resonance spectrum of silver nanowires

We investigate the plasmon resonances for silver nanowires with a non-regular cross section. To study the relationship between the cross section and the spectrum of the plasmon resonances, we consider cross sections evolving from a rectangular shape to a triangular one. In particular, we study the influence of the sharpness of a corner on the near-field enhancement at the vicinity of a particle and discuss its implications for surface-enhanced Raman scattering. We also investigate the influence of the absorption on the plasmon-resonance spectrum and on the near-field enhancement. Received: 15 August 2001 / Published online: 10 October 2001     

Active membranes studied by X-ray scattering

In view of recent theories of “active” membranes, we have studied multilamellar phospholipid membrane stacks with reconstituted transmembrane protein bacteriorhodopsin (BR) under different illumination conditions by X-ray scattering. The light-active protein is considered as an active constituent which drives the system out of equilibrium and is predicted to change the collective fluctuation properties of the membranes. Using X-ray reflectivity, X-ray non-specular (diffuse) scattering, and grazing incidence scattering, we find no detectable change in the scattering curves when changing the illumination condition. In particular the intermembrane spacing d remains constant, after eliminating hydration-related artifacts by design of a suitable sample environment. The absence of any observable non-equilibrium effects in the experimental window is discussed in view of the relevant parameters and recent theories.