Tunable optical tweezers for wavelength-dependent measurements

Optical trapping forces depend on the difference between the trap wavelength and the extinction resonances of trapped particles. This leads to a wavelength-dependent trapping force, which should allow for the optimization of optical tweezers systems, simply by choosing the best trapping wavelength for a given application. Here we present an optical tweezer system with wavelength tunability, for the study of resonance effects. With this system, the optical trap stiffness is measured for single trapped particles that exhibit either single or multiple extinction resonances. We include discussions of wavelength-dependent effects, such as changes in temperature, and how to measure them.     

Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light

A quadrant photodiode placed in the back-focal plane of the microscope of a laser trap provides a high-resolution position sensor. We show that in addition to the lateral displacement of a trapped sphere, its axial position can be measured by the ratio of the intensity of scattered laser light to the total amount of the light reaching the detector. The addition of the axial information offers true three-dimensional position detection in solution, creating, together with a position control, a photonic force microscope with nanometer spatial and microsecond temporal resolution. The measured position signals are explained as interference of the unscattered trapping laser beam with the laser light scattered by the trapped bead. Our model explains experimental data for trapped particles in the Rayleigh regime (radius a <0.2lambda) for displacements up to the focal dimensions. The cross-talk between the signals in the three directions is explained and it is shown that this cross-talk can be neglected for lateral displacements smaller than 75 nm and axial displacements below 150 nm. The advantages of three-dimensional single-particle tracking over conventional video-tracking are shown through the example of the diffusion of the GPI-anchored membrane protein Thy1.1 on a neurite.     

多局域空心光阱及其光场调控

本文通过一种产生多局域空心光阱的光学系统,研究了光阱位置与反射镜偏转角度的关系.基于衍射积分和矩阵光学的理论,分析并计算了入射光源经过光学元件后的光场分布,通过调控反射镜的偏转角度,可以实现光阱位置的任意变换,达到精确捕获和囚禁微粒的目的.当两个反射镜偏转角度的关系为θ1-θ2=90°时,变换的光阱位置在一条倾斜的直线...     

The MPD test beam setup for testing detectors with the Nuclotron beams

A new specialized MPD Test Beam setup was mounted at the extracted beam of the Nuclotron at the Joint Institute for Nuclear Research to carry out methodical research and test detectors produced for the MPD experiment at the NICA accelerating facility. The setup is described in detail. The results of the testing of fast detectors for the MPD time-of-flight system are presented as an example of the operation of the setup.     

A setup for measuring the refractive index of a plane-parallel ceramic plate using the optical beam shift method

A setup for measuring the refractive indices of transparent solid samples of optical ceramics using shifts of a beam by a plane-parallel plate in a wavelength range of 400–1200 nm was developed. The minimum cross-sectional sizes of the investigated objects are 5–12 mm, and their thicknesses are 0.3–1.0 mm. A standard sample and a precise system for forming and recording optical signals equipped with a stepping motor with a step discreteness of <1 μm were used to improve the measurement accuracy of the refractive index. The cross-sectional size of the measuring beam is ?1 mm. The accuracy in determining the refractive index is ±0.004.     

Combining optical tweezers and patch clamp for studies of cell membrane electromechanics

We have designed and implemented a novel experimental setup which combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. In this system, optical tweezers provide measurement of forces at piconewton scale, and the patch-clamp technique allows control of the cell transmembrane potential. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement from the trapping center is monitored by a quadrant photodetector for dynamic measurements of tether force. Fluorescent beads and the corresponding fluorescence imaging optics are used to eliminate the shadow of the cell projected on the quadrant photodetector. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. A unique feature of this setup is that the patch-clamp headstage and the manipulator for the recording pipette are mounted on a piezoelectric stage, preventing relative movements between the cell and the patch pipette during the process of tether pulling. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from mammalian cochlear outer hair cells and human embryonic kidney cells are presented.     

A computer-aided setup for gas-sensing measurements of sensors based on semiconductor nanocomposites

The design of the computer-aided setup for gas-sensing measurements of semiconductor sensors is described. The setup is intended to programmably control thetemperature and gas supply and measure the sensor resistances. The curves of the responses of the sensors manufactured by the sol-gel and hydropyrolysis methods to the gas-composition variations are obtained. They allow one to extend model concepts on the gas adsorption at these sensors.     

Automated three-dimensional analysis of particle measurements using an optical profilometer and image analysis software

The automated collection of topographic images from an optical profilometer coupled with existing image analysis software offers the unique ability to quantify three‐dimensional particle morphology. Optional software available with most optical profilers permits automated collection of adjacent topographic images of particles dispersed onto a suitable substrate. Particles are recognized in the image as a set of continuous pixels with grey‐level values above the grey level assigned to the substrate, whereas particle height or thickness is represented in the numerical differences between these grey levels. These images are loaded into remote image analysis software where macros automate image processing, and then distinguish particles for feature analysis, including standard two‐dimensional measurements (e.g. projected area, length, width, aspect ratios) and third‐dimensional measurements (e.g. maximum height, mean height). Feature measurements from each calibrated image are automatically added to cumulative databases and exported to a commercial spreadsheet or statistical program for further data processing and presentation. An example is given that demonstrates the superiority of quantitative three‐dimensional measurements by optical profilometry and image analysis in comparison with conventional two‐dimensional measurements for the characterization of pharmaceutical powders with plate‐like particles.     

A hybrid scanning force and light microscope for surface imaging and three-dimensional optical sectioning in differential interference contrast

The design of a scanned-cantilever-type force microscope is presented which is fully integrated into an inverted high-resolution video-enhanced light microscope. This set-up allows us to acquire thin optical sections in differential interference contrast (DIC) or polarization while the force microscope is in place. Such a hybrid microscope provides a unique platform to study how cell surface properties determine, or are affected by, the three-dimensional dynamic organization inside the living cell. The hybrid microscope presented in this paper has proven reliable and versatile for biological applications. It is the only instrument that can image a specimen by force microscopy and high-power DIC without having either to translate the specimen or to remove the force microscope. Adaptation of the design features could greatly enhance the suitability of other force microscopes for biological work.     

Pressure jump relaxation setup with IR detection and millisecond time resolution

An instrument is described that allows the use of Fourier transform infrared (FTIR) spectroscopy as a detection system for kinetic processes after a pressure jump of up to 100 bars. The pressure is generated using a high performance liquid chromatography (HPLC) pump and water as a pressure transducing medium. A flexible membrane separates the liquid sample in the IR cell from the pressure transducing medium. Two electromagnetic switching valves in the setup enable pressure jumps with a decay time of 4 ms. The FTIR spectrometer is configured to measure time resolved spectra in the millisecond time regime using the rapid scan mode. All components are computer controlled. For a demonstration of the capability of the method first results on the kinetics of a phase transition between two lamellar phases of an aqueous phospholipid dispersion are presented. This combination of FTIR spectroscopy with the pressure jump relaxation technique can also be used for other systems which display cooperative transitions with concomitant volume changes.     

Shack-Hartmann wave front measurements in cortical tissue for deconvolution of large three-dimensional mosaic transmitted light brightfield micrographs

We present a novel approach for deconvolution of 3D image stacks of cortical tissue taken by mosaic/optical‐sectioning technology, using a transmitted light brightfield microscope. Mosaic/optical‐sectioning offers the possibility of imaging large volumes (e.g. from cortical sections) on a millimetre scale at sub‐micrometre resolution. However, a blurred contribution from out‐of‐focus light results in an image quality that usually prohibits 3D quantitative analysis. Such quantitative analysis is only possible after deblurring by deconvolution. The resulting image quality is strongly dependent on how accurate the point spread function used for deconvolution resembles the properties of the imaging system. Since direct measurement of the true point spread function is laborious and modelled point spread functions usually deviate from measured ones, we present a method of optimizing the microscope until it meets almost ideal imaging conditions. These conditions are validated by measuring the aberration function of the microscope and tissue using a Shack‐Hartmann sensor. The analysis shows that cortical tissue from rat brains embedded in Mowiol and imaged by an oil‐immersion objective can be regarded as having a homogeneous index of refraction. In addition, the amount of spherical aberration that is caused by the optics or the specimen is relatively low. Consequently the image formation is simplified to refraction between the embedding and immersion medium and to 3D diffraction at the finite entrance pupil of the objective. The resulting model point spread function is applied to the image stacks by linear or iterative deconvolution algorithms. For the presented dataset of large 3D images the linear approach proves to be superior. The linear deconvolution yields a significant improvement in signal‐to‐noise ratio and resolution. This novel approach allows a quantitative analysis of the cortical image stacks such as the reconstruction of biocytin‐stained neuronal dendrites and axons.     

Potential measurements with heavy ion beam probe system on LHD

The heavy ion beam probe system in the Large Helical Device (LHD) was improved as follows. At first, the additional new sweeper was installed into the diagnostic port to extend the observable region. By using this sweeper, the potential profile was measured in a wider minor radius range than in previous experiments, in the case of outward shifted magnetic configuration of LHD. Next, the real time control system was installed to control the probe beam orbit for measuring the potential in plasma with large plasma current. In this system, a digital signal processor was used to control the probe beam in real time. The system worked well in the fixed position observation mode. In the sweeping mode for profile measurement, this control system became unstable. The details of this system and the experimental results are reported in this article.     

BioPhotonics workstation: a versatile setup for simultaneous optical manipulation, heat stress, and intracellular pH measurements of a live yeast cell

In this study we have modified the BioPhotonics workstation (BWS), which allows for using long working distance objective for optical trapping, to include traditional epi-fluorescence microscopy, using the trapping objectives. We have also added temperature regulation of sample stage, allowing for fast temperature variations while trapping. Using this modified BWS setup, we investigated the internal pH (pH(i)) response and membrane integrity of an optically trapped Saccharomyces cerevisiae cell at 5 mW subject to increasing temperatures. The pH(i) of the cell is obtained from the emission of 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, at 435 and 485 nm wavelengths, while the permeability is indicated by the fluorescence of propidium iodide. We present images mapping the pH(i) and permeability of the cell at different temperatures and with enough spatial resolution to localize these attributes within the cell. The combined capability of optical trapping, fluorescence microscopy and temperature regulation offers a versatile tool for biological research.     

A simple backscattered electrons and specimen current detection device for the scanning electron microscope

A simple, low-investment device has been developed that allows the collection of backscattered electrons (BSEs) and specimen current (SC) signals for imaging purposes and current measurement. Originally, this system was designed for detection, measurement, and display of specimen current, with a video signal output whose level was modulated by this current. Eventually, a BSE detector was developed, using a graphite disk (about 8 cm in diameter) to collect the BSEs. The disk was mounted on a Philips SEM 5O5, attached and concentrically to the final lens aperture. This configuration gives a large solid angle of collection. The collected charge is further processed by the same electronics used in the aforementioned SC detection system. Electron channeling, topographic contrast with BSE, and material contrast with BSE and SC images can be obtained with reasonably good edge definition.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.     

基于光锥扩束机理的单脉冲激光近程静态探测方法

针对激光近程全向探测问题,在激光近程动态周向扫描探测机理研究基础上,提出了基于光锥扩束机理的单脉冲激光 近程静态周向探测方法。 基于激光近场探测理论和静态探测场空间几何分布,推导出基于光锥扩束机理的单脉冲激光近程静 态探测回波方程。 构建了单脉冲激光近程测距概率分布模型并搭建了实验室静态探测实验平台,研究了脉冲激光发射功率、倒 置反射光锥角、脉冲激光束发散角和目标尺寸投影面积对激光近程周向探测概率分布的影响机制。 结果表明:随着发射功率和 目标投影尺寸分别从 10 W 和 0. 01 m 2 增加到 30 W 和 0. 25 m 2 ,回波信号幅值亦随之从 0. 16 和 0. 43 μV 提升到 4. 22 和 5. 95 μV,随着倒置反射光锥角和光束发散角分别从 30° 和 10 mrad 增加到 120° 和 30 mrad,回波信号幅值随之从 3. 18 和 2. 52 μV 降低到 0. 88 和 1. 92 μV;周向探测概率分布随着发射功率和目标投影尺寸的增加而半宽减小且峰值增加并向左偏离、 随着倒置反射光锥角和光束发散角的增加半宽增大且峰值降低并向右偏离;探测分布对称性并不受以上 4 种因素影响。     

X-ray reflectometer for optical efficiency and scatter measurements

An instrument has been developed to determine the reflection efficiency and scatter characteristics of optical samples at x-ray wavelengths from 1.5 to 113 A. The reflectometer operates in an oil-free vacuum chamber and measures the reflection efficiency and scatter characteristics as a function of the angle of incidence. The reflection efficiency is given for lambda=8.34 A incident on a fused silica sample finished to a flatness of lambda/10. The experimental reflection efficiency is compared to the theoretical data. The scatter curves are given for the direct x-ray beam and for the beam reflected from the fused silica sample at theta=50 arc minutes. The full-width-at-half-maximum (FWHM) resolution of the instrument is approximately 13 arc seconds as determined by a least-squares smoothing of the experimental data.     

A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope

A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry.     

Quasianamorphic optical imaging system with tomographic reconstruction for electron beam imaging

We have developed a quasianamorphic optical tomography system coupled to a streak camera to provide continuous recording of the electron beam profile of an intense, long-pulse induction accelerator. A tomographic reconstruction method based on a maximum-entropy algorithm is used to reconstruct the images. The system has simplified the calculation of beam moments, eliminated ambiguity due to beam motion, and contributed to accelerator tuning.