Particle classification from light scattering with the scanning flow cytometer.

BACKGROUND:The differential light-scattering pattern, an indicatrix, provides the most complete characterization of the optical properties of a particle. Particle classification can be performed on the basis of particle parameters retrieved from the indicatrices. This classification extends the ability of flow cytometry in particle recognition. METHODS:The scanning flow cytometer (SFC) permits an acquisition of traces of light scattering signals, i.e., native SFC traces, from single particles. The acquired native SFC traces are transformed into indicatrices. The performance of the SFC in measurements of indicatrices has been demonstrated for the following particles: lymphocytes, erythrocytes, polystyrene particles, and milk-fat particles. RESULTS:The structure and profile of the indicatrix for each particle type have been found to be unique. Classification of polystyrene particles has been performed on the basis of the map formed by particle refractive index and size. The polystyrene particles were classified using this map into different size categories ranging from 1.4-7 microm, with a size deviation of 0.07 microm. CONCLUSIONS:The method based on analysis of native SFC traces shows better performance in particle classification than the method based on the particle refractive index and size map. The classification performance of the SFC will be useful, for example, for particle sorting and particle identification, and with additional fluorescent measurements may have applications in multiparameter particle-based immunoassay.     

Measuring the folding landscape of a harmonically constrained biopolymer

Pioneering studies have shown that the probability distribution of opening length for a DNA hairpin, recorded under constant force using an optical trap, can be used to reconstruct the energy landscape of the transition. However, measurements made under constant force are subject to some limitations. Under constant force a system with a sufficiently high energy barrier spends most of its time in the closed or open conformation, with relatively few statistics collected in the transition state region. We describe a measurement scheme in which the system is driven progressively through the transition by an optical trap and an algorithm is used to extract the energy landscape of the transition from the fluctuations recorded during this process. We illustrate this technique in simulations and demonstrate its effectiveness in experiments on a DNA hairpin. We find that the combination of this technique with the use of short DNA handles facilitates a high-resolution measurement of the hairpin's folding landscape with a very short measurement time.     

A method for matching the refractive index and kinematic viscosity of a blood analog for flow visualization in hydraulic cardiovascular models

In this work, we propose a simple method to simultaneously match the refractive index and kinematic viscosity of a circulating blood analog in hydraulic models for optical flow measurement techniques (PIV, PMFV, LDA, and LIF). The method is based on the determination of the volumetric proportions and temperature at which two transparent miscible liquids should be mixed to reproduce the targeted fluid characteristics. The temperature dependence models are a linear relation for the refractive index and an Arrhenius relation for the dynamic viscosity of each liquid. Then the dynamic viscosity of the mixture is represented with a Grunberg-Nissan model of type 1. Experimental tests for acrylic and blood viscosity were found to be in very good agreement with the targeted values (measured refractive index of 1.486 and kinematic viscosity of 3.454 milli-m2/s with targeted values of 1.47 and 3.300 milli-m2/s).     

Vesicle size distributions measured by flow field-flow fractionation coupled with multiangle light scattering.

The separation method, flow field-flow fractionation (flow FFF), is coupled on-line with multiangle laser light scattering (MALLS) for simultaneous measurement of the size and concentration of vesicles eluting continuously from the fractionator. These size and concentration data, gathered as a function of elution time, may be used to construct both number- and mass-weighted vesicle size distributions. Unlike most competing, noninvasive methods, this flow FFF/MALLS technique enables measurement of vesicle size distributions without a separate refractive index detector, calibration using particle size standards, or prior assumptions about the shape of the size distribution. Experimentally measured size distributions of vesicles formed by extrusion and detergent removal are non-Gaussian and are fit well by the Weibull distribution. Flow FFF/MALLS reveals that both the extrusion and detergent dialysis vesicle formation methods can yield nearly size monodisperse populations with standard deviations of approximately 8% about the mean diameter. In contrast to the rather low resolution of dynamic light scattering in analyzing bimodal systems, flow FFF/MALLS is shown to resolve vesicle subpopulations that differ by much less than a factor of two in mean size.     

Cancer cell viscoelasticity measurement by quantitative phase and flow stress induction

Cell viscoelastic properties are affected by the cell cycle, differentiation, and pathological processes such as malignant transformation. Therefore, evaluation of the mechanical properties of the cells proved to be an approach to obtaining information on the functional state of the cells. Most of the currently used methods for cell mechanophenotyping are limited by low robustness or the need for highly expert operation. In this paper, the system and method for viscoelasticity measurement using shear stress induction by fluid flow is described and tested. Quantitative phase imaging (QPI) is used for image acquisition because this technique enables one to quantify optical path length delays introduced by the sample, thus providing a label-free objective measure of morphology and dynamics. Viscosity and elasticity determination were refined using a new approach based on the linear system model and parametric deconvolution. The proposed method allows high-throughput measurements during live-cell experiments and even through a time lapse, whereby we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters such as circularity or cell mass. Additionally, the proposed method provides a simple approach to measure cell refractive index with the same setup, which is required for reliable cell height measurement with QPI, an essential parameter for viscoelasticity calculation. Reliability of the proposed viscoelasticity measurement system was tested in several experiments including cell types of different Young/shear modulus and treatment with cytochalasin D or docetaxel, and an agreement with atomic force microscopy was observed. The applicability of the proposed approach was also confirmed by a time-lapse experiment with cytochalasin D washout, whereby an increase of stiffness corresponded to actin repolymerization in time.     

Direct laser trapping of single DNA molecules in the globular state.

A sharply focused laser is able to trap small particles at the laser focal point due to the difference in refractive index of the particles and that of the surrounding medium. This technique, called laser trapping, can be used to manipulate animal or bacterial cells without any contact and has been widely applied in biological research. However, it has been difficult to trap biological macromolecules such as DNA molecules, because these molecules give a low difference in refractive index and cannot overcome Brownian motion. DNA molecules can be transformed to a condensed globular state. This transformation results in a higher refractive index of DNA due to its increased density. We demonstrate in this paper that a single DNA molecule can be optically trapped using a Nd:YAG laser (1064 nm wavelength) upon transformation from the coiled state to the globular state.     

Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy.

BACKGROUND: The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen. METHODS: The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI. RESULTS: Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 +/- 0.004. The uncertainty in the final RI value represents the technique measurement error. CONCLUSIONS: The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology.     

Calibration of Optical Tweezers for In Vivo Force Measurements: How do Different Approaches Compare?

There is significant interest in quantifying force production inside cells, but since conditions in vivo are less well controlled than those in vitro, in vivo measurements are challenging. In particular, the in vivo environment may vary locally as far as its optical properties, and the organelles manipulated by the optical trap frequently vary in size and shape. Several methods have been proposed to overcome these difficulties. We evaluate the relative merits of these methods and directly compare two of them, a refractive index matching method, and a light-momentum-change method. Since in vivo forces are frequently relatively high (e.g., can exceed 15 pN for lipid droplets), a high-power laser is employed. We discover that this high-powered trap induces local temperature changes, and we develop an approach to compensate for uncertainties in the magnitude of applied force due to such temperature variations.     

Effects of mismatched refractive indices in aquatic flow cytometry.

BACKGROUND: Forward-angle light scatter, as measured by flow cytometry, can be used to estimate the size spectra of cell assemblages from natural waters. The refractive index of water samples from aquatic environments can differ because of a variety of factors such as dissolved organic content, aldehyde preservative, sample salinity, and temperature. In flow cytometric analyses, mismatch between the refractive indices of the sheath fluid and the sample causes distortion of the forward-angle light scatter signal. We measured the effect of this mismatch on cell size measurements. METHODS: We examined the error by measuring the scatter signal of a variety of particle types and sizes and changing the sheath-to-sample salinity ratio. The effects were characterized for standard microspheres, cultured phytoplankton cells of different sizes, and natural populations from an estuarine river. RESULTS: We found that the distorted scatter signals resulted in an increase in the apparent size of small cells (1--2 microm) by a factor of 4.5 times. Cells in the size range of 3--5 microm were less affected by the salinity differences, and cells larger than 5 microm were not affected. Chlorophyll and phycoerythrin fluorescences and 90 degrees light scatter signals were not changed by sheath and sample salinity differences. CONCLUSIONS: Care must be taken to ensure that the sheath and sample refractive index are matched when using forward light scatter to measure cell size spectra, especially in estuarine studies, where salinity can vary greatly. Of the factors considered that can change the sample refractive index, salinity gradients in an estuary cause the largest index mismatch and, consequently, the largest error in scatter.     

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.Download video file.^{(62M, mov)}     

Plasmonic Optical Trapping in Biologically Relevant Media

We present plasmonic optical trapping of micron-sized particles in biologically relevant buffer media with varying ionic strength. The media consist of 3 cell-growth solutions and 2 buffers and are specifically chosen due to their widespread use and applicability to breast-cancer and angiogenesis studies. High-precision rheological measurements on the buffer media reveal that, in all cases excluding the 8.0 pH Stain medium, the fluids exhibit Newtonian behavior, thereby enabling straightforward measurements of optical trap stiffness from power-spectral particle displacement data. Using stiffness as a trapping performance metric, we find that for all media under consideration the plasmonic nanotweezers generate optical forces 3–4x a conventional optical trap. Further, plasmonic trap stiffness values are comparable to those of an identical water-only system, indicating that the performance of a plasmonic nanotweezer is not degraded by the biological media. These results pave the way for future biological applications utilizing plasmonic optical traps.     

Calibration of Optical Tweezers for In?Vivo Force Measurements: How do Different Approaches Compare?

There is significant interest in quantifying force production inside cells, but since conditions in vivo are less well controlled than those in vitro, in vivo measurements are challenging. In particular, the in vivo environment may vary locally as far as its optical properties, and the organelles manipulated by the optical trap frequently vary in size and shape. Several methods have been proposed to overcome these difficulties. We evaluate the relative merits of these methods and directly compare two of them, a refractive index matching method, and a light-momentum-change method. Since in vivo forces are frequently relatively high (e.g., can exceed 15 pN for lipid droplets), a high-power laser is employed. We discover that this high-powered trap induces local temperature changes, and we develop an approach to compensate for uncertainties in the magnitude of applied force due to such temperature variations.     

Tunable pores for measuring concentrations of synthetic and biological nanoparticle dispersions

Scanning ion occlusion sensing (SIOS), a technique that uses a tunable pore to detect the passage of individual nano-scale objects, is applied here for the rapid, accurate and direct measurement of synthetic and biological nanoparticle concentrations. SIOS is able to characterize smaller particles than other direct count techniques such as flow cytometry or Coulter counters, and the direct count avoids approximations such as those necessary for turbidity measurements. Measurements in a model system of 210-710 nm diameter polystyrene particles demonstrate that the event frequency scales linearly with applied pressure and concentration, and that measured concentrations are independent of particle type and size. Both an external-calibration and a calibration-free measurement method are demonstrated. SIOS is then applied to measure concentrations of Baculovirus occlusion bodies, with a diameter of ~1 μm, and the marine photosynthetic cyanobacterium Prochlorococcus, with a diameter of ~600 nm. The determined concentrations agree well with results from counting with microscopy (a 17% difference between the mean concentrations) and flow cytometry (6% difference between the mean concentrations), respectively.     

Compact, high performance surface plasmon resonance imaging system

We report the construction and characterization of a new compact surface plasmon resonance imaging instrument. Surface plasmon resonance imaging is a versatile technique for detection, quantification and visualization of biomolecular binding events which have spatial structure. The imager uses a folded light path, wide-field optics and a tilted detector to implement a high performance optical system in a volume 7 in. x 4 in. x 2 in. A bright diode light source and an image detector with fast frame rate and integrated digital signal processor enable real-time averaging of multiple images for improved signal-to-noise ratio. Operating angle of the imager is adjusted by linear translation of the light source. Imager performance is illustrated using resolution test targets, refractive index test solutions, and competition assays for the antiepileptic drug phenytoin. Microfluidic flowcells are used to enable simultaneous assay of three sample streams. Noise level of refractive index measurements was found to decrease proportional to the square root of the number of pixels averaged, reaching approximately 5 x 10(-7) refractive index units root-mean-square for 160 x 120 pixels image regions imaged for 1s. The simple, compact construction and high performance of the imager will allow the device to be readily applied to a wide range of applications.     

Cell cytometry with a light touch: sorting microscopic matter with an optical lattice

Lab-on-a-chip design is a key technology for increasing both the reliability and the functionality of many different preparation and diagnostic techniques in biomedicine. The drive towards ever more integrated lab-on-a-chip designs requires increasingly complex microfluidic systems. In order to build these systems, non-invasive actuators such as pumps, filters and mixers are required. We have demonstrated microfluidic sorting based on a 3D interference pattern, formed from multiple coherent laser beams, which has the potential to fulfil all the above criteria. By interfering five laser beams from a fibre laser at 1070 nm, we have formed a 3D optical lattice. When particles flow through the optical lattice their trajectories depend upon the force exerted on the particle by the optical lattice, in combination with the drag force exerted by the fluid flow. Hence, with the strength of a particle's interaction with the lattice determining the total force exerted upon it, its trajectory is determined by its physical properties. These properties include refractive index, size and shape, giving a range of criteria with which to sort an analyte. We have shown separation at 45 degrees of polymer from silica microspheres (by refractive index), the separation of protein microcapsules and the sorting of erythrocytes from lymphocytes. The interference pattern can be tailored to the particles and if a blockage occurs, the laser can simply be switched off, unlike solid-state micro-sorters, so that no jamming occurs. Efficiencies in excess of 95% have been achieved.     

The relationship between the light scattering properties of zymogen granules and the release of their contained proteins

The optical density of suspensions of the digestive enzyme-containing zymogen granule, a roughly spherical 1 micron diameter membrane-enclosed subcellular structure isolated from the exocrine pancreas of mammals, is reduced greatly when they are suspended in physiological media. This reduction in optical density is accompanied by the release of the granule's protein contents. It has traditionally been assumed that this property is due to granule lysis; that is, dissolution of the particle and its consequent disappearance as a strongly scattering object. Thus, lysis would decrease optical density by decreasing the number density of suspended spheres (N) according to Beer's law. However, as a general matter, changes in the optical density of suspensions of spheres may be a function of changes in the refractive index (m) or radius (r) of the objects as well. In this study, we apply Mie theory of scattering by small particles, which, in conjunction with Beer's law, allows us to evaluate whether changes in the scattering properties of granule suspensions are due to changes in N, m or r. Scattering by granule suspensions was reduced in three ways-pH, calcium ion concentration, and detergent concentration. A simple reduction in particle number did not account for decreased scattering and protein release in any of these circumstances. Instead, the changes appear attributable to decreases in particle size and refractive index.     

Direct measurement of oxygen consumption rate on the nematode Caenorhabditis elegans by using an optical technique

It is well known that aging and longevity strongly correlate with energy metabolism. The nematode Caenorhabditis elegans is widely used as an ultimate model of experimental animals. Thus, we developed a novel tool, which is constructed from an optical detector, using an indirect method that can measure simply the energy metabolism of C. elegans. If we measure the oxygen consumption rate using this optical tool, we can easily evaluate the activity of mitochondria as an index in the aging process. However, a direct measurement of the oxygen consumption rate of C. elegans exposed in air is thought to be impossible because of the high concentration of atmospheric oxygen and the small size of the animals. We demonstrate here that we can directly detect the oxygen consumption with a small number of animals (相似文献   

Microring Switching Control Using Plasmonic Ring Resonator Circuits for Super-Channel Use

The multi-wavelength selection and switching system using the hybrid plasmonic add-drop ring resonator (HPARR) for optical communication is proposed for multi-carrier super-channel-based designed. The plasmonic polariton technique applied in the ring resonator mode to the alternate waveguide interferometer switches the multi-wavelength laser emission in the various ranges. The combination of curvature-coupled plasmon ring and substances with different refractive index allows switching the multi-wavelength emission to shorter the free spectrum range (FSR) and specific wavelengths, without an applied pump signal or adjusted the ring size. It is suitable for the super-channel of wavelength division multiplex (WDM) in the future optical network.

     

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam¹. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles¹. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec¹, which has serious implications for biological matter^2,3.A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime⁴. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton''s Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.The double-nanohole structure has been shown to give a strong local field enhancement^5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres⁷ and 3.4 nm hydrodynamic radius bovine serum albumin proteins⁸. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.     

Integration of single-cell trapping and impedance measurement utilizing microwell electrodes

The ability to research individual cells has been seen as important in many kinds of biological studies. In the present study, cell impedance analysis is integrated into a single-cell trapping structure. For the purpose of precise positioning, a cell manipulation and measurement microchip, which uses an alternating current electrothermal effect (ACET) and a negative dielectrophoresis (nDEP) force to move a particle and cell on measurement electrodes, is developed. An ACET and an nDEP can be easily combined with subsequent analyses based on electric fields. A microwell presented in a previous study is separated into two parts, which are regarded as the measurement electrodes. The original structure is modified for precise positioning. Numerical simulations and analyses are conducted to compute and analyze the effects of the structural parameters. The results of simulations and analyses are used to obtain the optimum structure for the cell. The capture range of the microwell can be designed for cells of various sizes. In order to demonstrate the precision of the positioning, a particle is captured, measured, and released twice. The results show that the impedance error of the particle is about 3%. Finally, the developed structure is applied to trap and measure the impedance of a HeLa cell.