Simulation of ultrasound backscattering by red cell aggregates: effect of shear rate and anisotropy

Tissue characterization using ultrasound (US) scattering allows extraction of relevant cellular biophysical information noninvasively. Characterization of the level of red blood cell (RBC) aggregation is one of the proposed application. In the current paper, it is hypothesized that the microstructure of the RBCs is a main determinant of the US backscattered power. A simulation model was developed to study the effect of various RBC configurations on the backscattered power. It is an iterative dynamical model that considers the effect of the adhesive and repulsive forces between RBCs, and the effect of the flow. The method is shown to be efficient to model polydispersity in size, shape, and orientation of the aggregates due to the flow, and to relate these variations to the US backscattering properties. Three levels of aggregability at shear rates varying between 0.05 and 10 s(-1) were modeled at 40% hematocrit. The simulated backscattered power increased with a decrease in the shear rate or an increase in the RBC aggregability. Angular dependence of the backscattered power was observed. It is the first attempt to model the US power backscattered by RBC aggregates polydisperse in size and shape due to the shearing of the flow.     

Rhythmic Conformational Change in a SinglePolymer Chain Induced by Laser Irradiation

We have investigated a rhythmic conformational change in a single polymer chain (T4DNA, 166 kbp, the contour length = 56 m) between a folded state and a elongated state under thermodynamically open conditions using a focused continuons wave (cw) Nd:YAG laser beam (wavelength = 1064 nm), where the focused laser beam plays dual roles both of trapping of a polymer chain at the focus and making temperature gradient there. The oscillatory phenomenon is discuseed in terms of a limit-cycle oscillation with dissipation of photon energy.     

Insertion of microscopic objects through plant cell walls using laser microsurgery

A detailed protocol is presented for precisely inserting microscopic objects into the periplasmic region of plant callus cells using laser microsurgery. Ginkgo biloba and Agrobacterium rhizogenes were used as the model system for developing the optical tweezers and scalpel techniques using a single laser. We achieved better than 95% survival after plasmolyzing G. biloba cells, ablating a 2-4-μm hole through the cell wall using a pulsed UV laser beam, trapping and translating bacteria into the periplasmic region using a pulsed infrared laser beam, and then deplasmolyzing the cells. Insertion of bacteria is also described. A thermal model for temperature changes of trapped bacteria is included. Comparisons with other methods, such as a reverse-pressure gradient technique, are discussed and additional experiments on plants using laser microsurgery are suggested. Copyright 1998 John Wiley & Sons, Inc.     

Determination of red blood cell shape recovery time constant in a couette system by the analysis of light reflectance and ektacytometry

Red blood cell (RBC) shape change under shear is generally reversible, with the time course of shape recovery a function of the elastic and viscous properties of the RBC membrane. RBC shape recovery can be investigated, using several different techniques, to provide information about the membrane material properties that are not directly accessible by frequently used methods to assess RBC deformability (e.g., micropore filtration). In the present study, RBC shape recovery was studied in a Couette system after abrupt cessation of shear, either by analyzing the time course of laser light reflection or by serial measurements of elongation indexes from laser diffraction patterns. The time course of shape recovery monitored with both techniques can be described with an exponential equation. Calculated time constants for normal human RBC were 119 ± 17 msec and 97 ± 15 msec as measured by light reflection and ektacytometry, respectively. Treatment of RBC with glutaraldehyde resulted in dose-dependent decreases in the shape recovery time constant. Heat treatment (48 ° C, 20 min), which is known to increase mainly the shear elastic modulus of the membrane, decreased the time constant by 65%. In contrast, wheat germ agglutinin treatment increased the shape recovery time constant by 22%, presumably by increasing membrane surface viscosity. Our results indicate that the shape recovery time constant of RBC can be measured easily and accurately by computerized light reflection analysis.     

Enhancing the Strength of an Optical Trap by Truncation

Optical traps (tweezers) are beginning to be used with increasing efficacy in diverse studies in the biological and biomedical sciences. We report here results of a systematic study aimed at enhancing the efficiency with which dielectric (transparent) materials can be optically trapped. Specifically, we investigate how truncation of the incident laser beam affects the strength of an optical trap in the presence of a circular aperture. Apertures of various sizes have been used by us to alter the beam radius, thereby changing the effective numerical aperture and intensity profile. We observe significant enhancement of the radial and axial trap stiffness when an aperture is used to truncate the beam compared to when no aperture was used, keeping incident laser power constant. Enhancement in trap stiffness persists even when the beam intensity profile is modulated. The possibility of applying truncation to multiple traps is explored; to this end a wire mesh is utilized to produce multiple trapping that also alters the effective numerical aperture. The use of a mesh leads to reduction in trap stiffness compared to the case when no wire mesh is used. Our findings lead to a simple-to-implement and inexpensive method of significantly enhancing optical trapping efficiency under a wide range of circumstances.     

Orientation of Red Blood Cells and Rouleaux Disaggregation in Interference Laser Fields

The effect of interference laser fields on red blood cells (RBCs) was investigated both theoretically and experimentally. The optical trapping and orientation of individual RBC in interference fringes were observed. It was found that RBC rouleaux undergo disaggregation under the action of interference laser fields. To describe the effect of RBC orientation in interference fringes, we used the equation for torque exerted on a discoid dielectric particle in a gradient light field. The experimental results are in agreement with the predictions of the developed theoretical model.     

Wavelength dependence of cell cloning efficiency after optical trapping.

A study on clonal growth in Chinese hamster ovary (CHO) cells was conducted after exposure to optical trapping wavelengths using Nd:YAG (1064 nm) and tunable titanium-sapphire (700-990 nm) laser microbeam optical traps. The nuclei of cells were exposed to optical trapping forces at various wavelengths, power densities, and durations of exposure. Clonal growth generally decreased as the power density and the duration of laser exposure increased. A wavelength dependence of clonal growth was observed, with maximum clonability at 950-990 nm and least clonability at 740-760 nm and 900 nm. Moreover, the most commonly used trapping wavelength, 1064 nm from the Nd:YAG laser, strongly reduced clonability, depending upon the power density and exposure time. The present study demonstrates that a variety of optical parameters must be considered when applying optical traps to the study of biological problems, especially when survival and viability are important factors. The ability of the optical trap to alter either the structure or biochemistry of the process being probed with the trapping beam must be seriously considered when interpreting experimental results.     

Controlled rotation of biological microscopic objects using optical line tweezers

Controlled, continuous rotation of cells or intracellular objects was achieved using optical tweezers with an elliptic beam profile (line tweezers), which was generated by placing a cylindrical lens in the path of the trapping beam. By rotating the cylindrical lens, rotation of the elliptic trapping beam and hence of the object trapped therein was achieved. Compared to previously reported techniques for rotation of microscopic objects, this approach is much simpler, gives better utilization of available laser power and also allows much easier control of the trap beam profile. We have used this approach for rotation of biological objects varying in size from 2 to 40 m. At 25 mW trapping beam power at the object plane E. coli bacteria could be rotated at speeds approaching 10 Hz and an intracellular object (presumably a calcium oxalate crystal) trapped inside Elodea densa plant cell could be rotated with speeds of up to 4 Hz. To our knowledge, this is the first report for rotation of an intracellular object.     

Radiation pressure on a biconcave human Red Blood Cell and the resulting deformation in a pair of parallel optical traps

We calculated the three‐dimensional optical stress distribution and the resulting deformation on a biconcave human red blood cell (RBC) in a pair of parallel optical trap. We assumed a Gaussian intensity distribution with a spherical wavefront for each trapping beam and calculated the optical stress from the momentum transfer associated with the reflection and refraction of the incident photons at each interface. The RBC was modelled as a biconcave thin elastic membrane with uniform elasticity and a uniform thickness of 0.25 μm. The resulting cell deformation was determined from the optical stress distribution by finite element software, Comsol Structure Mechanics Module, with Young's modulus (E) as a fitting parameter in order to fit the theoretical results for cell elongation to our experimental data. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Forces of a single-beam gradient laser trap on a dielectric sphere in the ray optics regime

We calculate the forces of single-beam gradient radiation pressure laser traps, also called “optical tweezers,” on micron-sized dielectric spheres in the ray optics regime. This serves as a simple model system for describing laser trapping and manipulation of living cells and organelles within cells. The gradient and scattering forces are defined for beams of complex shape in the ray-optics limit. Forces are calculated over the entire cross-section of the sphere using TEM₀₀ and TEM₀₁^* mode input intensity profiles and spheres of varying index of refraction. Strong uniform traps are possible with force variations less than a factor of 2 over the sphere cross-section. For a laser power of 10 mW and a relative index of refraction of 1.2 we compute trapping forces as high as ~ 1.2 × 10^-6 dynes in the weakest (backward) direction of the gradient trap. It is shown that good trapping requires high convergence beams from a high numerical aperture objective. A comparison is given of traps made using bright field or differential interference contrast optics and phase contrast optics.     

Mitochondrial import driving forces: enhanced trapping by matrix Hsp70 stimulates translocation and reduces the membrane potential dependence of loosely folded preproteins

The mitochondrial heat shock protein Hsp70 (mtHsp70) is essential for driving translocation of preproteins into the matrix. Two models, trapping and pulling by mtHsp70, are discussed, but positive evidence for either model has not been found so far. We have analyzed a mutant mtHsp70, Ssc1-2, that shows a reduced interaction with the membrane anchor Tim44, but an enhanced trapping of preproteins. Unexpectedly, at a low inner membrane potential, ssc1-2 mitochondria imported loosely folded preproteins more efficiently than wild-type mitochondria. The import of a tightly folded preprotein, however, was not increased in ssc1-2 mitochondria. Thus, enhanced trapping by mtHsp70 stimulates the import of loosely folded preproteins and reduces the dependence on the import-driving activity of the membrane potential, directly demonstrating that trapping is one of the molecular mechanisms of mtHsp70 action.     

Probing Red Blood Cell Morphology Using High-Frequency Photoacoustics

A method that can rapidly quantify variations in the morphology of single red blood cells (RBCs) using light and sound is presented. When irradiated with a laser pulse, an RBC absorbs the optical energy and emits an ultrasonic pressure wave called a photoacoustic wave. The power spectrum of the resulting photoacoustic wave contains distinctive features that can be used to identify the RBC size and morphology. When particles 5–10 μm in diameter (such as RBCs) are probed with high-frequency photoacoustics, unique periodically varying minima and maxima occur throughout the photoacoustic signal power spectrum at frequencies >100 MHz. The location and distance between spectral minima scale with the size and morphology of the RBC; these shifts can be used to quantify small changes in the morphology of RBCs. Morphological deviations from the normal biconcave RBC shape are commonly associated with disease or infection. Using a single wide-bandwidth transducer sensitive to frequencies between 100 and 500 MHz, we were able to differentiate healthy RBCs from irregularly shaped RBCs (such as echinocytes, spherocytes, and swollen RBCs) with high confidence using a sample size of just 21 RBCs. As each measurement takes only seconds, these methods could eventually be translated to an automated device for rapid characterization of RBC morphology and deployed in a clinical setting to help diagnose RBC pathology.     

Theoretical model and experimental study of red blood cell (RBC) deformation in microchannels

The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.     

He–Ne laser (632.8 nm) pre‐irradiation gives protection against DNA damage induced by a near‐infrared trapping beam

We report the results of a study carried out to investigate the effect of He–Ne laser (632.8 nm) pre‐irradiation on DNA damage induced by continuous wave 1064 nm trapping beam exposure in MCF‐7 cells. A significant decrease in % tail DNA (p < 0.05) was observed in MCF‐7 cells pre‐exposed to He–Ne laser beam. The dependence of the induced protection against 1064 nm trapping beam irradiation induced DNA damage on the time interval between the two irradiations as well as the He–Ne laser pre‐irradiation parameters is presented. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Self-rotation of red blood cells in optical tweezers: prospects for high throughput malaria diagnosis

A simple and sensitive approach for detection of malarial parasite in blood samples is demonstrated. The approach exploits our finding that, in hypertonic buffer, a normal red blood cell (RBC) rotates by itself when trapped by an optical tweezers. The rotational speed increases linearly at lower trap-beam powers and more rapidly at higher powers. In contrast, under the same experimental conditions, RBC having a malarial parasite does not rotate. The rotational speeds of other RBCs from malaria-infected sample are of an order of magnitude less than that for normal RBC and also increase much more slowly with an increase in trap beam power than that for normal RBC. The difference in rotational speeds could be exploited for the diagnosis of malaria.     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

A practical optical trap for manipulating and isolating bacteria from complex microbial communities

A wide variety of naturally occurring bacteria cannot be isolated by classical microbiological methods, hindering study of microbial communities. To partially remove this limitation, a method using optical trapping was developed. It allows for one-step isolation of bacteria from a complex community to a pure culture. The method transfers a single bacterium to sterile culture medium using a non-destructive laser beam. The ease with which a previously unculturable cell was cultured by trapping suggests that competition may limit growth in some natural communities of bacteria.     

Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.

In the Escherichia coli class Ia ribonucleotide reductase (RNR), the best characterized RNR, there is no spectroscopic evidence for the existence of the postulated catalytically essential thiyl radical (R-S(*)) in the substrate binding subunit R1. We report first results on artificially generated thiyl radicals in R1 using two different methods: chemical oxidation by Ce(IV)/nitrilotriacetate (NTA) and laser photolysis of nitric oxide from nitrosylated cysteines. In both cases, EPR spin trapping at room temperature using phenyl-N-t-butylnitrone, and controls with chemically blocked cysteines, has shown that the observed spin adduct originates from thiyl radicals. The EPR line shape of the protein-bound spin adduct is typical for slow motion of the nitroxide moiety, which indicates that the majority of trapped thiyl radicals are localized in a folded region of R1. In aerobic R1 samples without spin trap that were frozen after treatment with Ce(IV)/NTA or laser photolysis, we observed sulfinyl radicals (R-S(*)=O) assigned via their g-tensor components 2.0213, 2.0094, and 2.0018 and the hyperfine tensor components 1.0, 1.1, and 0.9 mT of one beta-proton. Sulfinyl radicals are the reaction products of thiyl radicals and oxygen and give additional evidence for generation of thiyl radicals in R1 by the procedures used.     

Mixed Exciton-Charge-Transfer States in Photosystem II: Stark Spectroscopy on Site-Directed Mutants

Sickle erythrocytes exhibit abnormal morphology and membrane mechanics under deoxygenated conditions due to the polymerization of hemoglobin S. We employed dissipative particle dynamics to extend a validated multiscale model of red blood cells (RBCs) to represent different sickle cell morphologies based on a simulated annealing procedure and experimental observations. We quantified cell distortion using asphericity and elliptical shape factors, and the results were consistent with a medical image analysis. We then studied the rheology and dynamics of sickle RBC suspensions under constant shear and in a tube. In shear flow, the transition from shear-thinning to shear-independent flow revealed a profound effect of cell membrane stiffening during deoxygenation, with granular RBC shapes leading to the greatest viscosity. In tube flow, the increase of flow resistance by granular RBCs was also greater than the resistance of blood flow with sickle-shape RBCs. However, no occlusion was observed in a straight tube under any conditions unless an adhesive dynamics model was explicitly incorporated into simulations that partially trapped sickle RBCs, which led to full occlusion in some cases.     

Reflection‐mode switchable subwavelength Bessel‐beam and Gaussian‐beam photoacoustic microscopy in vivo

We have developed a reflection‐mode switchable subwavelength Bessel‐beam (BB) and Gaussian‐beam (GB) photoacoustic microscopy (PAM) system. To achieve both reflection‐mode and high resolution, we tightly attached a very small ultrasound transducer to an optical objective lens with numerical aperture of 1.0 and working distance of 2.5 mm. We used axicon and an achromatic doublet in our system to obtain the extended depth of field (DOF) of the BB. To compare the DOF performance achieved with our BB‐PAM system against GB‐PAM system, we designed our system so that the GB can be easily generated by simply removing the lenses. Using a 532 nm pulse laser, we achieved the lateral resolutions of 300 and 270 nm for BB‐PAM and GB‐PAM, respectively. The measured DOF of BB‐PAM was approximately 229 μm, which was about 7× better than that of GB‐PAM. We imaged the vasculature of a mouse ear using BB‐PAM and GB‐PAM and confirmed that the DOF of BB‐PAM is much better than the DOF of GB‐PAM. Thus, we believe that the high resolution achieved at the extended DOF by our system is very practical for wide range of biomedical research including red blood cell (RBC) migration in blood vessels at various depths and observation of cell migration or cell culture.