Alignment of biological microparticles by a polarized laser beam

The optical alignment of biological samples is of great relevance to microspectrometry and to the micromanipulation of single particles. Recently, Bayoudh et al. (J. Mod. Opt. 50:1581–1590, 2003) have shown that isolated, disk-shaped chloroplasts can be aligned in a controlled manner using an in-plane-polarized Gaussian beam trap, and suggested that this is due to their nonspherical shape. Here we demonstrate that the orientation of various micrometer-sized isolated biological particles, trapped by optical tweezers, can be altered in a controlled way by changing the plane of linear polarization of the tweezers. In addition to chloroplasts, we show that subchloroplast particles of small size and irregular overall shape, aggregated photosynthetic light-harvesting protein complexes as well as chromosomes can be oriented with the linearly polarized beam of the tweezers. By using a laser scanning confocal microscope equipped with a differential polarization attachment, we also measured the birefringence of magnetically oriented granal chloroplasts, and found that they exhibit strong birefringence with large local variations, which appears to originate from stacked membranes. The size and sign of the birefringence are such that the resulting anisotropic interaction with the linearly polarized laser beam significantly contributes to the torque orienting the chloroplasts.     

Directed Positioning of Micronuclei in Paramecium tetraurelia with Laser Tweezers: Absence of Detectible Damage After Manipulation

Possible covert damage from the use of the laser optical force trap (laser tweezers) to reposition micronuclei in Paramecium tetraurelia was assessed by measuring proliferation rates and postautogamous survival and mutation rates of cells after laser manipulations. No differences in subsequent daily proliferation rates among laser manipulated and various control classes of cells were seen. Similarly, the rates of postautogamous lethality and of “slow growth mutations” after repositioning of both micronuclei were not different from such rates in unmanipulated controls. In spite of extensive manipulations of micronuclei by the laser tweezers, there is no evidence of any damage induced by these manipulations. The laser tweezers therefore appears to be a tool of benign effect upon living cells, with tremendous potential use in many cell and developmental biological investigations.     

激光捕捉和悬浮生物微粒的原理

利用不均匀分布光束的横向梯度,对微粒产生光学梯度力,建立光学势阱,可用来捕捉和悬浮细胞一类的生物微粒,起到光镊作用。     

Mechanical manipulation of bone and cartilage cells with 'optical tweezers'.

The single beam optical gradient trap (optical tweezers) uses a single beam of laser light to non-invasively manipulate microscopic particles. Optical tweezers exerting a force of approximately 7 pN were applied to single bone and cartilage derived cells in culture and changes in intracellular calcium levels were observed using Fluo-3 labelling. Human derived osteoblasts responded to optical tweezers with an immediate increase in [Ca2+]i that was inhibited by the addition of a calcium channel blocker nifedipine. Force applied to different regions of cells resulted in a variable response. [Ca2+]i elevation in response to load was lower in rat femur derived osteoblasts, and not apparent in primary chondrocytes and the osteocytic cell line (MLO Y4).     

Physiological monitoring of optically trapped cells: assessing the effects of confinement by 1064-nm laser tweezers using microfluorometry.

We report the results of microfluorometric measurements of physiological changes in optically trapped immotile Chinese hamster ovary cells (CHOs) and motile human sperm cells under continuous-wave (CW) and pulsed-mode trapping conditions at 1064 nm. The fluorescence spectra derived from the exogenous fluorescent probes laurdan, acridine orange, propidium iodide, and Snarf are used to assess the effects of optical confinement with respect to temperature, DNA structure, cell viability, and intracellular pH, respectively. In the latter three cases, fluorescence is excited via a two-photon process, using a CW laser trap as the fluorescence excitation source. An average temperature increase of < 0.1 +/- 0.30 degrees C/100 mW is measured for cells when held stationary with CW optical tweezers at powers of up to 400 mW. The same trapping conditions do not appear to alter DNA structure or cellular pH. In contrast, a pulsed 1064-nm laser trap (100-ns pulses at 40 microJ/pulse and average power of 40 mW) produced significant fluorescence spectral alterations in acridine orange, perhaps because of thermally induced DNA structural changes or laser-induced multiphoton processes. The techniques and results presented herein demonstrate the ability to perform in situ monitoring of cellular physiology during CW and pulsed laser trapping, and should prove useful in studying mechanisms by which optical tweezers and microbeams perturb metabolic function and cellular viability.     

光镊和拉曼光镊在生物医学研究中的应用

光镊是由美国科学家Arthur Ashkin于1986年发明的,是一种利用高度汇聚的激光束产生的三维梯度势阱来俘获、操纵微小粒子的技术。因其可俘获、操纵单个细胞,并在细胞和亚细胞层次上为生物医学研究提供方便,近年来,已越来越多地被应用于生物医学研究中。本文在介绍光镊的原理和特点的基础上,阐述了光镊(尤其是拉曼光镊)技术在生物医学领域中的研究进展、现状和展望。     

高斯光束中细胞横向受力分析

按照几何光学的原理建立了高斯光束中细胞受力的力学模型,并利用数值计算,得到了细胞偏离光轴受到的向轴回复力大小与细胞的离轴距离X0、直径2R等的关系.结果表明高斯光束对大小不同的细胞有相同的光学势阱宽度,对较大细胞势阱较深所以较容易稳定俘获.讨论了光钳设计中注意的问题.     

Quantitative measurements of force and displacement using an optical trap.

We combined a single-beam gradient optical trap with a high-resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and piconewton forces with millisecond resolution. When an external force is applied to a micron-sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to the applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond time scale, and thus a trapped bead can be used as a force transducer. The performance can be enhanced by a feedback circuit so that the position of the trap moves by means of acousto-optic modulators to exert a force equal and opposite to the external force applied to the bead. In this case the position of the trap can be used to measure the applied force. We consider parameters of the trapped bead such as stiffness and response time as a function of bead diameter and laser beam power and compare the results with recent ray-optic calculations.     

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.     

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.     

Versatile laser‐based cell manipulator

Here we describe a two‐photon microscope and laser ablation setup combined with optical tweezers. We tested the setup on the fission yeast Schizosaccharomyces pombe, a commonly used model organism. We show that long‐term imaging can be achieved without significant photo‐bleaching or damage of the sample. The setup can precisely ablate sub‐micrometer structures, such as microtubules and mitotic spindles, inside living cells, which remain viable after the manipulation. Longer exposure times lead to ablation, while shorter exposures lead to photo‐bleaching of the target structure. We used optical tweezers to trap intracellular particles and to displace the cell nucleus. Two‐photon fluorescence imaging of the manipulated cell can be performed simultaneously with trapping. The combination of techniques described here may help to solve a variety of problems in cell biology, such as positioning of organelles and the forces exerted by the cytoskeleton. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

All-optical constant-force laser tweezers

Optical tweezers are a powerful tool for the study of single biomolecules. Many applications require that a molecule be held under constant tension while its extension is measured. We present two schemes based on scanning-line optical tweezers to accomplish this, providing all-optical alternatives to force-clamp traps that rely on electronic feedback to maintain constant-force conditions for the molecule. In these schemes, a laser beam is rapidly scanned along a line in the focal plane of the microscope objective, effectively creating an extended one-dimensional optical potential over distances of up to 8 microm. A position-independent lateral force acting on a trapped particle is created by either modulating the laser beam intensity during the scan or by using an asymmetric beam profile in the back focal plane of the microscope objective. With these techniques, forces of up to 2.69 pN have been applied over distances of up to 3.4 microm with residual spring constants of <26.6 fN/microm. We used these techniques in conjunction with a fast position measurement scheme to study the relaxation of lambda-DNA molecules against a constant external force with submillisecond time resolution. We compare the results to predictions from the wormlike chain model.     

Preferential attachment of membrane glycoproteins to the cytoskeleton at the leading edge of lamella

The active forward movement of cells is often associated with the rearward transport of particles over the surfaces of their lamellae. Unlike the rest of the lamella, we found that the leading edge (within 0.5 microns of the cell boundary) is specialized for rearward transport of membrane-bound particles, such as Con A-coated latex microspheres. Using a single-beam optical gradient trap (optical tweezers) to apply restraining forces to particles, we can capture, move and release particles at will. When first bound on the central lamellar surface, Con A-coated particles would diffuse randomly; when such bound particles were brought to the leading edge of the lamella with the optical tweezers, they were often transported rearward. As in our previous studies, particle transport occurred with a concurrent decrease in apparent diffusion coefficient, consistent with attachment to the cytoskeleton. For particles at the leading edge of the lamella, weak attachment to the cytoskeleton and transport occurred with a half-time of 3 s; equivalent particles elsewhere on the lamella showed no detectable attachment when monitored for several minutes. Particles held on the cell surface by the laser trap attached more strongly to the cytoskeleton with time. These particles could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/- 14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere on the lamella. Fluorescent succinylated Con A staining showed no corresponding concentration of general glycoproteins at the leading edge, but cytochalasin D-resistant filamentous actin was found at the leading edge. Our results have implications for cell motility: if the forces used for rearward particle transport were applied to a rigid substratum, cells would move forward. Such a mechanism would be most efficient if the leading edge of the cell contained preferential sites for attachment and transport.     

Development of a dual joystick‐controlled laser trapping and cutting system for optical micromanipulation of chromosomes inside living cells

A multi‐joystick robotic laser microscope system used to control two optical traps (tweezers) and one laser scissors has been developed for subcellular organelle manipulation. The use of joysticks has provided a “user‐friendly” method for both trapping and cutting of organelles such as chromosomes in live cells. This innovative design has enabled the clean severing of chromosome arms using the laser scissors as well as the ability to easily hold and pull the severed arm using the laser tweezers. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

Detection of forces and displacements along the axial direction in an optical trap

We present measurements of the forces on, and displacements of, an optically trapped bead along the propagation direction of the trapping laser beam (the axial direction). In a typical experimental configuration, the bead is trapped in an aqueous solution using an oil-immersion, high-numerical-aperture objective. This refractive index mismatch complicates axial calibrations due to both a shift of the trap center along the axial direction and spherical aberrations. In this work, a known DNA template was unzipped along the axial direction and its characteristic unzipping force-extension data were used to determine 1), the location of the trap center along the axial direction; 2), the axial displacement of the bead from the trap center; and 3), the axial force exerted on the bead. These axial calibrations were obtained for trap center locations up to approximately 4 microm into the aqueous solution and with axial bead displacements up to approximately 600 nm from the trap center. In particular, the axial trap stiffness decreased substantially when the trap was located further into the aqueous solution. This approach, together with conventional lateral calibrations, results in a more versatile optical trapping instrument that is accurately calibrated in all three dimensions.     

Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle's displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads.     

不同微生物的单光束激光陷阱操纵

本文报导了分别采用Ｈｅ－Ｎｅ和Ａｒ＾＋激光器与光不显微镜构成的单光束激光陷阱操纵酵母菌、青霉等不同微生物的实验观察结果,讨论了操纵条件。研究表明：用单光束高会聚激光产生的梯度力操纵微生物体,其有效作用力的大小不仅与激光功率、波长、束腰半径和光束会聚角有关,还与微生物的大小、吸收系数、菌龄及培养方法等因素有关。     

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.     

光捕捉活细胞染色体

本文综合报道了作者近数年来以PTK_2细胞为实验材料,用Nd:YAG激光器所发射的1.06微米波长和氩离子泵浦Titanium-Sapphire激光所发射的700—760毫微米波长的连续激光微光束作为光捕捉在显微操作染色体方面的一些主要实验结果。所得结果表明光捕捉可诱发中期细胞的落后染色体向中期板加速移动,抓住后期细胞的一对染色体,使其停留在中期板保持静止不动,而其余的染色体对照常进行染色单体的分离並移向两极,在后期一直用光捕捉抓住的那对染色单体,最终在胞质分裂时将进入一个子细胞,或丢失在分裂沟中或两染色单体分开,各自分别进入原相对的子细胞。作为光捕捉Titanium-Sapphire激光器发射的700—760毫微米波长的激光束,比Nd:YAG激光的1.06微米波长能在更高的输出能量水平下操作而产生较小的对细胞损伤的副作用,从而更容易操作染色体。在适宜的输出能量水平下操作,光捕捉不会对细胞造成损伤,受光捕捉的细胞一般都能继续分裂直至形成两个子细胞。实验结果证明光捕捉技术是一项研究活细胞纺锤体、染色体运动等细胞生物学问题而又不损伤细胞的良好工具。光捕捉技术也可能对诱发单体、三体细胞,研究细胞遗传提供新的手段。