Fast position measurements with scanning line optical tweezers

Scanning line optical tweezers are a powerful tool for the study of colloidal or biomolecular systems in the low-force regime. We present a fast, high-resolution particle position measurement scheme that extends the capabilities of these instruments into the realm of dynamic measurements. The technique is based on synchronous detection of forward-scattered laser light during a line scan. We demonstrate a position resolution of better than 50 nm for bandwidths of as much as 40 kHz for pairs of microspheres trapped in a flat line potential at center-to-center separations of 1.7-6 microm.     

Speckle in confocal scanning transmission optical microscopes

The formation of speckle patterns in confocal scanning transmission optical microscopes is examined. Using a gaussian approximation, expressions for the contrast in both in and out of focus planes are derived in terms of the statistical parameters of thin phase screens. The results are illustrated by examples in which the formulae are evaluated numerically and compared to experimental data.     

Optical tweezers for confocal microscopy

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

Imaging biological specimens with the confocal scanning laser microscope/macroscope

A new confocal scanning laser microscope/macroscope (cslm/M) has recently been developed. It combines in one instrument the high resolution capability of a confocal scanning beam microscope for imaging small specimens, with good resolution confocal imaging of macroscopic specimens. Some of its main features include: (a) 0.25 μm lateral resolution in the microscope mode and 5 μm lateral resolution in the macroscope mode; (b) a field of view that can vary from 25 μm × 25 μm to 75,000 μm × 75,000 μm; (c) capability for acquiring large data sets from 512 × 512 pixels to 2048 × 2048 pixels; (d) 0.5 μm depth resolution in the microscope mode and 200 μm depth resolution in the macroscope mode.

In this work the cslm/M was used to image whole biological specimens (> 5 m diameter), including insects which are ideal specimens for the macroscope. Specimens require no preparation, unlike scanning electron microscope (SEM) specimens which require a conductive coating. The specimens described in this paper are too large to be imaged in their entirety by a scanning beam laser microscope, however they can be imaged by slower scanning stage microscopes. In the macroscope mode the cslm/M was used to acquire a large number (e.g. 20–40) of confocal image slices which were then used to reconstruct a three-dimensional image of the specimen. High resolution images were collected by the cslm/M by switching to the microscope mode where high numerical aperture (NA) objectives were used to image a small area of interest. Reflected-light and fluorescence images of plant and insect specimens are presented which demonstrate the morphological details obtained in various imaging modes. A process for three-dimensional visualization of the data is described and images are shown.     

Resolution and contrast in confocal optical scanning microscopes

A theoretical study is made of a simple method of obstructing an amplitude pupil which results in improved microscope resolution with only a little degradation of the image contrast. Using a confocal scanning optical microscope the resultant point spread function is calculated, from which the image of a point object is deduced. The calculations are made for two different apertures — the first has four-fold symmetry and the second has eight-fold symmetry — and are compared with full circular and annular apertures.     

共焦扫描光学显微镜的高分辨率

讨论了共焦扫描光学显微镜的高分辨率性质,指出共焦扫描显微镜由于采用点探测器,因而视场大大减小,信噪比大大提高,同时每幅图像逐点扫描形成,在光学系统信息能力不变的前提下,系统的空间域通带宽度增加和时域通带宽度减小。因而可成高分辨率的像,特别是其独特的深度分辨率特性使得可以实现光学断层扫描成像。给出了所研制的共焦扫描荧光显微镜所获得光学断层扫描图像     

远场光钳与近场光钳的发展

以经典光学为基础的光钳技术(又称"光镊")在生物、物理和化学等领域得到了广泛的发展和应用,但是该技术受到高倍显微物镜的尺寸和光学衍射极限等多种因素的制约,从而限制了其进一步发展。而远场光纤光钳和近场光钳技术,从不同方面克服了传统光钳的局限。回顾了传统光钳、远场光纤光钳和近场光钳的发展,着重讨论了各种方法的工作原理、实验方法和技术性能,对这几种光钳技术进行了深入地总结和细致地比较。     

Three-dimensional orientation of microscopic objects using combined elliptical and point optical tweezers

We report the development of a simple approach for controlled three-dimensional orientation of microscopic objects. The approach makes use of the fact that whereas in an elliptical trap, an object lacking spherical symmetry orients with its long axis along the major axis of the elliptical tweezers, in point tweezers it orients with its long axis along the trapping beam. Therefore, a combined use of point and elliptical tweezers and a control over the relative power of the two trapping beams can be used for three-dimensional orientation of the object. The use of this approach for three-dimensional orientation of biological objects varying in size from 2 to 20 μm is discussed. PACS 07.60.-j; 87.80.Cc     

Imaging properties and applications of scanning optical microscopes

This review paper is concerned with the imaging properties and major uses of scanning optical microscopes. It is shown that the confocal scanning microscope exhibits a form of super-resolution and that the instrument in general has great application in nonlinear microscopy and the inspection of electronic devices.     

A confocal laser scanning microscopic study on thermoresponsive binary microgel dispersions incorporated with CdTe quantum dots

Monodisperse poly(N-isopropylacrylamide) (PNIPAM) particles loaded with cadmium telluride (CdTe) quantum dots (QDs) of two different sizes (4.7 nm and 5.6 nm) were synthesized in aqueous medium by bonding the capping agent on the quantum dots to the amide groups of PNIPAM and incubating the samples at 45°C. A huge increase in the photoluminescence (PL) intensity (green and red regions) is observed for the PNIPAM-CdTe QDs composites compared to the parent CdTe QDs. We report here for the first time the imaging of binary dispersion of green and red luminescent PNIPAM-CdTe QDs composites using a fluorescence confocal laser scanning microscope. These composites have potential applications both in material science and biology.     

Potential mapping of optical tweezers

Optical tweezers are very often used for measurement of piconewton range forces. Depending on the displacement of the trapped bead, the trap may become stiffer which causes considerable underestimation of the measured force. We have shown, both by theory and experiment, that such a stiffening occurs for beads larger than 0.5 μm in radius. For the first time, we have shown that the displacement at which the stiffening starts is size dependent and that the stiffening starts at higher forces for larger beads. We have shown that for the applications, which simultaneous force measurement and position sensing are on demand (such as biopolymer stretching), mid-range sized (～1.5 μm in radius) beads could be the best choice.     

Optical doughnut for optical tweezers

We describe novel optical doughnuts for optical tweezers. With new phase functions, the proposed doughnut beams have dark cores in specified shapes. The technique can offer a simple method for creating a variety of beam shapes to match the trapped objects. One can rotate the beams directly by revolving their phase structures about their axes on the initial plane. The technique for generating the traditional Laguerre-Gaussian beam can be used to create these novel beams.     

利用光镊技术演示光的自旋角动量

阐述了光与物体相互作用时自旋角动量的传递与扭力矩原理.基于光镊光致旋转原理,利用能够悬浮单个粒子的光镊技术并采用具有双折射特性的CaCO3晶体粒子,设计了微粒在不同偏振光场中的旋转运动实验内容,研究光与双折射晶体粒子相互作用产生的光致旋转效应,观察和测量由自旋角动量引起粒子的扭转力矩的大小、方向以及旋转速度等力学效应.     

Phase-transition-like properties of double-beam optical tweezers

We report on double-beam optical tweezers that undergo previously unknown phase-transition-like behavior resulting in the formation of more optical traps than the number of beams used to create them. We classify the optical force fields which produce multiple traps for a double-beam system including the critical behavior. This effect is demonstrated experimentally in orthogonally polarized (noninterfering) dual-beam optical tweezers for a silica particle of 2.32 μm diameter. Phase transitions of multiple beam trapping systems have implications for hopping rates between traps and detection of forces between biomolecules using dual-beam optical tweezers. It is an example of a novel dynamic system with multiple states where force fields undergo a series of sign inversions as a function of parameters such as size and beam separation.     

Polarization-induced stiffness asymmetry of optical tweezers

A tightly focused, linearly polarized laser beam, so-called optical tweezers, is proven to be a useful micromanipulation tool. It is known that there is a stiffness asymmetry in the direction perpendicular to the optical axis inherited from the polarization state of the laser. In this Letter, we report our experimental results of stiffness asymmetry for different bead sizes measured at the optimal trapping condition. We also provide the results of our generalized Lorenz-Mie based calculations, which are in good agreement with our experimental results. We also compare our results with previous reports.     

Planar optical tweezers using tapered-waveguide junctions

We demonstrate planar optical tweezers using the evanescent field of a silicon nitride tapered-waveguide junction between a singlemode waveguide and a multimode waveguide. Our experiments show that the junction embedded in a fluidic channel holds up to one and two polystyrene particles of sizes of 2.2 μm and 1 μm, respectively. The trapped particles are successively substituted by the incoming particles. Our experiments and numerical modeling reveal that the junction particle trapping depends on particle size and number.     

Dual-wedge scanning confocal reflectance microscope

A confocal reflectance microscope has been developed that incorporates a dual-wedge scanner to reduce the size of the device relative to current raster scanning instruments. The scanner is implemented with two prisms that are rotated about the optical axis. Spiral and rosette scans are performed by rotating the prisms in the same or opposite directions, respectively. Experimental measurements show an on-axis lateral resolution of 1.6 microm and optical sectioning of 4.7 microm, which compares with a diffraction-limited resolution of 0.8 and 1.9 microm, respectively.     

New calibration method for position detector for simultaneous measurements of force constants and local viscosity in optical tweezers

We present a new method to calibrate a quadrant photodiode used as position detector to monitor latex beads trapped in optical tweezers. The method combines the dragging Stoke’s force with the thermal noise analysis (power spectral density (PSD)) of the Brownian motion of the trapped bead. Position detector calibrations used in other similar methods normally utilise a bead attached to the coverslip: the voltage-position calibration factor is found by translating the bead across the waist of a laser beam. The so determined calibration factor is then assumed to be the same when beads are investigated in the optical trap. This procedure presents some drawbacks since attached beads can be affected by proximity effects due to the coverslip glass surface which alter the position sensor response itself. On the contrary, our method is able to provide, simultaneously, the calibration factor, the trap stiffness, and the local viscosity of the medium making use of a single trapped bead.     

Micromachined transmissive scanning confocal microscope

We have developed a miniature scanning confocal microscope that uses electrostatically actuated microlenses for focusing and scanning. Objective lenses, scanners, a pupil, and a pinhole of the confocal microscope are microfabricated and integrated into a volume smaller than 2 mm3 by stacking these components. Objective lenses are composed of two vertically cascaded polymer microlenses integrated into micromachined comb actuators. Raster scanning is implemented by electrostatically actuating each microlens in orthogonal directions. We have demonstrated reflection confocal imaging with 3-microm transverse resolution over a 100-microm field of view and a 0.38-mm working distance at lambda = 633 nm.