A hybrid total internal reflection fluorescence and optical tweezers microscope to study cell adhesion and membrane protein dynamics of single living cells

The dynamics of cell surface membrane proteins plays an important role in cell–cell interactions. The onset of the interaction is typically not precisely controlled by current techniques, making especially difficult the visualization of early-stage dynamics. We have developed a novel method where optical tweezers are used to trap cells and precisely control in space and time the initiation of interactions between a cell and a functionalized surface. This approach is combined with total internal reflection fluorescence microscopy to monitor dynamics of membrane bound proteins. We demonstrate an accuracy of ∼2 s in determining the onset of the interaction. Furthermore, we developed a data analysis method to determine the dynamics of cell adhesion and the organization of membrane molecules at the contact area. We demonstrate and validate this approach by studying the dynamics of the green fluorescent protein tagged membrane protein activated leukocyte cell adhesion molecule expressed in K562 cells upon interaction with its ligand CD6 immobilized on a coated substrate. The measured cell spreading is in excellent agreement with existing theoretical models. Active redistribution of activated leukocyte cell adhesion molecule is observed from a clustered to a more homogenous distribution upon contact initiation. This redistribution follows exponential decay behaviour with a characteristic time of 35 s.     

A terahertz scanning near-field optical microscope with an attenuated total internal reflection module

The first terahertz scanning near-field optical microscope with an attenuated total internal reflection module and a free-electron laser (FEL) as the radiation source was developed. A scanning system with positioning using a confocal sensor with chromatic coding and a surface-subwavelength probe touch sensor were developed and tested. A new technique for sensing the distance between the probe and a conducting surface via corona-discharge current measurement was developed. A specific lock-in system for detection of probe-scattered pulse-periodic radiation, which includes a hot-electron superconducting bolometer and an electronic signal-storage circuit, was developed to operate with the Novosibirsk terahertz FEL. All elements of the microscope were tested, and their working capacity was demonstrated. Experiments on the detection of microscope-probe-scattered terahertz radiation have been initiated.     

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K⁺ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.     

Use of the approximations in cell studies by total internal reflection fluorescence microscopy (TIRF)

In determining cell parameters by the use of total internal reflectance fluorescence microscopy it is necessary to evaluate the electric field strength in the neighbourhood of the cell. It has been suggested that the true field distribution be assumed to be of exponential form. In some circumstances, this approximation gives rise to errors and seriously incorrect results are obtained. The true field distribution is easily obtained numerically so the use of an exponential approximation is unnecessary and errors are avoided.     

A prism combination for near isotropic fluorescence excitation by total internal reflection

Total internal reflection fluorescence (TIRF) microscopy is finding increasing application for selectively detecting molecules at or near a glass–water surface. As with all fluorescence methods, the efficiency of excitation of a fluorophore is potentially sensitive to the polarization state of the source. In TIRF, s‐polarized excitation produces an evanescent field that is perpendicular to the incident plane (y direction), whereas p‐polarized light generates a more complex pattern but one dominated by a field that is vertical to the surface (z direction). Thus, fluorophores whose absorption dipoles are fixed in the x direction are not favourably aligned for excitation. Here we describe a beam‐splitting prism arrangement that allows excitation by two orthogonal beams, thus giving isotropic excitation in the x–y plane with s‐polarized light. With linearly polarized light at the magic angle, near isotropic excitation in three dimensions should be achieved. This prism design should find application in polarized fluorescence microscopy to investigate the rotational motions of macromolecules or to minimize flickering of fluorescence emission arising from molecular rotations in single molecule studies.     

Time-gated total internal reflection fluorescence spectroscopy (TG-TIRFS): application to the membrane marker laurdan

A novel setup for total internal reflection fluorescence microscopy with spectral and temporal (nanosecond) resolution was used to measure the emission spectra of the membrane marker laurdan either selectively within the plasma membrane or in whole living cells, depending on the incident angle of the excitation light. With increasing temperature, the intensity of the fluorescence band around 490 nm increased in comparison with the band around 440 nm, which has previously been assigned to a phase transition of membrane lipids from gel to liquid crystalline phase. For a better separation of the overlapping spectral bands, time‐gated detection with a delay of 10–15 ns with respect to the exciting laser pulse was used. As a parameter of membrane dynamics the so‐called generalized polarization GP = (I₄₄₀ ? I₄₉₀)/(I₄₄₀ + I₄₉₀) was evaluated at temperatures between 24 and 41 °C and variable angles of the incident light permitting to excite laurdan molecules either within the plasma membrane or in the whole cell. A decrease of the GP values by ≈ 0.2 units between 28 and 41 °C indicated an increase in membrane fluidity or a decrease in membrane stiffness with increasing temperature. In addition, higher GP values were observed for the plasma membrane as compared with intracellular membranes, probably due to a higher amount of cholesterol. Because properties of the plasma membrane have a large influence on the uptake or release of certain pharmaceutical agents or metabolites, the direct assessment of the dynamics of the plasma membrane by total internal reflection fluorescence spectroscopy appears to be important for pharmacology.     

Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64°, both the dorsal and ventral membranes were illuminated, while images excited above 66° illuminated only focal contacts. Above 74° the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 ± 13 nm. Focal contact areas estimated by TIRFM (1·3 ± 0·7 μm²) agreed closely with areas observed by immunofluorescence staining of vinculin (1·5 ± 0·3 μm²).     

Elimination of the effects of stray light in measurements by total internal reflection aqueous fluorescence (TIRAF)

Total internal reflection aqueous fluorescence has been shown to be capable of achieving spatial resolution in surface contours of about 1 nm. When used with highly structured objects, errors in measurements can arise from light scattered either by the object or within the body of the microscope. We describe how these errors can be eliminated when studying surface contours of human platelets.     

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device

A novel compact illumination device in variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell–substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four‐layer model, as well as a Gaussian‐shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell–substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time‐resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).     

A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy

Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water-immersion objective lens and (4) a prismless arrangement using a high NA oil-immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal-to-background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil-immersion lens gave the brightest image for sample depths < 30 µm but above this limit, a water-immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.     

High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar

Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 10(8) Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.     

Total internal reflection fluorescence imaging using an upconverting cover slip for multicolour evanescent excitation

Total internal reflection fluorescence microscopy is well known as a means of studying surface‐bound structures in cell biology. It is usually measured either by coupling a light source to the sample using a prism or with a special objective where light passing through the periphery of the lens illuminates the contact region beyond the critical angle. In this study we present a new and simple approach to total internal reflection fluorescence microscopy where the sample is mounted on a cover slip prepared from a high‐index upconverting glass‐ceramic. Excitation of the cover slip with a low‐cost near‐infrared laser diode generates intense narrow‐band visible emission within the cover slip, some of which is totally internally reflected. This emission gives rise to an evanescent wave at the interface and hence can excite surface‐bound fluorescent species. Depending on the excitation conditions the cover slip can generate violet, green and red emission and hence can excite a wide range of fluorescent labels. Fluorescence emission from the sample can be detected in spectral regions where the direct emission from the cover slip is very weak. The advantages and limitations of the technique are discussed in comparison with conventional total internal reflection fluorescence microscopy measurements and prospects for novel total internal reflection fluorescence microscopy geometries are considered.     

Non-optically probing near-field microscopy with illumination of total internal reflection

We have developed a non-optically probing near-field microscope with illumination of total internal reflection. Because the illumination light does not pass through the specimens, it is possible to observe thick specimens or highly absorptive materials. It reduces the background noise because the decay length of the evanescent wave is a few hundred nanometres. We found that although in the total internal reflection illumination system the light passed through the photosensitive film and illuminated the specimen, it did not affect the photosensitive film severely and did not limit the resolution. The imaging properties of reflection illumination and transmission illumination are analysed using a finite-differential time domain method.     

Maximum allowable load of atomic force microscope (AFM) nanorobot

In this paper, maximum load-carrying capacity algorithm is developed for atomic force microscope (AFM) nanorobot. Considering real-time control limitation for micro nano manufacture process, predicting possibility of this manufacture process is really important. Main limitation in this method is cantilever’s geometry. Finite element method is used for finding effect of variation in geometry parameters. Cantilever’s twist angle has a relationship with cantilever geometry; we use this point as a criterion for finding maximum load-carrying capacity. Changing in this parameter can be realized by optic sensor that is used in AFM nanorobot. Finite element result is used for modeling geometrical effect. Finally, an algorithm to determine maximum load-carrying capacity with considering finite element result and for different cantilever geometries is presented; thus, with using these simulations, particle size can be estimated.     

基于受抑全反射的碱金属气室镀膜厚度测量

针对原子自旋器件的碱金属气室镀膜层厚度的精确测量,提出了一种基于受抑全反射的膜层厚度测量方法。根据该方法搭建了膜厚测量系统,并进行了实验测试。分析了受抑全反射的基本理论和基于受抑全反射的膜厚测量原理,介绍了基于该方法的膜厚测量系统的构成及工作原理并分析了影响系统测量精度的主要因素和解决方案。通过分析和仿真激光器波长的波动、入射角变化以及折射率参数的不准确等对膜厚测量结果的影响评价了系统的性能。最后,利用该系统对镀膜样品进行了测量实验,并利用薄膜分析仪做了对比试验。实验结果表明:该方法的测量结果存在一个2.6nm左右的常值偏差,对其补偿后能够较为准确地对镀膜层厚度进行测量,测量精度接近1nm,基本满足碱金属气室镀膜质量检测的需求,且具有较高的稳定性和可靠性。     

原子力显微镜(AFM)在光盘检测及其质量控制中的应用

综述了原子力显微镜(AFM)在光盘质量检测中的应用.AFM能够在nm尺度上直接对光盘及其模板上的信息位几何结构的特征尺寸及其误差进行三维测量,从而可以建立生产工艺参数和信息位几何结构之间、信息位几何结构和盘片电气性能之间的关系,进而找出影响光盘质量的直接原因.用AFM进行光盘质量检测主要有三方面：盘片和模板表面的定性观测;信息位几何结构的半定量分析;信息位特征尺寸的统计分析.定性观测和半定量分析可以对盘片播放的高误差率、凹坑形态和块出错率、凸台形态及其表面粗糙度等参数进行有针对性的检测;而信息位特征尺寸的统计分析则可以对信息位几何结构的关键参数进行面向生产过程的统计分析.所得结论表明AFM在光盘质量检测过程中具有独特的优势.     

Dynamic analysis of tapping mode atomic force microscope (AFM) for critical dimension measurement

Transient dynamics of tapping mode atomic force microscope (AFM) for critical dimension measurement are analyzed. A simplified nonlinear model of AFM is presented to describe the forced vibration of the micro cantilever-tip system with consideration of both contact and non-contact transient behavior for critical dimension measurement. The governing motion equations of the AFM cantilever system are derived from the developed model. Based on the established dynamic model, motion state of the AFM cantilever system is calculated utilizing the method of averaging with the form of slow flow equations. Further analytical solutions are obtained to reveal the effects of critical parameters on the system dynamic performance. In addition, features of dynamic response of tapping mode AFM in critical dimension measurement are studied, where the effects of equivalent contact stiffness, quality factor and resonance frequency of cantilever on the system dynamic behavior are investigated. Contact behavior between the tip and sample is also analyzed and the frequency drift in contact phase is further explored. Influence of the interaction between the tip and sample on the subsequent non-contact phase is studied with regard to different parameters. The dependence of the minimum amplitude of tip displacement and maximum phase difference on the equivalent contact stiffness, quality factor and resonance frequency are investigated. This study brings further insights into the dynamic characteristics of tapping mode AFM for critical dimension measurement, and thus provides guidelines for the high fidelity tapping mode AFM scanning.     

Application of frustrated total reflection for studying cells in vitro

A mathematical analysis of the dependence of the light flux scattered upon frustrated total reflection (FTR) on the angle of incidence of the light beam is presented for monolayers of spread and spherical cells. It is shown that using these relationships it is possible to estimate the refractive index of hyaloplasm at cell-substrate contacts and to study the flattening of the cell surface facing the glass. Analysis of FTR for single cells is presented. A condition is given that must be fulfilled for the brightly shining regions of the cell to be interpreted as the sites of attachment of the cell surface to glass. The condition defines the range of angles of incidence at which FTR does not depend on the optical properties of the cytoplasm portion being at a distance from the glass. The fulfillment of this condition is necessary for spectroscopic and ellipsometric studies of cell surface contacting the glass, by means of FTR. The conclusions are confirmed by experimental data. A peculiarity of light scattering by cells, associated with the coherence of the incident light beam is discussed.     

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high‐resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non‐polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR‐FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP‐vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR‐FM and wide‐field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR‐FSM affords major improvements in these parameters compared with wide‐field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low‐level GFP‐fusion protein expression is a useful tool for FSM imaging. When used in time‐lapse mode, TIR‐FSM of actin and GFP‐conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.