Development of a standing-wave fluorescence microscope with high nodal plane flatness

This article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness. As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible. The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique. The instrument developed uses a green-emitting helium–neon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces. The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods. To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655, 1996, 18–25). To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen. A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.     

Method of calibration of a fluorescence microscope for quantitative studies

Confocal microscopy is based on measurement of intensity of fluorescence originating from a limited volume in the imaged specimen. The intensity is quantized in absolute (albeit arbitrary) units, producing a digital 3D micrograph. Thus, one may obtain quantitative information on local concentration of biomolecules in cells and tissues. This approach requires estimation of precision of light measurement (limited by noise) and conversion of the digital intensity units to absolute values of concentration (or number) of molecules of interest. To meet the first prerequisite we propose a technique for measurement of signal and noise. This method involves registration of a time series of images of any stationary microscope specimen. The analysis is a multistep process, which separates monotonic, periodic and random components of pixel intensity change. This approach permits simultaneous determination of dark and photonic components of noise. Consequently, confidence interval (total noise estimation) is obtained for every level of signal. The algorithm can also be applied to detect mechanical instability of a microscope and instability of illumination source. The presented technique is combined with a simple intensity standard to provide conversion of relative intensity units into their absolute counterparts (the second prerequisite of quantitative imaging). Moreover, photobleaching kinetics of the standard is used to estimate the power of light delivered to a microscope specimen. Thus, the proposed method provides in one step an absolute intensity calibration, estimate of precision and sensitivity of a microscope system.     

Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope

Three levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu- and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi-parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co-localization of nuclear components. DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi-stranded). We have assessed by laser scanning microscopy the presence of left-handed Z-DNA in polytene chromosomes of Diptera as well as the spatio-temporal distribution of Z-DNA binding proteins in whole-mount Drosophila embryos and ovaries. We have determined the 3-D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti-BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.     

Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements

A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.     

Development of a shear force scanning near-field fluorescence microscope for biological applications

In this paper, a shear force scanning near-field fluorescence microscope combined with a confocal laser microspectrofluorometer is described. The shear force detection is realized based on a bimorph cantilever, which provides a very sensitive, reliable, and easy to use method to control the probe-sample distance during scanning. With the system, high-quality shear force imaging of various samples has been carried out. Furthermore, simultaneous shear force and near-field fluorescence imaging of biological cells has also been realized. As an example, we especially present the result on the distribution of P-glycoprotein in the plasma membrane of human small cell lung cancer cells, suggesting that the system would be a promising tool for biological applications.     

A standard video-rate confocal laser-scanning reflection and fluorescence microscope

A confocal laser-scanning microscope (CLSM) differs from a conventional microscope by affording an extreme depth discrimination, as well as a slightly improved resolution. These features afford improved imaging, and make possible new imaging techniques. The CLSM developed at TNO has standard video-rate imaging, and is capable of working in reflection and in fluorescence mode simultaneously. Nonconfocally the laser-scanning microscope can also be used in transmission mode. In addition to the evident advantages of a fast system when searching objects or studying living objects, the time needed to produce an image of extended depth of focus and high resolution is very short. Furthermore, the high-speed averaging of many images at low laser-power levels, and the short dwelling time of the focused laser beam (60 ns) obviate quenching effects in fluorescence microscopy and prevent damage to the object. In this article the TNO-CLSM system is outlined. The most important specifications are summarized, and some representative micrographs obtained with the system are shown. Furthermore, the performance of the system is illustrated by some experimental results.     

光电测量设备光学系统的像面照度均匀性检测

提出了一种光电测量设备光学系统像面照度均匀性的检测方法.该方法首先对被检设备的CCD进行标定,然后用给出的均匀性检测系统对整机光学系统进行检测.该检测系统利用积分球产生均匀背景光信号,将光测设备置于积分球通光口处,利用计算机采集CCD图像.根据CCD的标定结果,用两点多段校正算法对采集到的图像进行校正,通过对校正后的图像进行分析,得到像面照度的不均匀度.此外,将图像分为多个区域,计算各个区域的灰度与像面中心区域灰度的比值,得出图像的灰度分布,实现了光电测量设备光学系统像面照度均匀性的室内检测.利用研制的光电测量设备光学系统像面照度均匀性检测系统对某型号光电测量设备进行了检测,得到了该设备光学系统像面照度不均匀度达到18.7%,不满足不均匀度≤10%的要求,应对该光测设备进行重新装调.在重新装调后对该设备进行复检,得到重新装凋后的不均匀度为6.7%,像面照度均匀性得到了提高,验证了该方法的有效性.     

Combination of AFM with an objective-type total internal reflection fluorescence microscope (TIRFM) for nanomanipulation of single cells

A new instrument was constructed by combining an objective-type total internal reflection fluorescence microscope with an atomic force microscope (AFM). Our purpose of constructing such an instrument is to detect and confirm the result of cellular level manipulations made with the AFM part through the detection system of the highly sensitive fluorescence microscope part. In this combination, manipulations are now possible from the nanometer to the micrometer scales and the fluorescence detection system is sensitive enough even for localizing single molecules. In this paper, we applied the system as a precise intracellular injector (nanoplanter). Fluorescent beads were first chemically immobilized onto a ZnO whisker that was glued to an AFM tip and were injected into a living BALB/3T3 cell together with the whisker. It was demonstrated that the system could clearly show the result of injection, that is, the presence of a small number of fluorescent beads in the cell.     

LED illumination for video-enhanced DIC imaging of single microtubules

In many applications high‐resolution video‐enhanced differential interference contrast microscopy is used to visualize and track the ends of single microtubules. We show that single ultrabright light emitting diodes from Luxeon can be used to replace conventional light sources for these kinds of applications without loss of function. We measured the signal‐to‐noise ratio of microtubules imaged with three different light emitting diode colours (blue, red, green). The blue light emitting diode performed best, and the signal‐to‐noise ratios were high enough to automatically track the ends of dynamic microtubules. Light emitting diodes as light sources for video‐enhanced differential interference contrast microscopy are high performing, low‐cost and easy to align alternatives to existing illumination solutions.     

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 scanning near-field optical microscope having scanning electron tunnelling microscope capability using a single metallic probe tip

A new microscope system that has the combined capabilities of a scanning near-field optical microscope (SNOM) and a scanning tunnelling microscope (STM) is described. This is achieved with the use of a single metallic probe tip. The distance between the probe tip and the sample surface is regulated by keeping the tunnelling current constant. In this mode of operation, information about the optical properties of the sample, such as its refractive index distribution and absorption characteristics, can be disassociated from the information describing its surface structure. Details of the surface structure can be studied at resolutions smaller than the illumination wavelength. The performance of the microscope is evaluated by analysing a grating sample that was made by coating a glass substrate with gold. The results are then compared with the corresponding SNOM and STM images of the grating.     

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.     

Evanescent field excitation and measurement of dye fluorescence in a metallic probe near-field scanning optical microscope

We introduce a method of dye fluorescence excitation and measurement that utilizes a near-field scanning optical microscope (NSOM). This NSOM uses an apertureless metallic probe, and an optical system that contains a high numerical aperture (NA) objective lens (NA= 1.4). When the area which satisfies NA < 1 is masked, the objective lens allows for the rejection of possible transmitted light (NA < 1) through the sample. In such conditions, the focused spot consists of only the evanescent field. We found that this NSOM system strongly reduces the background of the dye fluorescence and allows for the measurement of the fluorescence intensity below the diffraction limit of the excitation source.     

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).     

Basic parameters of front-end microelectronic units of detector electronics

Results of the analysis of literature sources on development of microelectronic signal acquisition and processing units for ionizing radiation detectors in large-scale physical experiments are given. The basic parameters of these units, such as the channel number, power consumption per channel, conversion coefficient and noise level are summarized in the generalizing table. Conclusions on the performed analysis are made and trends in the design and implementation of the considered units manufactured as applications specific integrated circuits are noted.     

Real time computer simulation of electron microscope images with tilted illumination: Grain boundary applications

Computer programs have been developed to simulate electron microscope images from digitized graphically represented model structures. Via a television rate image processing system, these programs allow real time, interactive modification of the microscope objective lens parameters, incident beam inclination, and incident beam energy. In addition to explaining the computational methods, the need for using tilted beam illumination is explored to extend microscope resolution. For this study, the subject of grain boundary imaging is analyzed for a copper σ = 5,36.9°,(310) tilt boundary with a [001] common rotation axis. The Cu {200} lattice spacings of ～1.8Å on both sides of the interface cannot be reliably resolved under axial illumination conditions in a 200 kV microscope. Therefore, either tilted beam modes or higher incident beam energies were explored and the types of image features correlated with atomic position data through the digital frame store system.     

Verification of cell viability at progressively higher scanning forces using a hybrid atomic force and fluorescence microscope

Dry 40× and 60× microscope objectives were fitted with opaque black masks in order to eliminate reflection and scattering of light off the objective front lens assembly during oblique incidence reflection (OIR) microscopy. The reflection and scattering are shown to induce background glare that leads to degradation in the quality of the OIR images. Mask prototypes were designed and machined to snap onto the spring-loaded retractable front lens assembly of each objective. OIR images of live cells and normalized intensity line profiles are used to demonstrate that, if these alterations to the housing of the objective are implemented, background glare is significantly reduced with the 60× objective, and virtually eliminated with the 40× objective.     

Observation of atomic steps on single crystal surfaces by a commercial scanning electron microscope.

Atomic steps on (111) and (100) crystal surfaces of Pt were observed using a commercial scanning electron microscope (SEM) in secondary electron mode. By comparing the SEM images and those by reflection electron microscopy (REM), the observed contrast was confirmed to be that from atomic steps on crystal surfaces. The contrast mechanism is briefly discussed. One application of this imaging technique is also shown.     

Routine fluorescence microscopy of single untethered protein molecules confined to a planar zone

To bypass limitations of ensemble averaging biochemical analysis, microscopy‐based detection and tracking are needed for single protein molecules that are diffusing in aqueous solution. Confining the molecules to a planar zone dramatically assists tracking. Procedures of microscopy should be routine enough so that effort is focused on the biochemistry. Fluorescence microscopy and partial planar confinement of single, untethered, aqueous protein molecules have been achieved here by use of a routine procedure. With this procedure, multiple thermally diffusing Alexa 488‐stained bovine serum albumin molecules were observed during partial confinement to a thin aqueous zone next to a cover slip. The procedure produces confinement by partial re‐swelling of a previously dried agarose gel on the microscope slide. Confinement was confirmed through analysis that revealed thermal motion lower in the third dimension than it was in the plane of observation.