Aperture Objective Research Articles

Tight focusing of femtosecond elliptically polarised vortex light pulses

This paper studies the tight focusing properties of femtosecond elliptically polarised vortex light pulses. Based on Richards—Wolf vectorial diffraction integral, the expressions for the electric field, the velocity of the femtosecond light pulse and the total angular momentum of focused pluses are derived. The numerical calculations are also given to illustrate the intensity distribution, phase contour, the group velocity variation and the total angular momentum near the focus. It finds that near the focus the femtosecond elliptically polarised vortex light pulse can travel at various group speeds, that is, slower or faster than light speed in vacuum, depending on the numerical aperture of the focusing objective system. Moreover, it also studies the influence of the numerical aperture of the focusing objective and the time duration of the elliptically polarised vortex light pulse on the total angular momentum distribution in the focused field.

Imaging mechanisms analysis of compact digital holographic microscope for microparticles measurement

Conventional optical microscopy suffers from small depth of focus due to its high numerical aperture and magnification of the microscope objective. In comparison, digital in-line holographic microscopy (DIHM) provides information about the entire 3D volume through numerical reconstruction of the single hologram at several depths. This advantage makes DIHM an effective tool for the measurement of microparticles in suspension. Recently, our group has demonstrated the potential of DIHM for accurate measurement of particles with sizes ranging from 40 microns to a few millimetres. In this paper, the applicability of DIHM is extended for measurement of near-micron sized particles. A compact digital holographic microscope with a single microscope objective is presented. The system imaging mechanisms of the microscope is analyzed first and the recording distance of digital hologram is calculated using spatial frequency analysis. Then the system magnification, lateral resolution and depth resolution are analyzed in terms of the hologram recording distance. Finally, the characterization of microparticles with a diameter of 1 micron and 10 microns is demonstrated with the compact setup. The experimental results show the efficiency and accuracy of this method with a measured error less than 1.55% in the diameter of certified particles.

Intrinsic Linear Heterogeneity of Amyloid β Protein Fibrils Revealed by Higher Resolution Mass-per-length Determinations

Amyloid β proteins spontaneously form fibrils in vitro that vary in their thermodynamic stability and in morphological characteristics such as length, width, shape, longitudinal twist, and the number of component filaments. It is vitally important to determine which variant best represents the type of fibril that accumulates in Alzheimer disease. In the present study, the nature of morphological variation was examined by dark-field and transmission electron microscopy in a preparation of seeded amyloid β protein fibrils that formed at relatively low protein concentrations and exhibited remarkably high thermodynamic stability. The number of filaments comprising these fibrils changed frequently from two to six along their length, and these changes only became apparent when mass-per-length (MPL) determinations are made with sufficient resolution. The MPL results could be reproduced by a simple stochastic model with a single adjustable parameter. The presence of more than two primary filaments could not be discerned by transmission electron microscopy, and they had no apparent relationship to the longitudinal twist of the fibrils. However, the pitch of the twist was strongly affected by the pH of the negative stain. We conclude that highly stable amyloid fibrils may form in which a surprising amount of intrinsic linear heterogeneity may be obscured by MPL measurements of insufficient resolution, and by the negative stains used for imaging fibrils by electron microscopy.

Resolution improvement in digital holography by angular and polarization multiplexing

Angular and polarization multiplexing techniques are utilized in both object and reference arms in the digital holographic microscopy system to improve its resolution. The angular multiplexing provides on-axis and off-axis illumination and reference beams with different carrier frequencies. Polarization multiplexing prohibits the occurrence of interference between low and high object spatial frequencies and reference beams. The proposed system does not require special light sources or filtering masks. Experimental results show that the resolution of the synthesized image exceeds the resolution determined by the numerical aperture of the imaging microscope objective.

Focal modulation microscopy with annular apertures: A numerical study

Image formation of focal modulation microscopy with annular aperture (AFMM) is presented. The spatial resolution is discussed. Compared with confocal microscopy, AFMM can simultaneously enhance the axial and transverse resolution. By adjusting the width of the annular objective aperture, AFMM can be adjusted from best spatial resolution performance to highest signal level. The capability of background rejection is also investigated, showing AFMM has the potential to further increase the imaging penetration depth. Finally, an optimal configuration of AFMM is elaborated.

Determination of the linear attenuation range of electron transmission through film specimens

We have investigated the linear attenuation range of electron transmission through film specimens and its dependence on the electron energy, the acceptance half-angle of a detector or an objective aperture, and specimen properties, in the scanning transmission electron microscope (STEM) and the conventional transmission electron microscope (TEM). Electron transmission in the bright-field mode was calculated by the Monte Carlo simulation of electron scattering, and its range of the linear attenuation in film thickness was then determined by a linear least squares fit. The corresponding linear thickness range was shown to increase with the electron energy and the acceptance half-angle, although it decreased with the increase in the atomic number of specimen materials. Under the condition of a 300 kV STEM or a 3 MV ultra-high voltage electron microscope (ultra-HVEM), the linear attenuation range could extend to several microns for light specimen materials, and this was validated by experimental data in the ultra-HVEM. The presented results can be helpful for accurately measuring the specimen thickness or mass from electron transmission, and estimating the deviation of electron transmission from linearity when tilting a specimen in electron tomography.

Particle tracking stereomicroscopy in optical tweezers: Control of trap shape

We present an optical system capable of generating stereoscopic images to track trapped particles in three dimensions. Two-dimensional particle tracking on each image yields three dimensional position information. Our approach allows the use of a high numerical aperture (NA=1.3) objective and large separation angle, such that particles can be tracked axially with resolution of 3 nm at 340 Hz. Spatial Light Modulators (SLMs), the diffractive elements used to steer and split laser beams in Holographic Optical Tweezers, are also capable of more general operations. We use one here to vary the ratio of lateral to axial trap stiffness by changing the shape of the beam at the back aperture of the microscope objective. Beams which concentrate their optical power at the extremes of the back aperture give rise to much more efficient axial trapping. The flexibility of using an SLM allows us to create multiple traps with different shapes.

A TEM phase plate loading system with loading monitoring and nano-positioning functions

We present a phase plate loading system developed for a commercial transmission electron microscope (TEM). Our system can be installed without modifying the optical design of the TEM. This system is equipped with a loading monitoring set that allows users to easily and safely locate the phase plate between the pole pieces, and also comes with an airlock that permits quick loading of a phase plate without the need to re-vent the TEM column. The system uses a home-made three-axis nano-positioner to precisely position the phase plate hole at the desired location. Our system has a precision of ∼10 nm, an improvement of one order of magnitude compared with the precision of a phase plate holder modified from an objective aperture. We demonstrate the successful installation and the use of the loading system to place a phase plate at the desired position. Our phase plate loading system can be used to accommodate various types of phase plates and thus provides a good way to greatly speed up the development of TEM phase plates.

Focusing of a femtosecond vortex light pulse through a high numerical aperture objective

We investigate the focusing properties of a femtosecond vortex light pulse focused by a high numerical aperture objective. By using the Richards-Wolf vectorial diffraction method, the intensity distribution, the velocity variation and the orbital angular momentum near the focus are studied in great detail. We have discovered that the femtosecond vortex light pulse can travel at various speeds, that is, slower or faster than light with a tight focusing system. Moreover, we have found that the numerical aperture of the focusing objective and the duration of the vortex light pulse will influence the orbital angular momentum distribution in the focused field.

An Order of Magnitude Improvement in STEM Resolution: Wavelength High-Energy Electron Localization

There have been many attempts to improve the resolution of electron microscopes. Transmission electron microscopes are normally limited in resolution by a balance between the diffraction limit (which could be overcome by the use of a large objective aperture) and the spherical aberration of the lenses used (which could be overcome by the use of a small objective aperture). In recent years successful attempts have been made to achieve resolutions beyond this limit by holography and also by the development of aberration correctors.

Tight focusing of a partially coherent and partially polarized beam

The focusing properties of a partially coherent and partially polarized beam focused by a high numerical aperture (NA) objective are investigated. The influence of the degree of polarization and the correlation length of the incident beam and the numerical aperture of the objective on the intensity and degree of polarization in the focal region is studied. It is shown that the intensity and degree of polarization in the focal region change with the degree of polarization and the correlation length of the incident beam.

Calculation of optical trapping forces on dielectric spheres at an oil-water interface with ray-optics model

The transverse trapping forces on a dielectric sphere located at an oil-water interface are theoretically investigated with the ray-optics model. The transverse trapping forces rely on the internal property of the particle-interface system, and increase with either the decrease of three phase contact angles at the oil-water interface or the use of oil phase with low refractive index. The numerical results also show that the transverse trapping forces can be improved by either decreasing the numerical aperture of the microscope objective or shrinking the diameter of the trapping laser beam.

High contrast hollow-cone dark field transmission electron microscopy for nanocrystalline grain size quantification

In this paper, we describe hollow-cone dark field (HCDF) transmission electron microscopy (TEM) imaging, with a slightly convergent beam, as an improved technique that is suitable to form high contrast micrographs for nanocrystalline grain size quantification. We also examine the various factors that influence the HCDF TEM image quality, including the conditions of microscopy (alignment, focus and objective aperture size), the properties of the materials imaged (e.g., atomic number, strain, defects), and the characteristics of the TEM sample itself (e.g., thickness, ion milling artifacts). Sample preparation was found to be critical and an initial thinning by wet etching of the substrate (for thin film samples) or tripod polishing (for bulk samples), followed by low-angle ion milling was found to be the preferred approach for preparing high-quality electron transparent samples for HCDF imaging.

Method of improving the quality of nanopatterning in atomic image projection electron-beam lithography

The authors demonstrated successful nanopatterning using a high resolution lattice image obtained by transmission electron microscopy (TEM) as a mask signal [H. S. Lee et al., Adv. Mater. (Weinheim, Ger.) 19, 4198 (2007)]. In this process, the quality of the patterning result is critically dependent on the lattice image, which, in turn, is critically dependent on the quality of the mask. It is often noted that the quality of the mask prepared by the conventional TEM sample preparation technique is far from perfect, which critically limits the quality of patterning. In this work, they first discuss the various origins of the noise signal generated by the mask and then introduce a special type of objective aperture (noise reduction aperture) to remove the noise signal. The effect of the noise reduction aperture, designed for the Si [110] zone axis, is experimentally demonstrated.

Surface Plasmon Enhanced TIRF Imaging

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a powerful technique for imaging dynamic membrane events in living cells. However the information gained from TIRFM may be limited by the intensity of the florescence signal and by background noise emanating from the inner part of the cell. Here we describe how Surface Plasmon Mediated Fluorescence Microscopy (SPMFM) can enhance TIRFM by increasing the fluorescence signal 5-6 fold and by reducing background noise dramatically. The imaging configuration is that for TIRFM except that the coverglass is coated on one side with a nanometre film of silver. For SPMFM (as in TIRFM), a high numerical aperture (N.A.) objective is essential for achieving required illumination angles.

Interaction of TorsinA with Its Major Binding Partners Is Impaired by the Dystonia-associated ΔGAG Deletion

Early onset (DYT1) torsion dystonia is a dominantly inherited movement disorder associated with a three-base pair (DeltaGAG) deletion that removes a glutamic acid residue from the protein torsinA. TorsinA is an essential AAA(+) (ATPases associated with a variety of cellular activities) ATPase found in the endoplasmic reticulum and nuclear envelope of higher eukaryotes, but what it does and how changes caused by the DeltaGAG deletion lead to dystonia are not known. Here, we asked how the DYT1 mutation affects association of torsinA with interacting proteins. Using immunoprecipitation and mass spectrometry, we first established that the related transmembrane proteins LULL1 and LAP1 are prominent binding partners for torsinA in U2OS cells. Comparative analysis demonstrates that these two proteins are targeted to the endoplasmic reticulum or nuclear envelope by their divergent N-terminal domains. Binding of torsinA to their C-terminal lumenal domains is stabilized when residues in any one of three motifs implicated in ATP hydrolysis (Walker B, sensor 1, and sensor 2) are mutated. Importantly, the DeltaGAG deletion does not stabilize this binding. Indeed, deleting the DeltaGAG encoded glutamic acid residue from any of the three ATP hydrolysis mutants destabilizes their association with LULL1 and LAP1C, suggesting a possible basis for loss of torsinA function. Impaired interaction of torsinA with LULL1 and/or LAP1 may thus contribute to the development of dystonia.

Scattering information obtained by optical microscopy: Differential dynamic microscopy and beyond

We describe the use of a bright-field microscope for dynamic light scattering experiments on weakly scattering samples. The method is based on collecting a time sequence of microscope images and analyzing them in the Fourier space to extract the characteristic time constants as a function of the scattering wave vector. We derive a theoretical model for microscope imaging that accounts for (a) the three-dimensional nature of the sample, (b) the arbitrary coherence properties of the light source, and (c) the effect of the finite numerical aperture of the microscope objective. The model is tested successfully against experiments performed on a colloidal dispersion of small spheres in water, by means of the recently introduced differential dynamic microscopy technique [R. Cerbino and V. Trappe, Phys. Rev. Lett. 100, 188102 (2008)]. Finally, we extend our model to the class of microscopy techniques that can be described by a linear space-invariant imaging of the density of the scattering centers, which includes, for example, dynamic fluorescence microscopy.

Tight focusing of partially coherent and circularly polarized vortex beams

Based on the vectorial Debye theory, the tight focusing properties of partially coherent and circularly polarized vortex beams are investigated. The focused characteristics of right-circular and left-circular polarized partially coherent vortex beams in the focal region are presented and compared by some numerical calculation results. Furthermore, the influences of the source coherence and the numerical aperture of the focusing objective on the tight focusing properties are studied in great detail. It is shown that the coherence and polarization properties of the focused left-circular polarized beam is less influenced by the source coherence and the numerical aperture of the focusing objective than that of the focused right-circular polarized beam. By selecting certain parameters, the widely used flat top beam can be obtained.

High-quality virus images obtained by transmission electron microscopy and charge coupled device digital camera technology

The introduction of digital cameras has led to the publication of numerous virus electron micrographs of low magnification, poor contrast, and low resolution. Described herein is the methodology for obtaining highly contrasted virus images in the magnification range of approximately 250,000–300,000×. Based on recent advances in charged couple device (CCD) digital camera technology, methodology is described for optimal imaging parameters for using CCD cameras mounted in side- and bottom-mount position of electron microscopes and the recommendation of higher accelerating voltages, larger objective apertures, and small spot size. The authors are concerned with the principles of image formation and modulation, advocate a better use of imaging software to improve image quality, and recommend either pre- or post-acquisition adjustment for distributing pixel intensities of compressed histograms over the entire range of tonal values.

A light touch on nanotubes: femtonewton force sensing and nanometric spatial resolution

Light can exert a mechanical action onmatter. Although this simple concept has been known for centuries (Kepler’s explanation of comet tails is based on it), the advent of the laser age has led to tremendous experimental advances and understanding of this phenomenon. Optical tweezers, instruments based on a tightly focused laser beam, have been used to trap, manipulate, control, and assemble dielectric particles, single atoms, cells, metal, and semiconducting nanostructures, leading to a real optical revolution in physics, biology, and nanotechnology. When used as force transducer, optical tweezers can measure forces in the piconewton range. In this context, the concept of photonic force microscopy (PFM) has been developed by scanning a dielectric sphere over surfaces in a liquid environment and sensing the force interaction. While the lateral resolution in PFM applications is related to particle size, the extension of conventional optical trapping to nanoparticles is a difficult task, since the radiation force scales with the volume of the trapped particle. Recently, optical trapping of carbon nanotube bundles was realized in aqueous environments. The almost linear geometry of these nanostructures, which have a subwavelength cross section and very high aspect ratio, is of crucial importance for bridging the gap between the micro and nano-world. In our work we demonstrated that the nanotubes’ small transverse size is key to achieving nanometric resolution for PFM applications, while an axial dimension in the micron range ensures stable trapping and allows force sensing in the femtonewton regime. A critical element in force sensing with optical tweezers is Brownian motion—random fluctuations of particles in a fluid. Performing statistical analysis of an optically trapped particle’s fluctuations about equilibrium enables force measurements. The Figure 1. (a) Optical trapping geometry. A laser beam is expanded to over-fill the back aperture of a high numerical microscope objective. The laser light propagates along the z axis (oriented as k wavevector) while x is the polarization axis (oriented as E=electric field). The light is focused in a chamber containing the nanotube solution; here the cylinder represents a randomly oriented nanotube. (b) A laser trapped bundle oriented by radiation torque along the optical axis. (c) The same bundle un-trapped (laser is off) and randomly oriented by Brownian motion.