Aperture Objective Research Articles

Fluorescence Detector for Capillary Separations Fabricated by 3D Printing

A simple inexpensive light-emitting diode (LED)-based fluorescence detector for detection in capillary separations is described. The modular design includes a separate high power LED source, detector head, designed in the epifluorescence arrangement, and capillary detection cells. The detector head and detection cells were printed using a 3D printer and assembled with commercially available optical components. Optical fibers were used for connecting the detector head to the LED excitation source and the photodetector module. Microscope objective or high numerical aperture optical fiber were used for collection of the fluorescence emission from the fused silica separation capillary. As an example, mixture of oligosaccharides labeled by 8-aminopyrene-1,3,6-trisulfonate (APTS) was separated by capillary zone electrophoresis and detected by the described detector. The performance of the detector was compared with both a semiconductor photodiode and photomultiplier as light sensing elements. The main advantages of the 3D printed parts, compared to the more expensive alternatives from the optic component suppliers, include not only cost reduction, but also easy customization of the spatial arrangement, modularity, miniaturization, and sharing of information between laboratories for easy replication or further modifications of the detector. All information and files necessary for printing the presented detector are enclosed in the Supporting Information.

Nonlinear intensity attenuation with increasing thickness and quantitative TEM tomography of micron-sized materials.

Nowadays three-dimensional (3D) analyses of nanometer-sized and sub-micron-sized objects have been widely achieved by tomography in transmission electron microscopes (TEM). One of the next methodological targets should be quantitative 3D reconstructions in which not only the shape but also the internal density is correctly reproduced. This is, however, generally hindered by the nonlinearity between projection mass-thickness and image intensity. In the case of BF-TEM images, the ideal exponential attenuation with increasing thickness is disturbed by multiple scatterings. The nonlinearity in the tilt series should induce an inaccurate density distribution in the reconstructed volume.In the present study, the nonlinear attenuation effect has been analyzed using amorphous carbon microcoils (CMCs). Their well-defined shapes and compositional homogeneity are quite useful to estimate the mass-thickness. For measurements over a wide range of acceleration voltages (400, 600, 800 and 1000 kV), we used a high-voltage electron microscope (JEOL: JEM-1000KRS). The intensity attenuation with increasing thickness was measured in the BF-TEM images taken with various sizes of objective apertures (OAs). The results have shown that using a smaller OA and a lower voltage enhances the nonlinearity in the intensity attenuation.It is considered that such nonlinear attenuation should induce failures in conversion from intensity to thickness and thus inhibits correct 3D reconstructions. The influence of the nonlinearity on the tomographic reconstructions has been examined using a specially-developed 360°-tilt sample holder for elimination of the missing-wedge effect [1]. Figure1 shows the reconstruction results from the tilt series taken under three imaging conditions; 1000 kV with the smallest or the largest OA, and 400 kV with the largest OA. At a glance, the 3D shape of the CMC has been reconstructed well regardless of the differences in the imaging conditions. However, in the sliced image in (a), the interior region is so blurred that the inner walls are difficult to identify. If the reconstructed density is examined by the profiles between x and y in the sliced images, it is clear that the internal density is not uniform but has gradient from the center in (b). Moreover, there is a slight increase of the vacuum level at the inside region of the coil. The uniform densities in the material and vacuum have been reconstructed only in (c) taken at 1000 kV with the largest OA, which is the imaging condition achieving the perfect linearity in the intensity attenuation.jmicro;63/suppl_1/i5/DFU044F1F1DFU044F1Fig. 1.3D reconstructions of a carbon microcoil taken at (a) 1000 kV with the largest objective aperture (OA), (b) 400 kV with the largest OA and (c) 1000 kV with the smallest OA Based on analysis of the nonlinearity under other various imaging conditions, we found that the linearity tends to collapse when the electron transmittance falls below about 2/3 [1]. This information should be beneficial in actual tomography experiments because one can foresee quality of the reconstruction before acquiring numerous images for the tilt series.

Condenser-free contrast methods for transmitted-light microscopy.

Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser-free yet highly effective method of obtaining phase contrast in transmitted-light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light-path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser-free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser-free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour-contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser-free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next-generation transmitted-light microscopy designs. The condenser-free illumination method, using rings of independent or radially-scanned emitters, may be exploited in future in other electromagnetic wavebands, including X-rays or the infrared.

Vibrationally resonant sum-frequency generation microscopy with a solid immersion lens.

We use a hemispheric sapphire lens in combination with an off-axis parabolic mirror to demonstrate high-resolution vibrationally resonant sum-frequency generation (VR-SFG) microscopy in the mid-infrared range. With the sapphire lens as an immersed solid medium, the numerical aperture (NA) of the parabolic mirror objective is enhanced by a factor of 1.72, from 0.42 to 0.72, close to the theoretical value of 1.76 ( = nsapphire). The measured lateral resolution is as high as 0.64 μm. We show the practical utility of the sapphire immersion lens by imaging collagen-rich tissues with and without the solid immersion lens.

Analysis of nonlinear intensity attenuation in bright-field TEM images for correct 3D reconstruction of the density in micron-sized materials.

To obtain the correct tomographic reconstruction of micron-sized materials, the nonlinear intensity attenuation of bright-field transmission electron microscopy (BF-TEM) images was analyzed as a function of the sample thickness using a high-voltage electron microscope. The intensity attenuation was precisely measured relative to the projection thickness of carbon microcoils (CMCs) at acceleration voltages of 400-1000 kV using objective apertures (OAs) with radii of 2.1-28 nm(-1). The results show that the nonlinearity is strongly dependent on the OA size and the acceleration voltage. The influence of nonlinearity on tomographic reconstructions was also examined using a specially developed 360°-tilt sample holder. Sliced images of the reconstructed volumes indicated that an increase in the nonlinearity caused artificial fluctuations in the internal density of materials and inaccurate shapes of the objects in more significant cases. Conditions sufficient for reconstruction with the correct density have been estimated to be 0.67 of the minimum electron transmittance, and for reconstructions with correct shapes, 0.4. This information enables foreseeing the quality of the reconstruction from a single BF-TEM image prior to the tilt-series acquisition. As a result to demonstrate the appropriateness of these conditions, a CMC with a diameter of 3.7 µm was reconstructed successfully; i.e. not only the shape but also the internal density were correctly reproduced using electron tomography.

Fast Bits Recording in Photoisomeric Polymers by Phase-Modulated Femtosecond Laser

High storage capacity and recording-access rate are two critical factors that the current optical storage technology pursues. Efforts on shortening the laser wavelength, increasing the numerical aperture of objective, and increasing the optical drive speed have been made to reach this end. However, these methods have run their full course because of many existing difficulties on the physical and manufacturing aspect. People have to explore innovative ways to satisfy the requirement of both mass data and high rate. Multidimensional optical storage and holographic recording are two promising technologies to increase the capacity of memory and recording rate by orders of magnitude. We report on the fast bits recording in bisazobenzene functional polymer film by femtosecond laser holographic recording. The mechanism of the photo-induced anisotropy of the material is reviewed and discussed. The demonstration of multidimensional holographic recording is presented. Some influence factors in the holographic processing are discussed.

To construct a stable and tunable optical trap in the focal region of a high numerical aperture lens

Based on the diffraction theory, the focusing properties of a radially polarized quadratic Bessel–Gaussian beam (QBG) with on-axis radial phase variance wavefront are investigated theoretically in the focal region of a high numerical aperture (NA) objective lens. The phase wavefront C and pupil beam parameter μ of QBG are the functions of the radial coordinate. The detailed numerical calculation of the focusing property of a QBG beam is presented. The numerical calculation shows that the beam parameter μ and phase parameter C have greater effect on the total electric field intensity distribution. It is observed that under the condition of different μ, evolution principle of focal pattern differs very remarkably on increasing C. Also, some different focal shapes may appear, including rhombic shape, quadrangular shape, two-spherical crust focus shape, two-peak shape, one dark hollow focus, two dark hollow focuses pattern, and triangle dark hollow focus, which find wide optical applications such as optical trapping and nanopatterning.

Point spread function and lateral resolution analysis of digital holographic microscopy system

A major issue so far for digital holography is the low lateral resolution. In order to improve the resolution, we deduce the point spread function of the most common and practical digital holographic microscopy system. Based on the derived formulas, the determinant factor of lateral resolution of the system is analyzed. The results show that the optimal resolution of this system is limited by the numerical aperture of microscope objective, and the resolution can be further deteriorated by the recording media parameters and recording distance. By decreasing the recording distance the resolution can be improved dramatically. In addition, by choosing large photosensitive area and small pixel size camera the resolution of the holographic microscopy system can be improved a little. Moreover, the highest resolution and the best image quality can be achieved by image-plane digital holography. Experiment results demonstrate the correctness of the theoretical analysis.

Opposed-view dark-field digital holographic microscopy

Scattering and absorption belong to the major problems in imaging the internal layers of a biological specimen. Due to the structural inhomogeneity of the specimen, the distribution of the structures in the upper layers of a given internal structure of interest is different from the lower layers that may result in different interception of scattered light, falling into the angular aperture of the microscope objective, from the object in each imaging view. Therefore, different spatial frequencies of the scattered light can be acquired from different (top and bottom) views. We have arranged an opposed-view dark-field digital holographic microscope (DHM) to collect the scattered light concurrently from both views with the aim to increase the contrast of internal structures and improve the signal-to-noise ratio. Implementing a DHM system gives the possibility to implement digital refocusing process and obtain multilayer images from each side without a depth scan of the object. The method is explained and the results are presented exemplary for a Drosophila embryo.

Breaking the diffraction-limited resolution barrier in fiber-optical two-photon fluorescence endoscopy by an azimuthally-polarized beam

Although fiber-optical two-photon endoscopy has been recognized as a potential high-resolution diagnostic and therapeutic procedure in vivo, its resolution is limited by the optical diffraction nature to a few micrometers due to the low numerical aperture of an endoscopic objective. On the other hand, stimulated emission depletion (STED) achieved by a circularly-polarized vortex beam has been used to break the diffraction-limited resolution barrier in a bulky microscope. It has been a challenge to apply the STED principle to a fiber-optical two-photon endoscope as a circular polarization state cannot be maintained due to the birefringence of a fiber. Here, we demonstrate the first fiber-optical STED two-photon endoscope using an azimuthally-polarized beam directly generated from a double-clad fiber. As such, the diffraction-limited resolution barrier of fiber-optical two-photon endoscopy can be broken by a factor of three. Our new accomplishment has paved a robust way for high-resolution in vivo biomedical studies.

Comparative Analysis of Trapping Efficiencies between Gaussian and Laguerre-Gaussian beams for Optical Tweezers

Using numerical calculations based on a mathematical model, this paper shows how the axial trapping efficiency (Qz) and the radial trapping efficiency (Qr) of optical tweezers depend on specific parameters such as size of the dielectric particle (r), numerical aperture of the microscope objective (NA), refractive index of the dielectric particle (np) to be trapped and the refractive index of the surrounding medium (nm) in which the particle is being trapped. The model is used to calculate the trapping efficiencies and the forces acting on a particle. The calculations are done for two types of laser beams: Gaussian and LaguerreGausssian. Results for both types are then compared in terms of effective trapping. For all of the simulations, the particle being trapped is considered to be a micrometer-sized spherical polystyrene bead.

ツイン・フーコー法

A novel Lorentz electron microscopy —twin-Foucault imaging (TFI) method— is introduced. The twin-Foucault imaging method enables observation of two Foucault images simultaneously by using an electron biprism instead of an objective aperture. The electron biprism is installed between two electron beams deflected by magnetic domains. Potential applied to the biprism deflects the two electron beams further, and two Foucault images are then obtained in one visual field. The twin-Foucault images are able to extract the magnetic domain structures and to reconstruct an ordinary electron micrograph. The Foucault method was demonstrated with 180° domain and 90°/180° domain structures of manganite La1−αSrαMnO3.

Insights on the study of nafion nanoscale morphology by transmission electron microscopy.

Nafion is one of the most common materials used for polyelectrolyte membranes and is the standard to which novel materials are compared. In spite of great interest in Nafion’s nanostructure, it is still a subject of controversy. While multiple research efforts have addressed Nafion’s morphology with Transmission Electron Microscopy, the results of these efforts have often been inconsistent and cannot satisfactorily describe the membrane structure. One of the reasons for differences in the reported results is the lack of sufficient control over the damage caused by electron beam irradiation. In this work, we describe some aspects of damage in the material that have a strong influence on the results. We show that irradiation causes mass loss and phase separation in the material and that the morphologies that have been observed are, in many cases, artifacts caused by damage. We study the effect of the sample temperature on damage and show that, while working at low temperature does not prevent damage and mass loss, it slows formation of damage-induced artifacts to the point where informative low-dose images of almost undamaged material may be collected. We find that charging of the sample has a substantial effect on the damage, and the importance of charge neutralization under irradiation is also seen by the large reduction of beam induced movement with the use of an objective aperture or a conductive support film. To help interpret the low-dose images, we can apply slightly higher exposures to etch away the hydrophobic phase with the electron beam and reveal the network formed by the hydrophilic phase. Energy loss spectroscopy shows evidence that fluorine removal governs the beam damage process.

Abstract P2-03-03: Feasibility of evaluation of breast tissue using confocal microscopy with strip mosaicing

Abstract Background: Confocal microscopic strip mosaicing (CSM) provides noninvasive optical sectioning and high resolution, which allows for imaging of nuclear and morphological detail in freshly excised tissue. CSM can image large areas of tissue at micron-level resolution in minutes, which may offer an advantage over standard histology that requires days. We have conducted a preliminary investigation of the feasibility of this technology for the evaluation of breast tissue from surgical excision specimens. Design: In a prospective study, 80 fresh human breast tissue samples from surgical excision specimens of 24 patients were imaged using a prototype confocal strip scanner. Fresh tissue specimens were immersed in Acridine Orange (AO) for 45 seconds to stain the nuclei, then pressed against the glass imaging window and imaged with a 30X, 0.75 numerical aperture (measured) objective lens and a 488 nm laser. Images were acquired in two modes of contrast: in fluorescence (with AO), showing nuclear morphology, and in reflectance (endogenous), showing stroma. The use of fluorescence for nuclear staining mimics the use of hematoxylin in pathology, and the use of reflectance eosin. Use of two contrast modes allows the fluorescence image to be colorized purple and the reflectance image pink, producing confocal strip mosaics that mimic H&E histology in appearance. Specimens were subsequently fixed in formalin and routinely processed to obtain H&E stained sections. H&E and confocal images were compared by the study pathologist (M.M.) Results: Freshly excised breast tissue samples as large as 2 cm x 2 cm were imaged in less than five minutes, with 1-micron resolution and measured optical sectioning of 6 microns. We compared the CSM images against standard histopathology images. In our series we evaluated the following histologies: 12 invasive carcinoma (11 ductal, 1 lobular), 3 ductal carcinoma in-situ, 3 lobular carcinoma in situ, 1 atypical lobular hyperplasia, 1 atypical ductal hyperplasia and various benign lesions such as fat necrosis, fibrocystic changes, and ductal hyperplasia. In confocal images invasive and in situ carcinoma as well as benign ducts and lobules were distinguished from surrounding stromal tissue. Limitations that are typically encountered in standard histology, such as distinguishing low grade ductal carcinoma in situ (DCIS) from lobular carcinoma in situ (LCIS) or atypical proliferations were encountered in the grayscale confocal images as well. Conclusion: In this initial feasibility study, CSM produced images that could be diagnosed as benign or neoplastic by the study pathologist. Further study is needed to build an image library of breast histology and compare reproducibility of histologic diagnoses between CSM (grayscale and colorized images) and traditional optical microscopy, and assess interobserver reproducibility in diagnosis. CSM potentially provides rapid and noninvasive evaluation of breast parenchyma, and has a potential application for intraoperative margin assessment of resected breast specimens. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-03-03.

STUDY OF THE MAJOR CONDITIONS INFLUENCING STEP HEIGHT MEASUREMENT USING INTERFERENCE MICROSCOPE

Step height measurements in micro and nano scale are essential to the micro-technology. The interferometric measurement is carried out using two main techniques: white light scanning interferometry (WLI) and phase stepping interferometry (PSI). A step height standard sample of nominal step height value h of 90.1 nm has been studied using the PSI mode with an optical filter light of λ = 633.4 nm against three parameters: vertical distance (z) between objective and sample, intensity of the incident light and the inclination angles of the sample to the incident light. The effect of numerical aperture of objectives has been referenced to some literature. The total budget of studied parameters for the sample 90.1 nm leads to expanded uncertainty of 0.46 nm.

Optical fibers for miniaturized surface-enhanced Raman-scattering probes

A range of optical fibers with surface-enhanced Raman scattering (SERS) functionalized tips have been evaluated for use as micro-scale sensing devices. In order to optimize the sensitivity of the optical fiber probe, the relationship between SERS intensity and different fiber parameters was investigated. It was found that the numerical aperture, core size, mode structure, and core material have a major effect on the probe performance, as does the numerical aperture of the microscope objective. The results suggest that an ideal fiber for SERS sensing should be single mode at the excitation wavelength and have low-background core material.

Ion-specific Effects on Prion Nucleation and Strain Formation

Ordered, fibrous, self-seeding aggregates of misfolded proteins known as amyloids are associated with important diseases in mammals and control phenotypic traits in fungi. A given protein may adopt multiple amyloid conformations, known as variants or strains, each of which leads to a distinct disease pattern or phenotype. Here, we study the effect of Hofmeister ions on amyloid nucleation and strain generation by the prion domain-containing fragment (Sup35NM) of a yeast protein Sup35p. Strongly hydrated anions (kosmotropes) initiate nucleation quickly and cause rapid fiber elongation, whereas poorly hydrated anions (chaotropes) delay nucleation and mildly affect the elongation rate. For the first time, we demonstrate that kosmotropes favor formation of amyloid strains that are characterized by lower thermostability and higher frangibility in vitro and stronger phenotypic and proliferation patterns effectively in vivo as compared with amyloids formed in chaotropes. These phenomena point to inherent differences in the biochemistry of Hofmeister ions. Our work shows that the ionic composition of a solution not only influences the kinetics of amyloid nucleation but also determines the amyloid strain that is preferentially formed.

Vortex beam production and contrast enhancement from a magnetic spiral phase plate

Electron vortex beam probes offer the possibility of mapping magnetic moments with atomic resolution. In this work we consider using the stray magnetic field produced from a narrow ferromagnetic rod magnetised along its long axis to produce a vortex beam probe, as an alternative to the currently used holographic apertures or gratings. We show through numerical modelling, electron holography observations and direct imaging of the electron probe, that a long narrow ferromagnetic rod induces a phase shift in the wave-function of passing electrons that approximately describes a helix in the regions near its ends. Directing this rod towards the optical axis of a charged-particle beam probe forming system at a limiting aperture position, with the free-end sufficiently close to the axis, is shown to offer a point spread function composed of vortex modes, with evidence of this appearing in observations of the electron probe formed from inserting a micro-fabricated CoFe rod into the beam path of a 300 keV transmission electron microscope (TEM). If the rod is arranged to contain the magnetic flux of h/e, thus producing a maximum phase shift of 2π, it produces a simple spiral-like phase contrast transfer function for weak phase objects. In this arrangement the ferromagnetic rod can be used as a phase plate, positioned at the objective aperture position of a TEM, yielding enhanced image contrast which is simulated to be intermediate between comparable Zernike and Hilbert phase plates. Though this aspect of the phase plate performance is not demonstrated here, agreement between our observations and models for the probe formed from an example rod containing a magnetic flux of ~2.35h/e, indicate this phase plate arrangement could be a simple means of enhancing contrast and gaining additional information from TEM imaged weak phase samples, while also offering the capability to produce vortex beam probes. However, steps still need to be taken to either remove or improve the support membrane for the rod in our experiments to reduce any effects from charging in the phase plate.

Intensity modulation of a tightly focused partially coherent and radially polarized beam

Because of the circular symmetry of a completely coherent radially polarized beam, the azimuthal intensity component is zero when it is focused by a high numerical aperture (NA) objective. In this paper, we show that such a conclusion is not tenable under the illumination of partially coherent beam whose coherent property depends on the azimuthal angle. Taking the Gaussian Schell-model (GSM) beam for an example, the tight focusing property of a partially coherent beam is studied, and the intensity and its radial, azimuthal and longitudinal components are particularly researched. The results show that the percentage of the components on the total intensity depends on the correlation length of the partially coherent beam. The azimuthal intensity component is produced under the illumination of partially coherent beam.

Properties analysis of digital image-plane holographic microscopic system

The point spread functions (PSFs) of digital image-plane holographic microscopy (DIPHM) and digital holographic microscopy (DHM) are deduced. It can be known from PSFs that the lateral resolution of DIPHM is determined by the numerical aperture of the microscopic objective and the pixel size of CCD, and is independent of the size of CCD sensor, which is quite different in DHM. By comparing the PSFs of DIPHM with spherical and plane reference waves, it is known that the spherical reference wave is preferred in DIPHM setup. The corresponding experiments are conducted to verify the theoretical analysis. It is testified from both experimental results and theoretical analysis that DIPHM is an optimization holographic imaging technique, which can yield high-quality imaging.