An efficient way of high-contrast, quasi-3D cellular imaging: off-axis illumination

An imaging system enabling a convenient visualisation of cells and other small objects is presented. It represents an adaptation of the optical microscope condenser, accommodating a built-in edge (relief) diaphragm brought close to the condenser iris diaphragm and enabling high-contrast pseudo-relief (quasi-3D) imaging. The device broadens the family of available apparatus based on the off-axis (or anaxial, asymmetric, inclined, oblique, schlieren-type, sideband) illumination. The simplicity of the design makes the condenser a user-friendly, dedicated device delivering high-contrast quasi-3D images of phase objects. Those are nearly invisible under the ordinary (axial) illumination. The phase contrast microscopy commonly used in visualisation of phase objects does not deliver the quasi-3D effect and introduces a disturbing 'halo' effect around the edges. The performance of the device presented here is demonstrated on living cells and tissue replicas. High-contrast quasi-3D images of cell-free preparations of biological origin (paper fibres and microcrystals) are shown as well.     

Improving Imaging Contrast of Non-Contacted Plasmonic Lens by Off-Axis Illumination with High Numerical Aperture

Off-axis illumination plasmonic lens (OAIPL) is proposed and demonstrated to improve the imaging contrast in non-contacted application manner. The spatial Fourier components of light transmitted through the nano-patterns are greatly enhanced in the imaging process by shifting the wave vectors with high numerical aperture off-axis illumination. On the other hand, a reflector in the image area helps to tailor the ratio between electric field components in the tangential and normal directions. These two effects resultantly deliver significant improvement of imaging performance, including enhanced resolution, imaging contrast, and elongation of air gap thickness. In comparison to the case of normal illumination, the air gap thickness for 30 and 60 nm half-pitch resolution is extended to 25 and 100 nm by OAIPL with numerical aperture (NA)?=?1.55, respectively.     

Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or approximately 100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy.     

Off Axis Illumination Planar Hyperlens for Non-contacted Deep Subwavelength Demagnifying Lithography

Off axis illumination (OAI) technique combined with planar hyperlens is applied to achieve the non-contacted deep subwavelength demagnifying lithography. The designed OAI is confirmed to shift the spatial spectra of mask, leading to enhancement of the featured wavevectors components. On the other hand, the reflection effect of Ag cladding is necessary to obtain high contrast and high fidelity of nanopattern lithography. It is numerically demonstrated that the half-pitch resolution 32 nm with OAI (NA?=?1.37) is obtained under 80 nm air working distance, which is about six times than the case of the conventional configuration under normal illumination (NA?=?0). Further simulations show that the minimum half-pitch resolution of the planar hyperlens with OAI could reach 18 nm (～λ/20).     

Asymmetric flows of spherical particles in a cylindrical tube

To study the rheological behavior of blood cells in various flow patterns through narrow vessels, we analyzed numerically the motion of blood cells arranged in one row or two rows in tube flow, at low Reynolds numbers. The particles are assumed to be identical rigid spheres placed periodically along the vessel axis at off-axis positions with equal spacings. The flow field of the suspending fluid in a circular cylindrical tube is analyzed by a finite element method applied to the Stokes equations, and the motion of each particle is simultaneously determined by a force-free and torque-free condition. In both cases of single- and two-file arrangements of the particles, their longitudinal and angular velocities are largely affected by the radial position and the axial spacing between neighboring particles. The apparent viscosity of the asymmetric flows is higher than that of the symmetric flow where particles are located on the tube centerline, and this is more pronounced when particles are placed farther from the tube centerline and when the axial distance between neighboring particles is reduced.     

Quality assessment of confocal microscopy slide based systems: performance.

BACKGROUND: All fluorescence slide-based cytometry detections systems basically include the following components: (1) an excitation light source, (2) intermediate optics, and (3) a detection device consisting of a CCD camera or a PMT. The optical principles employed is slide-based systems are similar to those of confocal microscopes (CLSM). METHODS: The following tests evaluated confocal equipment performance: dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, PMT reliability, and system noise. RESULTS: Quality assurance tests provide a basis to determine if the equipment is operating correctly. Laser power, PMTs function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance and laser stability were tested colocalization of UV and visible peaks of a bead should be less than 210 nm. Interference contrast optics decrease fluorescence resolution. CONCLUSIONS: QA tests that assess CLSM system performance are also applicable to other slide-based systems. By utilization this type of testing approach, the subjective nature of assessing the CLSM may be eliminated. These tests serve as guidelines for other investigators to ensure that their machines are providing data that is accurate with the necessary resolution, sensitivity and precision.     

Electrophysiological evidence for interaction between retinula cells in the flesh-fly

Summary In the electrical response of retinula cells to polarized light in the flesh-flyBoettcherisca peregrina, the polarization plane which showed the maximum sensitivity (Pol_max phase) to illumination by a small spot of light just large enough to cover only one retinula cell was found to differ from that with illumination by a larger spot of light which included adjacent cells. There was a difference of about 30°.This difference in Pol_max phase was assumed to indicate the occurrence of interaction between retinula cells even in the fly photoreceptor having rhabdom of the open type. This assumption was confirmed by the following experiments. (1) Under selective adaptation by a large spot of polarized light so as to eliminate the interaction effect, the Pol_max phase was found to be the same as that measured by a small spot even though the measurement had been made using a large spot of light. (2) The responses to polarized light illuminated from along some restricted off-axes showed a 60° shift in the Pol_max phase in comparison to those obtained from along the other axes. (3) The spectral sensitivity curves to illumination from along off-axes were almost all the same and were for the peripheral retinula cells. (4) The receptor potentials were found to increase in amplitude in a certain limited off-axis area that corresponded to the specific off-axis direction of illumination which had resulted in a shift of the Pol_max phase.It is concluded from these results that the peripheral retinula cells in the flesh-fly demonstrate interaction between certain two adjacent retinula cells. This interaction is positive but not a simple algebraic sum of the activity of two cells.This work was partly supported by a grant from the Japan Education MinistryI wish to thank the Department of Biology, Faculty of Sciences, Kyushu University (Prof. H. Morita and Prof. H. Tateda) for the constant supply of flesh-flies.     

Circadian effects of light no brighter than moonlight

In mammals, light entrains endogenous circadian pacemakers by inducing daily phase shifts via a photoreceptor mechanism recently discovered in retinal ganglion cells. Light that is comparable in intensity to moonlight is generally ineffective at inducing phase shifts or suppressing melatonin secretion, which has prompted the view that circadian photic sensitivity has been titrated so that the central pacemaker is unaffected by natural nighttime illumination. However, the authors have shown in several different entrainment paradigms that completely dark nights are not functionally equivalent to dimly lit nights, even when nighttime illumination is below putative thresholds for the circadian visual system. The present studies extend these findings. Dim illumination is shown here to be neither a strong zeitgeber, consistent with published fluence response curves, nor a potentiator of other zeitgebers. Nevertheless, dim light markedly alters the behavior of the free-running circadian pacemaker. Syrian hamsters were released from entrained conditions into constant darkness or dim narrowband green illumination (~0.01 lx, 1.3 x 10(-9) W/cm(2), peak lambda = 560 nm). Relative to complete darkness, constant dim light lengthened the period by ~0.3 h and altered the waveform of circadian rhythmicity. Among animals transferred from long day lengths (14 L:10 D) into constant conditions, dim illumination increased the duration of the active phase (alpha) by ~3 h relative to complete darkness. Short day entrainment (8 L:16 D) produced initially long alpha that increased further under constant dim light but decreased under complete darkness. In contrast, dim light pulses 2 h or longer produced effects on circadian phase and melatonin secretion that were small in magnitude. Furthermore, the amplitude of phase resetting to bright light and nonphotic stimuli was similar against dimly lit and dark backgrounds, indicating that the former does not directly amplify circadian inputs. Dim illumination markedly alters circadian waveform through effects on alpha, suggesting that dim light influences the coupling between oscillators theorized to program the beginning and end of subjective night. Physiological mechanisms responsible for conveying dim light stimuli to the pacemaker and implications for chronotherapeutics warrant further study.     

Anisotropy of energy losses in high-current Z-pinches produced by the implosion of cylindrical tungsten wire arrays

Results are presented from measurements of the anisotropy of energy losses in high-current Z-pinches produced by the implosion of wire arrays at the ANGARA-5-1 facility at load currents of up to 4MA. The energy losses were measured in the radial direction and along the pinch axis from the anode side. The main diagnostics were time-integrated thermocouple calorimeters, nanosecond X-ray diodes (XRDs) with different filters, and a foil radiation calorimeter with a time resolution of 2 μs. The azimuthal anisotropy of energy losses was measured for different wire array configurations and different shapes of the high-voltage electrode. The presence of strong initial azimuthal inhomogeneity of the wire mass distribution (sectioned arrays), as well as the use of conical electrodes instead of plane ones, does not increase the azimuthal inhomogeneity of the total energy losses. For cylindrical wire arrays, energy losses in the radial direction are compared with those along the pinch axis. According to XRD and calorimetric measurements, the radiation yield per unit solid angle along the pinch axis is two to three times lower than that in the radial direction. In the axial direction, the energy flux density of the expanding plasma is two to three times lower than the radiation intensity. The measured radiation yield across the pinch is 2.5–5 kJ/sr, while that along the pinch axis is 1–2 kJ/sr. The results obtained by means of XRDs agree to within measurement errors with those obtained using the radiation calorimeter. It is found that the energy per unit solid angle carried by the expanding plasma in the radial direction does not exceed 10% of the soft X-ray yield. Analysis of the structure of time-integrated pinhole images and signals from the radial and axial XRDs shows that radiation emitted in the radial direction from the hot central region of the pinch is partially screened by the less dense surrounding plasma halo, whereas radiation emitted in the axial direction is a superposition of the emissions from the hot dense central region of the pinch and the colder less dense peripheral plasma.     

THE VISUAL ACUITY OF THE HONEY BEE

1. Bees respond by a characteristic reflex to a movement in their visual field. By confining the field to a series of parallel dark and luminous bars it is possible to determine the size of bar to which the bees respond under different conditions and in this way to measure the resolving power or visual acuity of the eye. The maximum visual acuity of the bee is lower than the lowest human visual acuity. Under similar, maximal conditions the fineness of resolution of the human eye is about 100 times that of the bee. 2. The eye of the bee is a mosaic composed of hexagonal pyramids of variable apical angle. The size of this angle determines the angular separation between adjacent ommatidia and therefore sets the structural limits to the resolving power of the eye. It is found that the visual angle corresponding to the maximum visual acuity as found experimentally is identical with the structural angular separation of adjacent ommatidia in the region of maximum density of ommatidia population. When this region of maximum ommatidia population is rendered non-functional by being covered with an opaque paint, the maximum visual acuity then corresponds to the angular separation of those remaining ommatidia which now constitute the maximum density of population. 3. The angular separation of adjacent ommatidia is much smaller in the vertical (dorso-ventral) axis than in the horizontal (anterio-posterior) axis. The experimentally found visual acuity varies correspondingly. From this and other experiments as well as from the shape of the eye itself, it is shown that the bee''s eye is essentially an instrument for uni-directional visual resolution, functional along the dorso-ventral axis. The resolution of the visual pattern is therefore determined by the vertical angular separation of those ocular elements situated in the region of maximum density of ommatidia population. 4. The visual acuity of the bee varies with the illumination in much the same way that it does for the human eye. It is low at low illuminations; as the intensity of illumination increases it increases at first slowly and then rapidly; and finally at high intensities it becomes constant. The resolving power of a structure like the bee''s eye depends on the distance which separates the discrete receiving elements. The data then mean that at low illuminations the distance between receiving elements is large and that this distance decreases as the illumination increases. Since such a moving system cannot be true anatomically it must be interpreted functionally. It is therefore proposed that the threshold of the various ommatidia are not the same but that they vary as any other characteristic of a population. The visual acuity will then depend on the distance apart of those elements whose thresholds are such that they are functional at the particular illumination under investigation. Taking due consideration of the angular separation of ommatidia it is possible to derive a distribution curve for the thresholds of the ommatidia which resembles the usual probability curves, and which describes the data with complete fidelity.     

Toward fluorescence nanoscopy

For more than a century, the resolution of focusing light microscopy has been limited by diffraction to 180 nm in the focal plane and to 500 nm along the optic axis. Recently, microscopes have been reported that provide three- to sevenfold improved axial resolution in live cells. Moreover, a family of concepts has emerged that overcomes the diffraction barrier altogether. Its first exponent, stimulated emission depletion microscopy, has so far displayed a resolution down to 28 nm. Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level. As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever.     

Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy

Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured illumination microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct an SR image. Using an alternative approach, we demonstrate that the additional spatial information encoded in the knowledge of the position of the illumination pattern can be efficiently decoded using a generalized version of separation of photon by lifetime tuning (SPLIT) that does not require lifetime measurements. In the resulting SPLIT-SIM, the SR image is obtained by isolating a fraction of the intensity corresponding to the center of the diffraction-limited point spread function. This extends the use of the SPLIT approach from stimulated emission depletion microscopy to SIM. The SPLIT-SIM algorithm is based only on phasor analysis and does not require deconvolution. We show that SPLIT-SIM can be used to generate SR images of chromatin organizational motifs with tunable resolution and can be a valuable tool for the imaging of functional sites in the nucleus.     

Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.     

Immunofluorescence stimulated emission depletion microscopy

We report immunofluorescence imaging with a spatial resolution well beyond the diffraction limit. An axial resolution of approximately 50 nm, corresponding to 1/16 of the irradiation wavelength of 793 nm, is achieved by stimulated emission depletion through opposing lenses. We have demonstrated not only that an antibody-tagged label is stable enough to be recorded in this microscopy mode, but also that subdiffraction resolution can be obtained using a standard immunofluorescence preparation.     

The relative importance of ultraviolet and longer wavelengths in the response properties of fly visual systems

A generalized analysis of the generator potential responses of R1-6 cells of Calliphora provides remarkable information on the visual properties for the Diptera. This shows that, although these cells have two peak response sensivities for monochromatic stimuli at 350 and 480 nm under single color stimulus conditions, and when the background illumination is either zero or in the region of 450–560 nm, the sensitivity to ultraviolet light is practically eliminated for background illumination in either the ultraviolet or the region around 600 nm or when any simultaneous dynamic stimulus in the region of 480–550 nm is also applied. These results seem somewhat perplexing to an understanding of the behavioral vision properties. It also is not consistant with the concept that the ultraviolet response is initiated by a sensitizing pigment within these cells that transfers energy to the rhodopsin-metarhodopsin process. However, it strengthens other evidence that the limited condition of ultraviolet responses comes from interaction from R7,8 cells but does not play an important behavioral role in the visual system fed from cells R1-6. As discussed in this paper, any high level pattern recognition controlling behavioral response to ultraviolet stimuli comes from the R7,8 cell system.     

Measurement of the screw-home motion of the knee is sensitive to errors in axis alignment

Measurements of joint angles during motion analysis are subject to error caused by kinematic crosstalk, that is, one joint rotation (e. g., flexion) being interpreted as another (e.g., abduction). Kinematic crosstalk results from the chosen joint coordinate system being misaligned with the axes about which rotations are assumed to occur. The aim of this paper is to demonstrate that measurement of the so-called "screw-home" motion of the human knee, in which axial rotation and extension are coupled, is especially prone to errors due to crosstalk. The motions of two different two-segment mechanical linkages were examined to study the effects of crosstalk. The segments of the first linkage (NSH) were connected by a revolute joint, but the second linkage (SH) incorporated gearing that caused 15 degrees of screw-home rotation to occur with 90 degrees knee flexion. It was found that rotating the flexion axis (inducing crosstalk) could make linkage NSH appear to exhibit a screw-home motion and that a different rotation of the flexion axis could make linkage SH apparently exhibit pure flexion. These findings suggest that the measurement of screw-home rotation may be strongly influenced by errors in the location of the flexion axis. The magnitudes of these displacements of the flexion axis were consistent with the inter-observer variability seen when five experienced observers defined the flexion axis by palpating the medial and lateral femoral epicondyles. Care should be taken when interpreting small internal-external rotations and abduction-adduction angles to ensure that they are not the products of kinematic crosstalk.     

Off-axis loads cause failure of the distal radius at lower magnitudes than axial loads: a finite element analysis

Distal radius (Colles') fractures are a common fall-related injury in older adults and frequently result in long-term pain and reduced ability to perform activities of daily living. Because the occurrence of a fracture during a fall depends on both the strength of the bone and upon the kinematics and kinetics of the impact itself, we sought to understand how changes in bone mineral density (BMD) and loading direction affect the fracture strength and fracture initiation location in the distal radius. A three-dimensional finite element model of the radius, scaphoid, and lunate was used to examine changes of +/-2% and +/-4% BMD, and both axial and physiologically relevant off-axis loads on the radius. Changes in BMD resulted in similar percent changes in fracture strength. However, modifying the applied load to include dorsal and lateral components (assuming a dorsal view of the wrist, rather than an anatomic view) resulted in a 47% decrease in fracture strength (axial failure load: 2752N, off-axis: 1448N). Loading direction also influenced the fracture initiation site. Axially loaded radii failed on the medial surface immediately proximal to the styloid process. In contrast, off-axis loads, containing dorsal and lateral components, caused failure on the dorsal-lateral surface. Because the radius appears to be very sensitive to loading direction, the results suggest that much of the variability in fracture strength seen in cadaver studies may be attributed to varying boundary conditions. The results further suggest that interventions focused on reducing the incidence of Colles' fractures when falls onto the upper extremities are unavoidable may benefit from increasing the extent to which the radius is loaded along its axis.     

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.     

Fluorescence microscopy with super-resolved optical sections

The fluorescence microscope, especially its confocal variant, has become a standard tool in cell biology research for delivering 3D-images of intact cells. However, the resolution of any standard optical microscope is at least 3 times poorer along the axis of the lens that in its focal plane. Here, we review principles and applications of an emerging family of fluorescence microscopes, such as 4Pi microscopes, which improve axial resolution by a factor of seven by employing two opposing lenses. Noninvasive axial sections of 80-160 nm thickness deliver more faithful 3D-images of subcellular features, providing a new opportunity to significantly enhance our understanding of cellular structure and function.     

Near-Infrared Super Resolution Imaging with Metallic Nanoshell Particle Chain Array

We propose a near-infrared super resolution near field imaging system with an array of metallic nanoshell particle chain. The imaging array can plasmonically transfer the near field components of dipole sources and the super resolution images can be reconstructed in the output plane. By decreasing the metallic nanoshell’s thickness of the fixed size nanoparticle, the plasmon resonance wavelength of the isolate nanoshell particle is red-shifted to the near-infrared region. The operation wavelength of the imaging array is correspondingly red-shifted to the near-infrared region. In this paper, we study the incoherent and coherent super resolution imaging. The field intensity distributions at the different planes of imaging process are calculated using the finite element method. The simulation results demonstrate that the array has super resolution imaging capability at near-infrared wavelengths in the incoherent and coherent manners. The results also show that the image formation highly depends on the source coherence. In the same structural parameters, the reconstructed images under the illumination of incoherent light source reach to the higher image quality and spatial resolution than the images under the illumination of coherent light source of in phase. By reasonably designing parameters of the imaging array, the approximate spatial resolutions of λ/13 in incoherent case and λ/10 in coherent case are obtained at the near-infrared wavelength of 764 nm. Furthermore, the image–array distance and the chains’ spacing also affect the image reconstruction.