Back Focal Plane Of Objective Research Articles

High-speed multicolor structured illumination microscopy using a hexagonal single mode fiber array.

Structured illumination microscopy (SIM) is a widely used imaging technique that doubles the effective resolution of widefield microscopes. Most current implementations rely on diffractive elements, either gratings or programmable devices, to generate structured light patterns in the sample. These can be limited by spectral efficiency, speed, or both. Here we introduce the concept of fiber SIM that allows for camera frame rate limited pattern generation and manipulation over a broad wavelength range. Illumination patterns are generated by coupling laser beams into radially opposite pairs of fibers in a hexagonal single mode fiber array where the exit beams are relayed to the microscope objective's back focal plane. The phase stepping and rotation of the illumination patterns are controlled by fast electro-optic devices. We achieved a rate of 111 SIM frames per second and imaged with excitation patterns generated by both 488 nm and 532 nm lasers.

Analysis of axial scanning range and magnification variation in wide‐field microscope for measurement using an electrically tunable lens

Inserting an electrically tunable lens (ETL), such as liquid lens or tunable acoustic gradient lens, into a microscope can enable fast axial scanning, autofocusing, and extended depth of field. However, placing the ETL at different positions has different influences on image quality. Specially, in a wide-field microscope for measurement, the magnification has to be constant when introducing an ETL, otherwise it will affect measurement accuracy. To determine the best position of ETL, axial scanning range and magnification variation are quantitatively analyzed and discussed in finite and infinite microscopes through theoretical analysis, optical simulation, and experiment for four configurations: when ETL is placed at the back focal plane of objective, at the conjugate plane of objective's back focal plane between two relay lenses, or behind two relay lenses, and at imaging detector plane. The obtained results are as follows. When ETL is placed at the back focal plane, the system has a large scanning range, but the magnification varies because the back focal plane is inside the objective. When ETL is placed between two relay lenses, the magnification stays constant, but the scanning range is small. When ETL is placed behind two relay lenses, the magnification keeps invariant and the scanning range is large, but ETL and two relay lenses are inside the microscope and the system has to be customized. Finally, when ETL is placed at imaging detector plane, the magnification stays constant, but the scanning range is 0, which means the system has no axial scanning capability. RESEARCH HIGHLIGHTS: An electrically tunable lens (ETL) is introduced into a wide-field microscope for measurement. Axial scanning range and magnification variation are analyzed and discussed. Theoretical analysis, ZEMAX optical simulation and experiments are performed.

Enhancing the performance of the light field microscope using wavefront coding.

Light field microscopy has been proposed as a new high-speed volumetric computational imaging method that enables reconstruction of 3-D volumes from captured projections of the 4-D light field. Recently, a detailed physical optics model of the light field microscope has been derived, which led to the development of a deconvolution algorithm that reconstructs 3-D volumes with high spatial resolution. However, the spatial resolution of the reconstructions has been shown to be non-uniform across depth, with some z planes showing high resolution and others, particularly at the center of the imaged volume, showing very low resolution. In this paper, we enhance the performance of the light field microscope using wavefront coding techniques. By including phase masks in the optical path of the microscope we are able to address this non-uniform resolution limitation. We have also found that superior control over the performance of the light field microscope can be achieved by using two phase masks rather than one, placed at the objective's back focal plane and at the microscope's native image plane. We present an extended optical model for our wavefront coded light field microscope and develop a performance metric based on Fisher information, which we use to choose adequate phase masks parameters. We validate our approach using both simulated data and experimental resolution measurements of a USAF 1951 resolution target; and demonstrate the utility for biological applications with in vivo volumetric calcium imaging of larval zebrafish brain.

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.

Single Bipyramid Plasmonic Antenna Orientation Determined by Direct Photoluminescence Pattern Imaging

Photoluminescence of single gold bipyramid due to the radiative decay of the longitudinal surface plasmon is used to determine its 3D orientation using a direct emission pattern imaging technique. The photoluminescence (PL) intensity from individual gold bipyramids is recorded at the objective's back focal plane, which directly maps the angular emission distribution. By correlating the PL spectra, dark‐field scattering, emission pattern, and atomic force microscopy data, the spatial orientation and the radiation characteristics of an individual nanoantenna can be unambiguously determined. The PL emission pattern imaging can be used as a stand‐alone technique for the fast and facile determination of nanoantenna orientations. The experimental data also agree well with a simple analytical model based on a far‐field angular distribution of an isolated dipole emitter on the interface. In situ measurements are also performed on the PL pattern of a single gold bipyramid during optical heating to monitor the photothermal reshaping of the nanoantenna. The results show that the PL emission pattern imaging method is highly sensitive to changes of the geometry and orientation of the nanoparticle. The PL direct imaging technique offers great advantages, including a facile full‐3D angle‐resolved capability, free of photobleaching and photoblinking. Additionally, the results reveal that the gold bipyramids can generate efficient PL emission, and the unique features of gold bipyramids would be outstanding candidates for orientation sensing. These findings have great potential for characterizing optical nanoantennas, for optical imaging and sensing in materials science and biological applications.

Full-Field Near-Field Optical Microscope for Cell Imaging

We report a new full-field fluorescence microscopy method for imaging live cell membranes based on supercritical near-field emission. This technique consists of extracting the self-interference between undercritical and supercritical light by simple image subtraction. In the objective back focal plane, this is equivalent to adding a virtual mask blocking the subcritical emission. We show that this virtual mask is radically different from a real physical mask, enabling a 100 nm axial confinement and enhancing the image sensitivity without damaging the lateral resolution. This technique is easy to implement and simultaneously provides images of the inner parts of the cell and its membrane with standard-illumination light.

Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle

Laser illumination used in both conventional widefield epi-fluorescence as well as in total internal reflection fluorescence (TIRF) microscopy is subject to nonuniformities in intensity that obscure true image details. These intensity variations are interference fringes arising from coherent light scattering and diffraction at every surface in the laser light's optical path, including the lenses, mirrors, and coverslip. We present an inexpensive technique for effectively eliminating these interference fringes based upon introduction of the excitation laser beam by oblique through-the-objective incidence coupled with rapid azimuthal rotation of the plane of incidence. Although this rotation can be accomplished in several ways, a particularly simple method applicable to a free laser beam is to use an optical wedge, spun on a motor, which diverts the beam into a hollow cone of fixed angle. A system of lenses converts this collimated beam cone into a focused spot that traces a circle at the objective's back focal plane. Consequently, a collimated beam with fixed polar angle and spinning azimuthal angle illuminates the sample. If the wedge is spun rapidly, then the different interference patterns at every particular azimuthal incidence angle average out over a single camera exposure to produce an effectively uniform field of illumination.

Fluorescence emission patterns near glass and metal-coated surfaces investigated with back focal plane imaging

Often fluorophores observed by microscopy are located close to a planar surface as in total internal reflection microscopy or single molecule studies. The optical properties of fluorescence excitation and emission near a planar surface (possibly metal film coated) between two dielectrics are well understood from the viewpoint of classical electromagnetic theory. We present an experimental method whereby the angular dependence of the emission pattern of a fluorophore near a bare surface or a metal film coated surface that supports surface plasmon resonance can be measured with microscope optics. The technique involves an alteration of the microscope optics to directly record (on a digital CCD camera) the intensity pattern at the objective's back focal plane, which directly maps the angular emission pattern of fluorescence. The experimental emission profile on both glass and aluminum-coated surfaces is anisotropic with a peak at either the critical angle or both the critical angle and the surface plasmon angle. The observed profiles agree well with computer calculations and suggest some optical modifications that are potentially useful in cell biophysics.

Evanescent field excitation of fluorescence by epi-illumination microscopy

By simple modification of the pattern of fluorescence excitation light in an epi-illumination inverted microscope, one can achieve conditions that produce total internal reflection fluorescence (TIRF) by evanescent wave excitation. Though traditionally requiring a collimated beam traversing through a special prism, TIRF also can be achieved by epi-illumination through the periphery of a 1.4 numerical aperture objective. An opaque disk of appropriate size is placed in the illumination path external to the microscope so as to cast a sharp, real-image shadow at the objective's back focal plane. This shadow allows a hollow cone of epiillumination rays traveling at only super-critical angles to reach the glass/water interface at the sample plane. Three kinds of TIRF illumination patterns can be produced by variations of this scheme: (1) a small spot of illumination of 1.5 microm radius by use of a laser light source, (2) a large region of illumination by use of a laser-illuminated diffusing screen located upbeam from the opaque disk, and (3) a large region of illumination by use of a conventional mercury arc.

On predicting the optical diffraction pattern from thermotropic specimens having a banded texture

Specimens of a sheared and annealed thermotropic copolyester were examined in the polarizing microscope. The optical diffraction pattern associated with the periodic microstructure (banded texture) was observed in the objective back focal plane. The significant features of the diffraction pattern can be predicted analytically in terms of the in-plane periodic variation of optical orientation in the specimen. We explain how the relative intensities of the different diffracted orders depend on the orientation of the crossed polars relative to the specimen shear direction. For specimens in which the optical orientation varies sinusoidally with position along the unique axis, we derive the diffraction pattern exactly, and we explain why second and higher orders are not normally observed.

Four Stage E.M. for Phase Contrast and Strioscopic Microscopy

Phase contrast electron microscopy should allow visualization of unstained biological structures. In an attempt to achieve this, we have modified a Siemens Elmiskop la microscope, in a way similar to that described by Faget, et al. (Proc. Fifth Int. Congr. Electron Microscopy, Philad., 1962, Abstract A-7). A number of phase contrast and strioscopic arrangements can be tested. A section consisting of phase plate chamber and additional intermediate lens (I2) was added to the column between the standard intermediate lens (I1) and the projector lens (Fig. 1). I1 was set, so as to project the back focal plane of the objective to the level of the phase plate holder (Fig. 2). I2 supplied final magnifications from 1,000 x to 80,000 x. Resolution was better than 15 Å. Two stigmators were used, one in the objective back focal plane and one in the plane of the phase plate. The source consisted of either a hairpin- or point- filament to allow variable coherence of illumination.

A Slotted Electrode Stigmator for Correction of Short Focal Length Objectives

One of the difficulties which must be overcome in the design of objective lens stigmators is that they necessarily take up space where it is most precious, in the vicinity of the specimen. The space following the specimen is needed for the contrast aperture while the space between magnetic poles in the gap is required for stage mechanism. To go appreciably below the objective back focal plane to position stigmators subjects the image to possible image distortion when correction is large. A further problem is caused by the trend to shorter objective focal lengths, e.g., 1.5 mm, thus further reducing the space available for the stigmator.To adapt the electrostatic stigmator to these physical restraints, a design has been completed which is only 0.75 mm thick and has a free bore diameter of the same size. Its sensitivity is 0.1 micron/volt with 100 kV electrons. The overall diameter is uncritical and may be adapted to the physical requirements of the system. It should be possible to make stigmators of 3 mm thickness with a simple reduction of critical dimensions (thickness and slot width).