Three-dimensional Optical Sectioning Research Articles

Dual-sided illumination light-sheet fluorescence microscopy with virtual single-pixel imaging deconvolution

In light-sheet fluorescence microscopy (LSFM) a thin light sheet is used to excite the specimen from the side and imaging is performed in the direction perpendicular to the light-sheet. It has the advantages of fast imaging speed, high optical sectioning capability and low photobleaching and phototoxicity to samples. Therefore, it is suitable for high-quality, long-term three-dimensional dynamic observation of large living biological samples. However, the traditional Gaussian light sheet illumination microscopy technology has the problems of small imaging field of view and low spatial resolution. Based on the existing dual-sided illumination LSFM, a large field of view and high resolution LSFM combined with virtual single-pixel imaging deconvolution is presented in this paper, which improves the field of view and resolution of LSFM simultaneously. The relevant microscope is designed and built, and three-dimensional optical sectioning imaging experiments on fluorescent beads and transgenic zebrafish standard samples are carried out. The experimental results prove the three-dimensional high resolution imaging capability of the microscope, which is of great significance in developing the large field of view and high resolution LSFM.

Fabrication and characterization of a multilayered optical tissue model with embedded scattering microspheres in polymeric materials

We report on a novel fabrication approach to build multilayered optical tissue phantoms that serve as independently validated test targets for axial resolution and contrast in scattering measurements by depth-resolving optical coherent tomography (OCT) with general applicability to a variety of three-dimensional optical sectioning platforms. We implement a combinatorial bottom-up approach to prepare monolayers of light-scattering microspheres with interspersed layers of transparent polymer. A dense monolayer assembly of monodispersed microspheres is achieved via a combined methodology of polyelectrolyte multilayers (PEMs) for particle-substrate binding and convective particle flux for two-dimensional crystal array formation on a glass substrate. Modifications of key parameters in the layer-by-layer polyelectrolyte deposition approach are applied to optimize particle monolayer transfer from a glass substrate into an elastomer while preserving the relative axial positioning in the particle monolayer. Varying the dimensions of the scattering microspheres and the thickness of the intervening transparent polymer layers enables different spatial frequencies to be realized in the transverse dimension of the solid phantoms. Step-wise determination of the phantom dimensions is performed independently of the optical system under test to enable precise spatial calibration, independent validation, and quantitative dimensional measurements.

Three-Dimensional Optical Sectioning by Scanning Confocal Electron Microscopy with a Stage-Scanning System

We evaluated the depth resolution of annular dark-field (ADF) scanning confocal electron microscopy (SCEM) with a stage-scanning system by observation of nanoparticles. ADF-SCEM is a three-dimensional (3D) imaging technique that we recently proposed. An ADF-SCEM instrument involves a pinhole aperture before a detector for rejecting electrons from the out-of-focal plane in a specimen and an annular aperture under the specimen for collecting only scattered electrons. The stage-scanning system enables us to directly obtain optical slice images perpendicular and parallel to an optical axis at a desired position. In particular, the parallel slices visualize the elongation of nanoparticles along the optical axis, which depends on the depth resolution. ADF-SCEM effectively reduced the elongation length of the nanoparticles sufficiently to demonstrate depth sectioning, in comparison with scanning transmission electron microscopy and bright-field SCEM. The experimentally obtained length was nearly equal to the theoretically estimated one from the probe size considering the experimental conditions. Furthermore, we applied this ADF-SCEM technique to analysis of the 3D position of catalytic nanoparticles on carbon nanostructures.

Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.

Super-resolution laser scanning microscopy through spatiotemporal modulation.

Super-resolution optical microscopy has attracted great interest among researchers in many fields, especially in biology where the scale of physical structures and molecular processes fall below the diffraction limit of resolution for light. As one of the emerging techniques, structured illumination microscopy can double the resolution by shifting unresolvable spatial frequencies into the pass-band of the microscope through spatial frequency mixing with a wide-field structured illumination pattern. However, such a wide-field scheme typically can only image optically thin samples and is incompatible with multiphoton processes such as two-photon fluorescence, which require point scanning with a focused laser beam. Here, we propose two new super-resolution schemes for laser scanning microscopy by generalizing the concept of a spatially nonuniform imaging system. One scheme, scanning patterned illumination (SPIN) microscopy, employs modulation of the excitation combined with temporally cumulative imaging by a nondescanned array detector. The other scheme, scanning patterned detection (SPADE) microscopy, utilizes detection modulation together with spatially cumulative imaging, in this case by a nondescanned single-element detector. When combined with multiphoton excitation, both schemes can image thick samples with three-dimensional optical sectioning and much improved resolution.

Biologically Inspired Artificial Compound Eyes

This work presents the fabrication of biologically inspired artificial compound eyes. The artificial ommatidium, like that of an insect's compound eyes, consists of a refractive polymer microlens, a light-guiding polymer cone, and a self-aligned waveguide to collect light with a small angular acceptance. The ommatidia are omnidirectionally arranged along a hemispherical polymer dome such that they provide a wide field of view similar to that of a natural compound eye. The spherical configuration of the microlenses is accomplished by reconfigurable microtemplating, that is, polymer replication using the deformed elastomer membrane with microlens patterns. The formation of polymer waveguides self-aligned with microlenses is also realized by a self-writing process in a photosensitive polymer resin. The angular acceptance is directly measured by three-dimensional optical sectioning with a confocal microscope, and the detailed optical characteristics are studied in comparison with a natural compound eye.

Cell Imaging and Manipulation by Nonlinear Optical Microscopy

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.

A fully integrated near-field optical, far-field optical, and normal-force scanned probe microscope

We present a near-field scanning optical microscope (NSOM) which fully integrates near-field and far-field optics with scanned probe microscopy (SPM). The instrument incorporates a novel, flat scanning system that allows the NSOM/SPM head to be placed directly on the sample stage of any upright or inverted optical microscope and fully integrates the two microscopes. The near-field optical microscope that is designed around this flat scanner utilizes cantilevered optical force sensing probes that we have developed. Thus, the instrument can simultaneously image in a number of topographic and optical modes. The normal force sensing capability of our probes and microscope provides a significant advantage over straight optical fibers and the shear force detection techniques that are employed. The piezoelectric scanner incorporates X, Y, and Z scan ranges of over 40 μm into a flat stage less than 7 mm high. The scanning stage also provides for inertial translation of a sample over many millimeters. This completely and transparently integrates the technologies of near-field optics and other scanned probe microscopies with conventional far-field optical microscopy. Furthermore, the axial positioning capability of the flat scanner enhances the characteristics of the far-field microscope by allowing for three-dimensional optical sectioning that is required in techniques such as confocal imaging, nonlinear optical microscopies, and charge coupled device (CCD) imaging. Finally, the X, Y, and Z precision movements also have the potential for being used in optical tweezer applications.

Association of p60c-src with endosomal membranes in mammalian fibroblasts.

We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three-dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI-MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking.

Three-dimensional light microscopy for the analysis of chromosome organization in Drosophila embryo

We are interested in the three-dimensional physical relationships among chromosomes in mitotically active nuclei. Using high-resolution, three-dimensional optical sectioning microscopy incorporating a charge-coupled device (CCD), we have analyzed the spatial arrangements of chromosomes in nuclei from Drosophila melanogaster embryos. During early stages of nuclear division, these embryos exist as syncytial blastoderms, with up to 5000 nuclei forming a single layer near the embryo's surface and dividing essentially synchronously. This greatly facilitates the analysis of chromosome organization because of the simple monolayer arrangement of nuclear structures at defined mitotic stages.From prophase to anaphase, three-dimensional paths of condensed chromosomes stained with a DNA-specific fluorescent dye can be traced using an interactive computer program. We have used this method to ask the specific biological question of whether pairs of homologous chromosomes have a consistent, ordered arrangement in diploid nuclei. Our results have shown that homologous chromosomes are not associated with each other from prophase to anaphase in syncytial blastoderm stage embryos.

Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos.

The spatial and temporal dynamics of diploid chromosome organization, microtubule arrangement, and the state of the nuclear envelope have been analyzed in syncytial blastoderm embryos of Drosophila melanogaster during the transition from prophase to metaphase, by three-dimensional optical sectioning microscopy. Time-lapse, three-dimensional data recorded in living embryos revealed that congression of chromosomes (the process whereby chromosomes move to form the metaphase plate) at prometaphase occurs as a wave, starting at the top of the nucleus near the embryo surface and proceeding through the nucleus to the bottom. The time-lapse analysis was augmented by a high-resolution analysis of fixed embryos where it was possible to unambiguously trace the three-dimensional paths of individual chromosomes. In prophase, the centromeres were found to be clustered at the top of the nucleus while the telomeres were situated at the bottom of the nucleus or towards the embryo interior. This polarized centromere-telomere orientation, perpendicular to the embryo surface, was preserved during the process of prometaphase chromosome congression. Correspondingly, breakdown of the nuclear envelope started at the top of the nucleus with the mitotic spindle being formed at the positions of the partial breakdown of the nuclear envelope. Our observation provide an example in which nuclear structures are spatially organized and their functions are locally and coordinately controlled in three dimensions.