Supercontinuum dynamically visualizes a dividing single cell

During cell division, various organelles behave dynamically. Visualization of these dynamic behaviors of organelles is a promising one step forward for understanding life at the molecular level. One- or two-photon excited fluorescence microscopy has so far been used for visualizing these cell dynamics. The fluorescent probe introduced into a living cell can visualize the spatial distribution of a target molecule in real time, enabling the tracing of cell dynamics at the molecular level. Introducing a fluorescent probe into a cell, however, may alter the physical and chemical conditions of the cell. Here we show a new method for direct (no need for staining cells) visualization of living cell processes with coherent anti-Stokes Raman scattering (CARS) spectroscopy. A new light source, supercontinuum generated from a photonic crystal fiber, has facilitated ultrabroadband (>3500 cm(-1)) multiplex CARS spectroscopy and imaging with high molecular specificity. Using this multiplex CARS technique, we have been successful in tracing the whole cell division process, the splitting of a mother cell into two daughter cells, appearance and disappearance of septum, and dynamic distribution changes of organelles consisting of lipid membrane. The supercontinuum has also facilitated simultaneous measurement of the CARS and two-photon excited fluorescence (TPEF) spectra, enabling what we call multiple nonlinear spectral imaging. Three-dimensional image reconstruction of a living cell with high speed is now possible to elucidate more detailed molecular-level dynamics inside a dividing living cell.     

Contrast enhancement of underwater images with coherent optical image processors

The use of coherent optical processors for improving the quality of degraded underwater images is discussed, and a holographic filter suitable for the enhancement of contrast in underwater images is described. The filter is a modified matched spatial filter, and it performs a nonlinear local contrast enhancement. The theory of the operation of the coherent optical image processor with this new filter is presented and experimentally verified. The removal of the backscattered light from underwater images is demonstrated under laboratory conditions. The results show an improvement in the overall image contrast.     

Determining the size of coherent nanoinclusions and the crystal lattice misfit parameter using dark-field transmission electron microscopy images

The task of identifying nanodimensional inclusions with a structure coinciding with that of the surrounding matrix in transmission electron microscopy (TEM) images encounters considerable difficulties. Analysis of high-resolution TEM images requires a large volume of calculations and the exact knowledge of a large number of various parameters. The well-known method of determining coherent inclusion characteristics using dark-field images can be used only for relatively large objects. We describe a new method for determining the size and the crystal lattice misfit parameter using dark-field TEM images, which is applicable to coherent inclusions with dimensions as small as several nanometers.     

Optical micromanipulations inside yeast cells

We present a combination of nonlinear microscopy and optical trapping applied to three-dimensional imaging and manipulation of intracellular structures in living cells. We use Titanium-sapphire laser pulses for nonlinear microscopy of the nuclear envelope and the microtubules marked with green fluorescent protein in fission yeast. The same laser source is also used to trap small lipid granules naturally present in the cell. The trapped granule is used as a handle to exert a pushing force on the cell nucleus. The granule is moved in a raster-scanning fashion to cover the area of the nucleus and hence displace the nucleus away from its normal position in the center of the cell. Such indirect manipulations of an organelle (e.g., nucleus) can be useful when direct trapping of the chosen organelle is disadvantageous or inefficient. We show that nonlinear microscopy and optical manipulation can be performed without substantial damage or heating of the cell. We present this method as an important tool in cell biology for manipulation of specific structures, as an alternative to genetic and biochemical methods. This technique can be applied to several fundamental problems in cell biology, including the mechanism of nuclear positioning and the spatial coordination of nuclear and cell division.     

Imaging theory of nonlinear Raman confocal microscopy

A novel nonlinear Raman confocal microscopy utilizing Raman induced Kerr effect spectroscopy (RIKES) is presented in this paper. The imaging theory of RIKES confocal microscopy with Gaussian beam is derived. The imaging properties of RIKES confocal microscopy and the impact of different beam waist widths of Gaussian beam on the lateral and axial resolution have been analyzed in detail. It is proved that RIKES confocal microscopy has high sensitivity and high resolution, besides capability to characterize inherent structural features, such as vibration mode, vibration orientation, and optically induced molecular reorientation etc. Therefore, nonlinear Raman confocal microscopy that is based on RIKES has potential to provide a novel characteristic imaging method comparable to the existing imaging techniques based on other nonlinear optical processes, such as two-photon fluorescence, second harmonic generation (SHG) and coherent anti-Stoke Raman scattering (CARS).     

Cancellation of coherent artifacts in optical coherence tomography imaging

Coherent artifacts in optical coherence tomography (OCT) images can severely degrade image quality by introducing false targets if no targets are present at the artifact locations. Coherent artifacts can also add constructively or destructively to the targets that are present at the artifact locations. This constructive or destructive interference will result in cancellation of the true targets or in display of incorrect echo amplitudes of the targets. We introduce the use of a nonlinear deconvolution algorithm, CLEAN, to cancel coherent artifacts in OCT images of extracted human teeth. The results show that CLEAN can reduce the coherent artifacts to the noise background, sharpen the air-enamel and enamel-dentin interfaces, and improve the image contrast.     

Two-dimensional imaging and three-dimensional reconstruction of low reflectivity surfaces by using the range-gating upconversion second-harmonic method

Three-dimensional images of objects with very low reflectivity are obtained through a nonlinear upconversion gating with amplified femtosecond laser pulses. The current sensitivity of 10(-10) of the incident pulse intensity can be improved by use of better nonlinear crystals and higher-intensity gating pulses. The intensity rejection ratio between two coherent pulses with a delay of a few millimeters between them is better than 2 orders of magnitude. The depth resolution is ~15 μm. The transverse resolution of 300 μm is mainly limited by the two-dimensional detector that was used.     

Restoration of bilinearly distorted images. 3: Constrained least-squares method

A constrained least-squares solution based on a second difference smoothing measure is obtained for images which are distorted by a bilinear (quadratic and with nonzero memory) system in the presence of additive signal-independent noise. Results are applied to a partially coherent diffraction-limited imaging system. It is found that the optimum weight given to the smoothness factor is larger for a coherent (nonlinear) system than for an incoherent (linear) system. However, the restoration quality improves as the imaging system approaches incoherence.     

Probing chirality of a lipid tubular by confocal Raman microscopy

The chiral phospholipids 1,2-bis-(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9 PC) can self assemble into lipid nanotubules. This hollow cylindrical supramolecular structure shows promise in a number of biotechnological applications. The mechanism of lipid tubule formation was initiated by assembling of lipid bilayer sheets from amphiphilic solution. Upon cooling, small ribbons were detached from the sheets and rolled up into helical tubules. The lipid tubules obtained were 0.6-0.8 microm in diameter and approximately 50 microm in length. Raman spectra of individual polymerized lipid tubules were measured by focused laser excitation of 532 nm leading to intense and reproducible Raman spectra. The chirality of lipid tubules was investigated by atomic force microscopy (AFM) and confocal Raman microscopy. We report the Raman mapping images revealing helical tubular profiles of C=C stretching and C[triple bond]C stretching of lipid tubules. Circular dichroism property of lipid tubules has also been probed with a 532 nm laser.     

Spatial distribution of fluorescence intensity within large droplets and its dependence on dye concentration

The dependence of fluorescence intensity distributions within droplets on added dye concentration has been calculated by extension of the geometrical-optics approximation and verified by experimental observations. With rising dye concentration, surface plots of the equatorial fluorescence pattern show decreasing relevance of intensity enhancement at focusing points of internal light rays and increasing effects of linear absorption on the characteristic features of the distribution. For comparison with experimentally obtained images of the fluorescence intensity distribution within droplets, a method for calculating volume-integrated intensity distributions was developed in which image distortion at the fluid-air interface is included. A comparison of the calculated and the experimentally determined fluorescence intensity distributions within a droplet confirmed the accuracy of the geometrical-optics approach at high dye concentrations. However, discrepancies from experimental results are visible at low dye concentrations owing to nonlinear optical effects.     

Linear size gradient single layers of polymer-dispersed liquid crystal micrometer-sized droplets for diffractive optics

An experimental study of coherent light diffraction by wedge-formed single layers composed of liquid crystal (LC) micro-sized droplets dispersed in a transparent solid polymer matrix is reported. The micrometer-sized polymer-dispersed liquid crystal (PDLC) material contains prolate-ellipsoid-like LC droplets with a linear-gradient size distribution along the wedge slope. The droplet diameter in the films reaches several tens of micrometers, defined by the wedge. Such a droplet organization in a two-dimensional layer provides both spatial and electrical control of the coherent light diffraction by the LC/polymer interface.     

Quantitative image analysis of broadband CARS hyperspectral images of polymer blends

We demonstrate that broadband coherent anti-Stokes Raman scattering (CARS) microscopy can be very useful for fast acquisition of quantitative chemical images of multilayer polymer blends. This is challenging because the raw CARS signal results from the coherent interference of resonant Raman and nonresonant background and its intensity is not linearly proportional to the concentration of molecules of interest. Here we have developed a sequence of data-processing steps to retrieve background-free and noise-reduced Raman spectra over the whole frequency range including both the fingerprint and C-H regions. Using a classical least-squares approach, we are able to decompose a Raman hyperspectral image of a tertiary polymer blend into quantitative chemical images of individual components. We use this method to acquire 3-D sectioned quantitative chemical images of a multilayer polymer blend of polystyrene, styrene-ethylene/propylene copolymer, and polypropylene that have overlapping spectral peaks.     

Combined scanning electrochemical/optical microscopy with shear force and current feedback

A technique that combines scanning electrochemical microscopy (SECM) and scanning optical microscopy (OM) was developed. Simultaneous scanning electrochemical/optical microscopy (SECM/OM) was performed by a special probe tip, which consists of an optical fiber core for light passage, surrounded by a gold ring electrode, and an outermost electrophoretic insulating sheath, with the tip attached to a tuning fork. To regulate the tip-substrate distance, either the shear force or the SECM tip current was employed as the feedback signal. The application of a quartz crystal tuning fork (32.768 kHz) for sensing shear force allowed simultaneous topographic, along with SECM and optical imaging in a constant-force mode. The capability of this technique was confirmed by obtaining simultaneously, for the first time, topographic, electrochemical, and optical images of an interdigitated array electrode. Current feedback from SECM also provided simultaneous electrochemical and optical images of relatively soft samples, such as a polycarbonate membrane filter and living diatoms in a constant-current mode. This mode should be useful in mapping the biochemical activity of a living cell.     

Discriminating and imaging different phosphatidylcholine species within phase-separated model membranes by principal component analysis of TOF-secondary ion mass spectrometry images

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) enables chemically imaging the distributions of various lipid species in model membranes. However, discriminating the TOF-SIMS data of structurally similar lipids is very difficult because the high intensity, low mass fragment ions needed to achieve submicrometer lateral resolution are common to multiple lipid species. Here, we demonstrate that principal component analysis (PCA) can discriminate the TOF-SIMS spectra of four unlabeled saturated phosphatidylcholine species, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) according to variations in the intensities of their low mass fragment ions (m/z ≤ 200). PCA of TOF-SIMS images of phase-separated DSPC/DLPC and DPPC/DLPC membranes enabled visualizing the distributions of each phosphatidylcholine species with higher contrast and specificity than that of individual TOF-SIMS ion images. Comparison of the principal component (PC) scores images to atomic force microscopy (AFM) images acquired at the same membrane location before TOF-SIMS analysis confirmed that the PC scores images reveal the phase-separated membrane domains. The lipid composition within these domains was identified by projection of their TOF-SIMS spectra onto PC models developed using pure lipid standards. This approach may enable the identification and chemical imaging of structurally similar lipid species within more complex membranes.     

Buckling of lipid tubules in shrinking liquid droplets

Self-assembled hollow lipid tubules are interesting and potentially useful supramolecular structures. Here, we study the deformation of lipid tubules of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) trapped inside liquid droplets on glass substrates. The interface tension of the shrinking liquid droplets exerts a compression force on the ends of the trapped lipid tubules, and causes them to buckle. This provides a method to measure their mechanical properties. The Young's modulus of the DC8,9PC lipid tubules is estimated to approximately 1.07 GPa. As the strain energy of the buckled tubules builds up, they poke through the interface of shrinking liquid droplets and then adhere onto glass substrates to form looplike shapes.     

Coherent use of opposing lenses for axial resolution increase. II. Power and limitation of nonlinear image restoration

We analyze the ability of nonlinear image restoration to remove interference artifacts in microscopes that enlarge the axial optical bandwidth through coherent counterpropagating waves. We calculate the images of an elaborate test object as produced by confocal, standing-wave, incoherent illumination interference image interference, and 4Pi confocal microscopes, and we subsequently investigate the extent to which the initial object can be restored by the information allowed by their optical transfer function. We find that nonlinear restoration is successful only if the transfer function is sufficiently contiguous and has amplitudes well above the noise level, as is mostly the case in a two-photon excitation 4Pi confocal microscope.     

Preparation and atomic force microscopy observation of lipid membranes on micro-patterned self-assembled monolayers

Using micro-contact printing (μCP) method, lipid membranes were deposited on the surface of micro-patterned self-assembled monolayers. Height and phase mode atomic force microscopy (AFM) images showed that the resulting deposited films were flat and that a micro-patterned composite bilayer system was constructed. It was also shown that the use of crystalline phase lipid membrane is effective for the preparation of the micro-patterned composite bilayer system of membranes with flat surface.     

Montage and Multiples in Hannah Maynard’s Self-Portraits

Hannah Maynard was a prolific photographic portraitist who opened her first commercial studio in Victoria, British Columbia, in 1862. While she maintained a conventional commercial practice, her creative photographic work began to assimilate and respond to significant societal shifts in unconventional ways. In the 1880s Maynard started to experiment with a variety of ‘avant-garde’ techniques including photo collage, photo montage, photo sculpture, and multiple and composite images. In her late series of multiple exposure self-portraits, the integrity of the self and of the image, an imperative of conventional photographic portraiture, comes into question. Through a metaphorical relationship with spirit photography, these self-reflexive images toy with the distance between realism and surrealism, between coherent self and spectral other, and between the living and the dead.     

Simultaneous imaging and chemical attack of a single living cell within a confluent cell monolayer by means of scanning electrochemical microscopy

Epithelial cell monolayers from rat kidney were imaged by scanning electrochemical microscopy (SECM) with sub-micrometer resolution in both lateral and vertical direction. Platinum disk ultra-microelectrodes (UMEs) with effective electrode radii between 200 and 600 nm were operated in the constant-height mode. The quality of the recorded SECM images compare favorably with those of phase contrast and confocal laser scanning microscopy. Besides the acquisition of SECM images, the UME was used to selectively attack a single living cell within the monolayer ensemble. Hydroxide ions were locally generated in the vicinity of a single target cell by the UME. The increase in pH induced cell necrosis that was subsequently imaged by SECM. It could be clearly demonstrated that the single target cell was selectively affected, whereas the adjacent reference cells remained unchanged.     

Imaging nanometer scale optical heterogeneities in phospholipid monolayers deposited on metal island films

Near-field scanning optical microscopy was applied to study the distribution of fluorescently labeled phospholipid monolayers deposited on the surface of gold island films by the Langmuir-Blodgett technique. Nanometer scale (approximately 50 nm) optical heterogeneities were observed in near-field fluorescence images of the monolayer deposited at 10 mN/m surface pressure. At higher surface pressure (30 mN/m) the heterogeneities became less pronounced. Overlaying of the near-field transmission and fluorescence images from the same area of the sample shows local transmission of gold island film is at a minimum where the fluorescence of the lipid monolayer is at a maximum. It was concluded that coverage of the metal island film by the Langmuir-Blodgett phospholipid monolayer is incomplete, and lipid molecules are preferentially localized in crevices of the film.