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At least 37 records · Page 2

Volumetric imaging of the 3D orientation of cellular structures with a polarized fluorescence light-sheet microscope

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations in biological samples, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the diffraction-limited three-dimensional distribution of the orientations and positions of ensembles of fluorescent dipoles that label biological structures. We share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model the distributions based on the polarization-dependent efficiency of excitation and detection of emitted fluorescence, using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labeled giant unilamellar vesicles, fast-scarlet-labeled cellulose in xylem cells, and phalloidin-labeled actin in U2OS cells. Additionally, we observe phalloidin-labeled actin in mouse fibroblasts grown on grids of labeled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

Science & Technology - Other Topics

Measurement of structure motion by means of a moving light sheet

The elaboration of an optical system which measures the displacement and velocity of large flexible space structures is reviewed. The motion-measuring system, comprised of a laser, optics, motorized mirror, two photodiodes and electronics, is designed to allow feedback for configuration control of flexible structures. A motor or scanner is employed to sweep the light sheet. The range of motion was shown to be 2 inches, and information was received at a rate of 30 Hz. The uncertainty in displacement measurement was better than +0.01 inch from a distance of 16 feet. The results are considered to be very good for two scanning light sheet systems. Higher sample rates and a more constant speed would improve the rotating mirror option, and the accuracy and range of the scanning mirror option could be upgraded as well. Both setups are shown to be viable noncontact measuring methods for the displacement and velocity of large space structures.

Tcheng, Ping

Development of high throughput light-sheet fluorescence lifetime imaging microscopy for 3D functional imaging of metabolic pathways in plant and microorganisms (Final Technical Report)

This research program will enable new biochemical contrast in the nanosecond lifetime domain through use of the recently demonstrated electro-optic fluorescence lifetime imaging technique (EO-FLIM) for wide-field lifetime imaging. The Stanford/Stanford Linear Accelerator Center multidisciplinary collaboration -- physics, applied physics, and structural biology -- will develop a light-sheet fluorescence lifetime imaging microscope for functional studies of microbial and plant metabolic pathways and dynamic interactions between plants and microorganisms in the rhizosphere. The proposed approach overcomes the imaging time bottleneck associated with existing fluorescence lifetime imaging methods. Initial demonstrations have shown a factor of 100,000 improvement in photon throughput compared to existing methods. High photon efficiency allowed the first wide-field fluorescence lifetime imaging of single molecules. Recent work has improved the technique’s repetition rate to enable compatibility with mode-locked lasers and demonstrated the combination of wide-field fluorescence lifetime imaging with super-resolution localization microscopy, observations of single molecule dynamics, and observation of donor lifetime quenching in single-molecule imaging. These results were achieved on standard camera sensors and would not have been possible with other wide-field approaches. The throughput and photon economy of the EO-FLIM method enables new BER-relevant imaging opportunities. In particular, scanned single- and two-photon light-sheet excitation will be used to achieve volumetric imaging with time-domain contrast.

47 OTHER INSTRUMENTATION

LITE-SM: A Light Sheet Illuminator Compatible with Super Resolution and Single Molecule Imaging

The LITE-SM (hereinafter referred to as the Tilt-SM) is a light sheet illumination system purpose-built for single molecule (SM) imaging. SM imaging is a fluorescence imaging technique where individual molecules can be tracked in living cells/tissues in real time allowing for unprecedented insights into the intracellular dynamics of these molecules. The innovative all-mirror optical design greatly reduces/eliminates the aberrations that plague lens-based designs. This iteration of the Tilt-SM has significantly better optical performance then its predecessor with over 10x increase in optical power at the sample, allowing for the use of much smaller and more cost-effective lasers then the original Phase 1 system. Excellent STORM imaging as well as SM tracking has been achieved, satisfying the key objectives of this study.

59 BASIC BIOLOGICAL SCIENCES

Laser Light Sheet Flow Visualization of the Space Launch System Booster Separation Test

Planar flow visualizations were obtained in a wind tunnel test in the NASA Langley Research Center’s Unitary Plan Wind Tunnel using the laser-light-sheet method. This method uses a laser to illuminate fine particles generated in the wind tunnel to visualize flow structures. The test article was designed to simulate the separation of the two solid rocket boosters (SRBs) from the core stage of the NASA Space Launch System (SLS) at Mach 4 using a scale model. The test was run on of the SLS Block 1B Cargo (27005) configuration and the SLS Block 1B Crew (28005) configuration. Planar flow visualization was obtained only on the crew configuration. Air at pressures up to 1500 psi was used to simulate plumes from the booster separation motors (BSMs) located at the nose, and aft skirt of the two boosters. The facility free stream was seeded with water vapor, which condensed and froze into small ice crystals in the tunnel nozzle expansion. A continuous wave green (532 nm) laser sheet was used to illuminate the ice crystals, and the resulting Mie-scattered light was collected with a camera. The resulting images clearly identify shock waves and other flow features including BSM plume shapes. Measurements were acquired for different BSM pressures and booster separation locations.

Danehy, Paul M.

A study of the rotor wake of a small-scale rotor model in forward flight using laser light sheet flow visualization with comparisons to analytical models

An experimental investigation was conducted in the 14 by 22 ft subsonic tunnel at NASA Langley Research Center to quantify the rotor wake behind a scale model helicopter rotor in forward flight (mu = 0.15 and 0.23) at one thrust level (C sub T = 0.0064). The rotor system used in the present test consisted of a four-bladed, fully articulated hub and utilized blades of rectangular planform with a NACA-0012 airfoil section. A laser light sheet, seeded with propylene glycol smoke, was used to visualize the flow in planes parallel and perpendicular to the freestream flow. Quantitative measurements of vortex location, vertical skew angle, and vortex particle void radius were obtained for vortices in the flow; convective velocities were obtained for blade tip vortices. Comparisons were made between the experimental results and the wake geometry generated by computational predictions. The results of these comparisons show that the interaction between wake vortex structures is an important consideration for correctly predicting the wake geometry.

Ghee, Terence A.

Spectral attenuation coefficients from measurements of light transmission in bare ice on the Greenland Ice Sheet

Light transmission into bare glacial ice affects surface energy balance, biophotochemistry, and light detection and ranging (lidar) laser elevation measurements but has not previously been reported for the Greenland Ice Sheet. We present measurements of spectral transmittance at 350–900 nm in bare glacial ice collected at a field site in the western Greenland ablation zone (67.15∘ N, 50.02∘ W). Empirical irradiance attenuation coefficients at 350–750 nm are ∼ 0.9–8.0 m−1 for ice at 12–124 cm depth. The absorption minimum is at ∼ 390–397 nm, in agreement with snow transmission measurements in Antarctica and optical mapping of deep ice at the South Pole. From 350–530 nm, our empirical attenuation coefficients are nearly 1 order of magnitude larger than theoretical values for optically pure ice. The estimated absorption coefficient at 400 nm suggests the ice volume contained a light-absorbing particle concentration equivalent to ∼ 1–2 parts per billion (ppb) of black carbon, which is similar to pre-industrial values found in remote polar snow. The equivalent mineral dust concentration is ∼ 300–600 ppb, which is similar to values for Northern Hemisphere warm periods with low aeolian activity inferred from ice cores. For a layer of quasi-granular white ice (weathering crust) extending from the surface to ∼ 10 cm depth, attenuation coefficients are 1.5 to 4 times larger than for deeper bubbly ice. Owing to higher attenuation in this layer of near-surface granular ice, optical penetration depth at 532 nm is 14 cm (20 %) lower than asymptotic attenuation lengths for optically pure bubbly ice. In addition to the traditional concept of light scattering on air bubbles, our results imply that the granular near-surface ice microstructure of weathering crust is an important control on radiative transfer in bare ice on the Greenland Ice Sheet ablation zone, and we provide new values of flux attenuation, absorption, and scattering coefficients to support model development and validation.

IceSat-2

Planar Particle Imaging Doppler Velocimetry Developed

Two current techniques exist for the measurement of planar, three-component velocity fields. Both techniques require multiple views of the illumination plane in order to extract all three velocity components. Particle image velocimetry (PIV) is a high-resolution, high accuracy, planar velocimetry technique that provides valuable instantaneous velocity information in aeropropulsion test facilities. PIV can provide three-component flow-field measurements using a two-camera, stereo viewing configuration. Doppler global velocimetry (DGV) is another planar velocimetry technique that can provide three component flow-field measurements; however, it requires three detector systems that must be located at oblique angles from the measurement plane. The three-dimensional configurations of either technique require multiple (DGV) or at least large (stereo PIV) optical access ports in the facility in which the measurements are being conducted. Optical access is extremely limited in aeropropulsion test facilities. In many cases, only one optical access port is available. A hybrid measurement technique has been developed at the NASA Glenn Research Center, planar particle image and Doppler velocimetry (PPIDV), which combines elements from both the PIV and DGV techniques into a single detection system that can measure all three components of velocity across a planar region of a flow field through a single optical access port. In the standard PIV technique, a pulsed laser is used to illuminate the flow field at two closely spaced instances in time, which are recorded on a "frame-straddling" camera, yielding a pair of single-exposure image frames. The PIV camera is oriented perpendicular to the light sheet, and the processed PIV data yield the two-component velocity field in the plane of the light sheet. In the standard DGV technique, an injection-seeded Nd:YAG pulsed laser light sheet illuminates the seeded flow field, and three receiver systems are used to measure three components of velocity. The receiver systems are oriented at oblique angles to the light sheet in order to accurately resolve the three-component velocity. Each DGV receiver system contains two cameras, which share a common view of the illuminated flow through a beam-splitting cube. One camera views the illuminated flow directly (reference camera) and the second camera images the illuminated flow through an iodine vapor cell (signal camera). The laser frequency (wavelength) is adjusted so that the Doppler-shifted light from particles in the flow falls on an iodine absorption feature, see the following graph. The iodine vapor cell acts as a frequency-to-velocity filter by modulating the intensity of the transmitted light as a function of the flow velocity (Doppler shift). The ratio of the signal and reference images yields the component of the flow velocity along the bisector of the laser sheet propagation direction and the receiver system observation direction. The hybrid system employs a single-component DGV receiver system configured to simultaneously acquire PIV image data, as shown in the following diagram. The cameras used in the DGV receiver are replaced with PIV frame-straddling cameras, and the receiver system views the illuminated light sheet plane at 90 (as in the standard PIV configuration).

Wernet, Mark P.

Concentration measurements in an axisymmetric jet using laser-induced fluorescence

Errors associated with the use of an image processing technique to correct images of an axisymmetric jet produced by laser-induced fluorescence are analyzed. In particular, attention is given to errors related to the alignment of the optical system, spreading of the laser light sheet, laser light attenuation due to light absorption by the fluorescent dye, and solubility effects. Although the above effects are negligible in the case of a small low-concentration jet, they are not negligible in all fluorescent dye experiments.

Shoe, B.

Boundary Layer Measurements in a Supersonic Wind Tunnel Using Doppler Global Velocimetry

A modified Doppler Global Velocimeter (DGV) was developed to measure the velocity within the boundary layer above a flat plate in a supersonic flow. Classic laser velocimetry (LV) approaches could not be used since the model surface was composed of a glass-ceramic insulator in support of heat-transfer measurements. Since surface flare limited the use of external LV techniques and windows placed in the model would change the heat transfer characteristics of the flat plate, a novel approach was developed. The input laser beam was divided into nine equal power beams and each transmitted through optical fibers to a small cavity within the model. The beams were then directed through 1.6-mm diameter orifices to form a series of orthogonal beams emitted from the model and aligned with the tunnel centerline to approximate a laser light sheet. Scattered light from 0.1-micron diameter water condensation ice crystals was collected by four 5-mm diameter lenses and transmitted by their respective optical fiber bundles to terminate at the image plane of a standard two-camera DGV receiver. Flow measurements were made over a range from 0.5-mm above the surface to the freestream at Mach 3.51 in steady state and heat pulse injected flows. This technique provides a unique option for measuring boundary layers in supersonic flows where seeding the flow is problematic or where the experimental apparatus does not provide the optical access required by other techniques.

Meyers, James F.

Exploratory flow visualization investigation of mast-mounted sights in presence of a rotor

A flow visualization investigation with a laser light sheet system was conducted on a 27-percent-scale AH-64 attack helicopter model fitted with two mast-mounted sights in the langley 14- by 22-foot subsonic tunnel. The investigation was conducted to identify aerodynamic phenomena that may have contributed to adverse vibration encountered during full-scale flight of the AH-64D apache/longbow helicopter with an asymmetric mast-mounted sight. Symmetric and asymmetric mast-mounted sights oriented at several skew angles were tested at simulated forward and rearward flight speeds of 30 and 45 knots. A laser light sheet system was used to visualize the flow in planes parallel to and perpendicular to the free-stream flow. Analysis of these flow visualization data identified frequencies of flow patterns in the wake shed from the sight, the streamline angle at the sight, and the location where the shed wake crossed the rotor plane. Differences in wake structure were observed between the sight configurations and various skew angles. Analysis of lateral light sheet plane data implied significant vortex structure in the wake of the asymmetric mast-mounted sight in the configuration that produced maximum in-flight vibration. The data showed no significant vortex structure in the wake of the asymmetric and symmetric configurations that produced no increase in in-flight adverse vibration.

Ghee, Terence A.

High-Resolution OH and CH2O Visualization in a Premixed Cavity-Anchored Ethylene-Air Flame in a M = 0.6 Flowfield

OH and CH2O were imaged in a premixed, cavity-anchored, ethylene-air turbulent flame using a high resolution planar laser-induced fluorescence (PLIF) system. The electrically-heated, continuous flow facility (UVa Supersonic Combustion Facility, Configuration E) consisted of a Mach 2 nozzle, an isolator with fuel injectors, a test section with a cavity flame holder and optical access, and an extender. Standard test conditions comprised total temperature 1200 K, total pressure 300 kPa, local equivalence ratio near 0.4, and local Mach number near 0.6. OH PLIF data was also collected for a case with reduced total temperature and another with reduced equivalence ratio. OH and CH2O were excited in separate experiments with light sheets at 283.55 nm and 352.48 nm, respectively. A light sheet of approximate thickness 25 ?m illuminated the stream-wise midplane. This plane was imaged for 120 mm downstream of the backward-facing step. The intensified camera system imaged OH with magnification 1.97, a square 6.67 mm field of view, and in-plane resolution of 39 ?m. The smallest observed OH structures observed were approximately 100 ?m wide. The CH2O PLIF image signal was much weaker; the smallest observed structures were approximately 200 ?m wide. Composite fluorescence images were computed for the observed area.

Geipel, Clayton M.

Fiber-optic-based laser vapor screen flow visualization system for aerodynamic research in larger scale subsonic and transonic wind tunnels

Laser vapor screen (LVS) flow visualization systems that are fiber-optic based were developed and installed for aerodynamic research in the Langley 8-Foot Transonic Pressure Tunnel and the Langley 7- by 10-Foot High Speed Tunnel. Fiber optics are used to deliver the laser beam through the plenum shell that surrounds the test section of each facility and to the light-sheet-generating optics positioned in the ceiling window of the test section. Water is injected into the wind tunnel diffuser section to increase the relative humidity and promote condensation of the water vapor in the flow field about the model. The condensed water vapor is then illuminated with an intense sheet of laser light to reveal features of the flow field. The plenum shells are optically sealed; therefore, video-based systems are used to observe and document the flow field. Operational experience shows that the fiber-optic-based systems provide safe, reliable, and high-quality off-surface flow visualization in smaller and larger scale subsonic and transonic wind tunnels. The design, the installation, and the application of the Langley Research Center (LaRC) LVS flow visualization systems in larger scale wind tunnels are highlighted. The efficiency of the fiber optic LVS systems and their insensitivity to wind tunnel vibration, the tunnel operating temperature and pressure variations, and the airborne contaminants are discussed.

Gary E Erickson

Application of optical correlation techniques to particle imaging velocimetry

Pulsed laser sheet velocimetry yields nonintrusive measurements of velocity vectors across an extended 2-dimensional region of the flow field. The application of optical correlation techniques to the analysis of multiple exposure laser light sheet photographs can reduce and/or simplify the data reduction time and hardware. Here, Matched Spatial Filters (MSF) are used in a pattern recognition system. Usually MSFs are used to identify the assembly line parts. In this application, the MSFs are used to identify the iso-velocity vector contours in the flow. The patterns to be recognized are the recorded particle images in a pulsed laser light sheet photograph. Measurement of the direction of the partical image displacements between exposures yields the velocity vector. The particle image exposure sequence is designed such that the velocity vector direction is determined unambiguously. A global analysis technique is used in comparison to the more common particle tracking algorithms and Young's fringe analysis technique.

Wernet, Mark P.

Application of optical correlation techniques to particle imaging

Pulsed laser sheet velocimetry yields noninstrusive measurements of velocity vectors across an extended 2-dimensional region of the flow field. The application of optical correlation techniques to the analysis of multiple exposure laser light sheet photographs can reduce and/or simplify the data reduction time and hardware. Here, Matched Spatial Filters (MSF) are used in a pattern recognition system. Usuallay MSFs are used to identify the assembly line parts. In this application, the MSFs are used to identify the iso-velocity vector contours in the flow. The patterns to be recognized are the recorded particle images in a pulsed laser light sheet photograph. Measurement of the direction of the particle image displacements between exposures yields the velocity vector. The particle image exposure sequence is designed such that the velocity vector direction is determined unambiguously. A global analysis technique is used in comparison to the more common particle tracking algorithms and Young's fringe analysis technique.

Wernet, Mark P.