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The HOLO Series: Critical Ground-Based Demonstrations of Holographic Scanning Lidars

Results of two lidar measurement campaigns are presented, HOLO-1 (Utah, March 1999) and HOLO-2 (New Hampshire, June 1999). These tests demonstrate the ability of lidars utilizing holographic optical elements (HOEs) to determine tropospheric wind velocity and direction at cloud altitude. Several instruments were employed. HOLO-1 used the 1,064 mm transmission-HOE lidar (HARLIE, Goddard Space Flight Center), a zenith-staring 532 nm lidar (AROL-2, Utah State University), and a wide-field video camera (SkyCam) for imagery of clouds overhead. HOLO-2 included these instruments plus the 532 nm reflection-HOE lidar (PHASERS, St. Anselm College). HARLIE and PHASERS scan the sky at constant cone angles of 45 deg. and 42 deg. from normal, respectively. The progress of clouds and entire cloud fields across the sky is tracked by the repetitive conical scans of the HOE lidars. AROL-2 provides the attitude information enabling the SkyCam cloud images to be analyzed for independent data on cloud motion. Data from the HOE lidars are reduced by means of correlations, visualization by animation techniques, and kinematic diagrams of cloud feature motion. Excellent agreement is observed between the HOE lidar results and those obtained with video imagery and lidar ranging.

Wilkerson, Thomas D.↗

Ground Based Operational Testing Of Holographic Scanning Lidars : The HOLO Experiments

Two aerosol backscatter lidar measurement campaigns were conducted using two holographic scanning lidars and one zenith staring lidar for the purposes of reliability testing under field conditions three new lidar systems and to develop new scanning measurement techniques and applications. The first campaign took place near the campus of Utah State University in Logan Utah in March of 1999 and is called HOLO-1. HOLO-2 was conducted in June of 1999 on the campus of Saint Anselm College, near the city of Manchester, New Hampshire. Each campaign covered a period of approximately one week of nearly continuous observation of cloud and aerosol backscatter in the visible and near infrared by lidar, and wide field visible sky images by video camera in the daytime. The scanning capability coupled with a high rep-rate, high average power laser enables both high spatial and high temporal resolution observations that Particularly intriguing is the possibility of deriving atmospheric wind profiles from temporal analysis of aerosol backscatter spatial structure obtained by conical scan without the use of Doppler techniques.

Schwemmer, Geary K.↗

On Holo-Hilbert Spectral Analysis: A Full Informational Spectral Representation for Nonlinear and Non-Stationary Data

The Holo-Hilbert spectral analysis (HHSA) method is introduced to cure the deficiencies of traditional spectral analysis and to give a full informational representation of nonlinear and non-stationary data. It uses a nested empirical mode decomposition and Hilbert-Huang transform (HHT) approach to identify intrinsic amplitude and frequency modulations often present in nonlinear systems. Comparisons are first made with traditional spectrum analysis, which usually achieved its results through convolutional integral transforms based on additive expansions of an a priori determined basis, mostly under linear and stationary assumptions. Thus, for non-stationary processes, the best one could do historically was to use the time- frequency representations, in which the amplitude (or energy density) variation is still represented in terms of time. For nonlinear processes, the data can have both amplitude and frequency modulations (intra-mode and inter-mode) generated by two different mechanisms: linear additive or nonlinear multiplicative processes. As all existing spectral analysis methods are based on additive expansions, either a priori or adaptive, none of them could possibly represent the multiplicative processes. While the earlier adaptive HHT spectral analysis approach could accommodate the intra-wave nonlinearity quite remarkably, it remained that any inter-wave nonlinear multiplicative mechanisms that include cross-scale coupling and phase-lock modulations were left untreated. To resolve the multiplicative processes issue, additional dimensions in the spectrum result are needed to account for the variations in both the amplitude and frequency modulations simultaneously. HHSA accommodates all the processes: additive and multiplicative, intra-mode and inter-mode, stationary and nonstationary, linear and nonlinear interactions. The Holo prefix in HHSA denotes a multiple dimensional representation with both additive and multiplicative capabilities.

Huang, Norden E.↗

Displacement analysis of the interior walls of a pipe using panoramic holo-interferometry

Standard phase-displacement equations, used for both double exposure and real time holo-interferometry, are combined with the geometrical mapping and optical characteristics of panoramic annular lenses to accurately measure surface displacement on the interior wall of a pipe. In this approach, one panoramic annular lens is used to illuminate the surface with coherent light, and the resulting intensity distribution is holographically recorded through a second panoramic annular lens. Interference fringes are obtained in real-time by comparing holograms recorded before and after the pipe is displaced. The annular images and the holographic fringes are acquired and stored digitally in a computer system. Image transformation algorithms are applied to remove optical distortions in the holographic pattern and fringe loci are compared to those predicted on the basis of theory.

Gilbert, John A.↗

Anaerobic Expression and Purification of Holo-CCIS, an Artificial Iron-sulfur Protein

Iron-sulfur proteins are ubiquitous among all living organisms and are indispensable for almost all metabolic pathways ranging from photosynthesis, respiration, nitrogen, and carbon dioxide cycles. The iron-sulfur clusters primarily serve as electron acceptors and donors and transfer electrons to active sites of various enzymes, thus driving the energy metabolism. Prokaryotes like E. coli have ISC and SUF pathways that help in the assembly and maturation of iron-sulfur proteins. These ironsulfur proteins, especially with [4Fe-4S] clusters, are highly sensitive to molecular oxygen, and it would be advantageous if the de novo proteins and native proteins having iron-sulfur binding sites are expressed and isolated under anaerobic conditions. Bacterially assembled iron-sulfur proteins, when isolated and purified anaerobically, exhibit improved biochemical and biophysical stabilities in comparison to the counterparts expressed and purified aerobically and reconstituted under anaerobic conditions. This protocol outlines the expression and purification of the artificial protein, Coiled-Coil IronSulfur (CCIS). It may be deployed to both natural and artificial [4Fe-4S] proteins when heterologously expressed in E. coli.

Bhanu P. Jagilinki↗

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗

Observation of Shear-Induced Turbulence Using HARLIE

Ground-based measurements of atmospheric aerosol structure were made using the Holographic Airborne Rotating Lidar Instrument Experiment (HARLIE) during the HOLO-1 field campaign. The scanning ability of HARLIE affords a unique opportunity to view various atmospheric phenomena. Shear-induced turbulence plays an important role in the transport of kinetic energy in the atmosphere and on March 10, 1999, several instances of shear-induced turbulence were observed via HARLIE. Using the data collected and upper-air wind profiles the nature of the instabilities is discussed.

Miller, David O.↗

Horizontal Wind Measurements using the HARLIE Holographic Lidar

We report the results of three campaigns in which the horizontal wind vector at cloud altitudes was measured using the holographic, conical-scan lidar HARLIE in its nadir-viewing mode. Measurements were made during the HOLO-1 and -2 tests in Utah and New Hampshire in March and June 1999, respectively, and at the DoE-ARM site in Oklahoma in September/October 2000. A novel algorithm facilitates the wind vector analysis of the HARLIE data. Observed wind velocity and direction were compared with radiosonde records and with other data obtained from video cloud imagery and independent lidar ranging. The results demonstrate good agreement between HARLIE data and the results of other methods. The conically scanning holographic lidar opens up new possibilities for obtaining the vertical profile of horizontal winds.

Wilkerson, Thomas↗

Large Aperture Scanning Lidar Based on Holographic Optical Elements

Lidar remote sensing instruments can make a significant contribution to satisfying many of the required measurements of atmospheric and surface parameters for future spaceborne platforms, including topographic altimeters, atmospheric profiles of, wind, humidity, temperature, trace molecules, aerosols, and clouds. It is highly desirable to have wide measurement swaths for rapid coverage rather than just the narrow ribbon of data that is obtained with a nadir only observation. For most applications global coverage is required, and for wind measurements scanning or pointing is required in order to retrieve the full 3-D wind vector from multiple line-of-sight Doppler measurements. Conventional lidar receivers make up a substantial portion of the instrument's size and weight. Wide angle scanning typically requires a large scanning mirror in front of the receiver telescope, or pointing the entire telescope and aft optics assembly, Either of these methods entails the use of large bearings, motors, gearing and their associated electronics. Spaceborne instruments also need reaction wheels to counter the torque applied to the spacecraft by these motions. NASA has developed simplified conical scanning telescopes using Holographic Optical Elements (HOEs) to reduce the size, mass, angular momentum, and cost of scanning lidar systems. NASA has developed two operating lidar systems based on 40 cm diameter HOEs. The first such system, named Prototype Holographic Atmospheric Scanner for Environmental Remote Sensing (PHASERS) was a joint development between NASA Goddard Space Flight Center (GSFC) and the University of Maryland College Park. PHASERS is based on a reflection HOE for use at the doubled Nd:YAG laser wavelength of 532 nm and has recently undergone a number of design changes in a collaborative effort between GSFC and Saint Anselm College in New Hampshire. The next step was to develop IR transmission HOEs for use with the Nd:YAG fundamental in the Holographic Airborne Rotating Lidar Instrument Experiment (HARLIE). The HOE spins like a compact disk in a large ring ball bearing. In an aircraft the HOE faces down, looking out through a window at an angle of 45 degrees off-nadir. The HOE diffracts 85% of the incident 532 nm light into a 160 micron spot at a focal length of 1 meter. HARLIE is a field deployable lidar measuring aerosol, cloud, and boundary layer backscatter for atmospheric research. It has flown several times and is also used from a ground-based trailer in an upward-looking mode. The HOE generates a 45 degree conical scan pattern by rotating at speeds up to 30 rpm. Like PHASERS, the HOE in HARLIE serves both as the laser collimating lens as well as the receiver telescope primary optic. The telescope is coupled to the receiver package via fiber optic. The transmitter is a diode pumped Nd:YAG laser operating at 1064 nm, delivering 1 mJ pulses at a 5 KHz rep-rate. The receiver has a 200 microradian field-of-view and a 0.5 nm optical bandpass. The photon counting data system utilizes a single Geiger-mode silicon avalanche photodiode detector, This new technology has also presented us with new data visualization challenges as well as new measurement techniques. The backscatter data obtained from a stationary (i.e. ground-based) scanning HOE lidar is on the surface of a cone, which when viewed over many consecutive scans can reveal atmospheric motions on this surface over time as the atmosphere advects over the site. In a moving platform such as an airplane or satellite, the data from consecutive scans cover different areas under the flight path, revealing atmospheric structure in 3-dimensions. An example of a visualization of HARLIE ground-based data is presented, showing aerosol backscatter on a 90 degree conical surface generated from one 360 degree scan of the lidar during the HOLO-1 field campaign on the afternoon of 10 March 1999. Higher backscatter levels are rendered as lighter signal against a dark background. Breaking Kelvin-Helmholtz waves are evident on the north side of the scan at an altitude of 10-11 km. Time series of successive scans made at regular intervals render unique views of atmospheric motions, from which vertical profiles of atmospheric wind vectors can be obtained using a unique data analysis approach. Wind vectors obtained from the lidar were compared with co-located radiosonde wind profiles during an intensive operating period in September-October 2000 at the Atmospheric Radiation Measurement Program's Southern Great Plains Central Facility.

Schwemmer, Geary K.↗

Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

Ciszak, Ewa↗

Structural Model for the Flip-Flop Action in Thiamin Pyrophosphate-Dependent Human Pyruvate Dehydrogenase

The derivative of vitamin B1 thiamin pyrophosphate (TPP) is a cofactor of enzymes performing catalysis in pathways of energy production, including (i) decarboxylation of alpha-keto acids followed by (ii) transketolation. These enzymes have shown a common mechanism of TPP activation by imposing an active V-conformation of this coenzyme that brings the N4 atom of the aminopyrimidine ring to the distance required for the intramolecular C-H N hydrogen-bonding with the C2- atom of the thiazolium ring. The reactive C2 atom of TPP is the nucleophile that attacks the carbonyl carbon of different substrates used by the TPP-dependent enzymes. The structure of the heterotetrameric human pyruvate dehydrogenase (Elp) recently determined in our laboratory (1) revealed the association pattern of the subunits and the specifics of two chemically equivalent cofactor binding sites. Dynamic nonequivalence of these two cofactor sites directs the flip-flop action of this enzyme, depending upon which two active sites effect each other (2). The crystal structure derived from the holo-form of Elp provided the basis for the model of the flip-flop action of Elp in which different steps of the catalytic reaction are performed in each of the two cofactor sites at any given moment, where these steps are governed by the concerted shuttle-like motion of the subunits. It is further proposed that balancing a hydrogen-bond network and related cofactor geometry determine the continuity of catalytic events.

Ciszak, Ewa↗

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha (sub 2) beta (sub 2)-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the C(sup alpha) -C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-Angstrom resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-Angstrom shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.

Ciszak, Ewa M.↗

Going to Extremes: Architecting Holographic Microscopes for Extreme Environments

Bacterial life exists on earth in extreme environments. These are environments described by large temperature excur- sions, large pressure excursions, and large radiation excursions from the nominal conditions near sea level which are largely populated by humans. These extreme locales represent such places as the ocean ice, the briny pools of Death Valley and deep mines, the acidic hot springs of the High Sierra or Yellowstone, or even the clouds of our upper atmosphere. The preponderance of bacterial life in these extreme environments here on earth suggests that bacterial life might likely exist in the extreme environments of our own solar system, such as the icy moons of Europa or Enceladus. Thus, architecting instruments for detect- ing life in these extreme environments on earth builds confidence that we can architect such instruments for flight missions. In this paper, we discuss our experience with designing digital holo- graphic microscope instruments to enable detection of bacteria in several extreme environments. In particular we discuss three different instruments. The first is our field instrument which is a small, portable instrument for examination of remote sites. The second is submersible instrument which enables exploration of deep aquatic environments and is deployed on a ocean-going drone. The third is a balloon-borne instrument to examine the bacterial content of the upper atmosphere. We will provide a review of each instrument and discuss aspects of instrument engineering for each particular application.

Ramirez, Alex↗