Real Time 3 D Holograms
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Next-Gen Holographic Microscope Offers Real-Time 3D Imaging
http://www.rdmag.com/news/2016/03/next-gen-holographic-microscope-offers-real-time-3d-imaging KAIST
3-D Images of representative biological cells taken with the HT-1
Researchers have developed a powerful method for 3D imaging of live cells without staining.
Professor YongKeun Park of the Physics Department at the Korea Advanced Institute of Science and Technology (KAIST) is a leading researcher in the field of biophotonics and has dedicated much of his research career to working on digital holographic microscopy technology. Park and his research team collaborated with the R&D team of a start-up that Park co-founded to develop a state-of-the-art, 2D/3D/4D holographic microscope that would allow a real-time label-free visualization of biological cells and tissues.
The HT is an optical analogy of X-ray computed tomography (CT). Both X-ray CT and HT share the same physical principle—the inverse of wave scattering. The difference is that HT uses laser illumination, whereas X-ray CT uses X-ray beams. From the measurement of multiple 2D holograms of a cell, coupled with various angles of laser illuminations, the 3-D refractive index (RI) distribution of the cell can be reconstructed. The reconstructed 3D RI map provides structural and chemical information of the cell including mass, morphology, protein concentration and dynamics of the cellular membrane.
The HT enables users to quantitatively and non-invasively investigate the intrinsic properties of biological cells, for example, dry mass and protein concentration. Some of the research team’s breakthroughs that have leveraged HT’s unique and special capabilities can be found in several recent publications, including a lead article on the simultaneous 3-D visualization and position tracking of optically trapped particles which was published in Optica on April 20, 2015.
Current fluorescence confocal microscopy techniques require the use of exogenous labeling agents to render high-contrast molecular information. Therefore, drawbacks include possible
- photo-bleaching
- photo-toxicity
- interference with normal molecular activities
Immune or stem cells that need to be reinjected into the body are considered particularly difficult to employ with fluorescence microscopy.
“As one of the two currently available, high-resolution tomographic microscopes in the world, I believe that the HT-1 is the best-in-class regarding specifications and functionality. Users can see 3D/4D live images of cells, without fixing, coating or staining cells. Sample preparation times are reduced from a few days or hours to just a few minutes,” said Park.
“Our technology has set a new paradigm for cell observation under a microscope. I expect that this tomographic microscopy will be more widely used in future in various areas of pharmaceuticals, neuroscience, immunology, hematology and cell biology,” Park added.
The researchers announced the launch of their new microscopic tool, the holotomography (HT)-1, to the global marketplace through the Korean start-up TomoCube. Two Korean hospitals, Seoul National University Hospital in Bundang and Boramae Hospital in Seoul, are currently using this microscope. The research team has also introduced the HT-1 at the Photonics West Exhibition 2016 that took place on February 16-18, 2016, in San Francisco, CA.
Chip-Based Atomic Physics Makes Second Quantum Revolution a Reality
http://www.rdmag.com/news/2016/03/chip-based-atomic-physics-makes-second-quantum-revolution-reality
A quartz surface above the electrodes used to trap atoms. The color map on the surface shows the electric field amplitude.
A University of Oklahoma-led team of physicists believes chip-based atomic physics holds promise to make the second quantum revolution—the engineering of quantum matter with arbitrary precision—a reality. With recent technological advances in fabrication and trapping, hybrid quantum systems are emerging as ideal platforms for a diverse range of studies in quantum control, quantum simulation and computing.
James P. Shaffer, professor in the Homer L. Dodge Department of Physics and Astronomy, OU College of Arts and Sciences; Jon Sedlacek, OU graduate student; and a team from the University of Nevada, Western Washington University, The United States Naval Academy, Sandia National Laboratories and Harvard-Smithsonian Center for Astrophysics, have published research important for integrating Rydberg atoms into hybrid quantum systems and the fundamental study of atom-surface interactions, as well as applications for electrons bound to a 2-D surface.
“A convenient surface for application in hybrid quantum systems is quartz because of its extensive use in the semiconductor and optics industries,” Sedlacek said. “The surface has been the subject of recent interest as a result of it stability and low surface energy. Mitigating electric fields near ‘trapping’ surfaces is the holy grail for realizing hybrid quantum systems,” added Hossein Sadeghpour, director of the Institute for Theoretical Atomic Molecular and Optical Physics, Harvard-Smithsonian Center for Astrophysics.
In this work, Shaffer finds ionized electrons from Rydberg atoms excited near the quartz surface form a 2-D layer of electrons above the surface, canceling the electric field produced by rubidium surface adsorbates. The system is similar to electron trapping in a 2-D gas on superfluid liquid helium. The binding of electrons to the surface substantially reduces the electric field above the surface.
“Our results show that binding is due to the image potential of the electron inside the quartz,” said Shaffer. “The electron can’t diffuse into the quartz because the rubidium adsorbates make the surface have a negative electron affinity. The approach is a promising pathway for coupling Rydberg atoms to surfaces, as well as for using surfaces close to atomic and ionic samples.”
A paper on this research was published in the American Physics Society’s Physical Review Letters. The OU part of this work was supported by the Defense Advanced Research Projects Agency Quasar program by a grant through the Army Research Office, the Air Force Office of Scientific Research and the National Science Foundation.
New Spin on Biomolecular Tags Lets MRI Catch Metabolic Wobbles
Duke scientists have discovered a new class of inexpensive and long-lived molecular tags that enhance MRI signals by 10,000-fold. To activate the tags, the researchers mix them with a newly developed catalyst (center) and a special form of hydrogen (gray), converting them into long-lived magnetic resonance “lightbulbs” that might be used to track disease metabolism in real time. [Thomas Theis, Duke University]
In principle, magnetic resonance imaging (MRI) could be used to track disease-related biomolecular processes. In practice, magnetic resonance signals die out too quickly. Also, these signals are detectable only with incredibly expensive equipment. The necessary devices, called hyperpolarizers, are commercially available, but they cost as much as $3 million each.
Yet magnetic resonance can be more practical, report scientists from Duke University. These scientists say that they have discovered a new class of molecular tags that enhance magnetic resonance signals by 10,000-fold and generate detectable signals that last over an hour, and not just a few seconds, as is the case with currently available tags. Moreover, the tags are biocompatible and inexpensive to produce, paving the way for widespread use of MRI to monitor metabolic process of conditions such as cancer and heart disease in real time.
According to the Duke team, which was led by physicist Warren S. Warren, Ph.D., and chemist Thomas Theis, Ph.D., the hyperpolarization window to in vitro and in vivo biochemistry can be opened by combining two advances: (1) the use of 15N2-diazirines as storage vessels for hyperpolarization, and (2) a relatively simple and inexpensive approach to hyperpolarization called SABRE-SHEATH.
The details appeared March 25 in the journal Science Advances, in an article entitled, “Direct and Cost-Efficient Hyperpolarization of Long-Lived Nuclear Spin States on Universal 15N2-Diazirine Molecular Tags.” The article explains that the promise of magnetic resonance in tracking chemical transformations has not been realized because of the limitations of existing techniques, such as dissolution dynamic nuclear polarization (d-DNP). Such techniques have lacked adequate sensitivity and are unable to detect small number of molecules without using unattainably massive magnetic fields.
MRI takes advantage of a property called spin, which makes the nuclei in hydrogen atoms act like tiny magnets. Applying a strong magnetic field, followed by a series of radio waves, induces these hydrogen magnets to broadcast their locations. Most of the hydrogen atoms in the body are bound up in water; therefore, the technique is used in clinical settings to create detailed images of soft tissues like organs, blood vessels, and tumors inside the body.
With greater sensitivity, however, magnetic resonance techniques could be used to track chemical transformations in real time. This degree of sensitivity, say the Duke scientists, could be within reach.
“We use a recently developed method, SABRE-SHEATH, to directly hyperpolarize 15N2 magnetization and long-lived 15N2 singlet spin order, with signal decay time constants of 5.8 and 23 minutes, respectively,” wrote the authors of the Science Advances article. “We find >10,000-fold enhancements generating detectable nuclear MR signals that last for over an hour.” The authors added that 15N2-diazirines represent a class of particularly promising and versatile molecular tags and can be incorporated into a wide range of biomolecules without significantly altering molecular function.
“This represents a completely new class of molecules that doesn’t look anything at all like what people thought could be made into MRI tags,” said Dr. Warren “We envision it could provide a whole new way to use MRI to learn about the biochemistry of disease.”
Qiu Wang, Ph.D., an assistant professor of chemistry at Duke and co-author on the paper, said the structure of 15N2-diazirine is a particularly exciting target for hyperpolarization because it has already been demonstrated as a tag for other types of biomedical imaging.
“It can be tagged on small molecules, macromolecules, amino acids, without changing the intrinsic properties of the original compound,” said Dr. Wang. “We are really interested to see if it would be possible to use it as a general imaging tag.” Magnetic resonance, added Dr. Theis, is uniquely sensitive to chemical transformations: “With magnetic resonance, you can see and track chemical transformations in real time.”
The scientists believe their SABRE-SHEATH catalyst could be used to hyperpolarize a wide variety of chemical structures at a fraction of the cost of other methods. “You could envision, in five or ten years, you’ve got the container with the catalyst, you’ve got the bulb with the hydrogen gas,” explained Dr. Warren. “In a minute, you’ve made the hyperpolarized agent, and on the fly you could actually take an image. That is something that is simply inconceivable by any other method.”
Hyperpolarization enables real-time monitoring of in vitro and in vivo biochemistry
Conventional magnetic resonance (MR) is an unmatched tool for determining molecular structures and monitoring structural transformations. However, even very large magnetic fields only slightly magnetize samples at room temperature and sensitivity remains a fundamental challenge; for example, virtually all MR images are of water because it is the molecule at the highest concentration in vivo. Nuclear spin hyperpolarization significantly alters this perspective by boosting nuclear MR (NMR) sensitivity by four to nine orders of magnitude (1–3), giving access to detailed chemical information at low concentrations. These advances are beginning to transform biomedical in vivo applications (4–9) and structural in vitro studies (10–16).
Current hyperpolarization technology is expensive and associated with short signal lifetimes
Still, two important challenges remain. First, hyperpolarized MR is associated with high cost for the most widespread hyperpolarization technology [dissolution dynamic nuclear polarization (d-DNP), $2 million to $3 million for commercial hyperpolarizers]. Second, hyperpolarized markers typically have short signal lifetimes: typically, hyperpolarized signals may only be tracked for 1 to 2 min in the most favorable cases (6), greatly limiting this method as a probe for slower biological processes.
The presented approach is inexpensive and produces long-lived signals
Here, we demonstrate that both of these challenges can be overcome simultaneously, setting the stage for hour-long tracking of molecular markers with inexpensive equipment. Specifically, we illustrate the potential of 15N2-diazirines as uniquely powerful storage vessels for hyperpolarization. We show that diazirine can be hyperpolarized efficiently and rapidly (literally orders of magnitude cheaper and quicker than d-DNP), and that this hyperpolarization can be induced in states that maintain hyperpolarization for more than an hour.
Our approach uses parahydrogen (p-H2) to directly polarize long-lived nuclear spin states. The first demonstration of parahydrogen-induced polarization (PHIP) was performed in the late 1980s (17–19). Then, PHIP was used to rely on the addition of p-H2 to a carbon double or triple bond, incorporating highly polarized hydrogen atoms into molecules. This approach generally requires specific catalyst-substrate pairs; in addition, hydrogen atoms usually have short relaxation times (T1) that cause signal decay within a few seconds. A more recent variant, SABRE (signal amplification by reversible exchange) (20, 21), uses p-H2 to polarize 1H atoms on a substrate without hydrogenation. In SABRE, both p-H2 and substrate reversibly bind to an iridium catalyst and the hyperpolarization is transferred from p-H2 to the substrate through J-couplings established on the catalytic intermediate. Recently, we extended this method to SABRE-SHEATH (SABRE in SHield Enables Alignment Transfer to Heteronuclei) for direct hyperpolarization of 15N molecular sites (22–24). This method has several notable features. Low-γ nuclei (13C, 15N) tend to have long relaxation times, particularly if a proton is not attached. In addition, conventional SABRE relies on small differences between four-bond proton-proton J-couplings (detailed in the Supplementary Materials), whereas SABRE-SHEATH uses larger two-bond heteronuclear J-couplings. It is extremely simple: SABRE-SHEATH requires nothing but p-H2, the catalyst, and a shield to reduce Earth’s field by about 99%. After 1 to 5 min of bubbling p-H2into the sample in the shield, we commonly achieve 10% nitrogen polarization, many thousands of times stronger than thermal signals (22). In contrast, d-DNP typically produces such polarization levels in an hour, at much higher cost.
Diazirines are small and versatile molecular tags
A general strategy for many types of molecular imaging is the creation of molecular tags, which ideally do not alter biochemical pathways but provide background-free signatures for localization. This strategy has not been very successful in MR because of sensitivity issues. Here, we demonstrate that SABRE-SHEATH enables a MR molecular beacon strategy using diazirines 
(three-membered rings containing a nitrogen-nitrogen double bond). They are highly attractive as molecular tags, primarily because of their small size. Diazirines have already been established as biocompatible molecular tags for photoaffinity labeling (25). They can be incorporated into many small molecules, metabolites, and biomolecules without drastically altering biological function. Diazirines share similarities with methylene (CH2) groups in terms of electronic and steric properties such that they can replace methylene groups without drastically distorting biochemical behavior. Furthermore, diazirines are stable at room temperature, are resistant to nucleophiles, and do not degrade under either acidic or alkaline conditions (25). With these attractive properties, diazirines have been used for the study of many signaling pathways. For example, they have been incorporated into hormones (26), epileptic drugs (27), antibiotics (28), hyperthermic drugs (29), anticancer agents (30), anesthetics (31), nucleic acids (32), amino acids (33), and lipids (34). They also have been introduced into specific molecular reporters to probe enzyme function and their binding sites such as in kinases (35), aspartic proteases (36), or metalloproteinases (37), to name a few. The nitrogen-nitrogen moiety is also intrinsically interesting, because the two atoms are usually very close in chemical shift and strongly coupled, thus suited to support a long-lived singlet state as described below.
Fig. 1The hyperpolarization mechanism.
(A) The precatalyst, 15N2-diazirine substrate, and p-H2 are mixed, resulting in the activated species depicted in (B). (B) Both p-H2 and the free 15N2-diazirine [2-cyano-3-(D3 methyl-15N2-diazirine)-propanoic acid] are in reversible exchange with the catalytically active iridium complex. The catalyst axial position is occupied by IMes [1,3-bis(2,4,6-trimethylphenyl)-imidazolium] and Py (pyridine) as nonexchanging ligands. The structure shown is a local energy minimum of the potential energy surface based on all-electron DFT calculations and the dispersion-corrected PBE density functional. In the complex, hyperpolarization is transferred from the parahydrogen (p-H2)–derived hydrides to the 15N nuclei (white, hydrogen; gray, carbon; blue, nitrogen; red, oxygen).
Density functional theory calculations shed light on polarization transfer catalyst
The Ir complex conformation shown in Fig. 1B was determined by all-electron density functional theory (DFT) calculations [semilocal Perdew-Burke-Ernzerhof (PBE) functional (38), corrected for long-range many-body dispersion interactions (39), in the FHI-aims software package (40, 41); see the Supplementary Materials for details]. The calculations indicate a η1 single-sided N attachment rather than η2-N=N attachment of the diazirines. In the Ir complex, hyperpolarization is transferred from p-H2 gas (~92% para-state, 7.5 atm) to the 15N2-diazirine. Both p-H2 and substrate are in reversible exchange with the central complex, which results in continuous pumping of hyperpolarization: p-H2 is continually refreshed and hyperpolarization accumulated on the diazirine substrate.
An alternate polarization transfer catalyst is introduced
As opposed to the traditional [Ir(COD)(IMes)(Cl)] catalyst (18), the synthesized [Ir(COD)(IMes)(Py)][PF6] results in a pyridine ligand trans to IMes, improving our hyperpolarization levels by a factor of ~3 (see the Supplementary Materials). We have found that this new approach, which avoids competition from added pyridine, makes it possible to directly hyperpolarize a wide variety of different types of15N-containing molecules (and even 13C). However, diazirines represent a particularly general and interesting class of ligands for molecular tags and are the focus here.
As depicted in Fig. 2A, the hyperpolarization proceeds outside the high-field NMR magnet at low magnetic fields, enabling SABRE-SHEATH directly targeting 15N nuclei (22). To establish the hyperpolarization, we bubble p-H2 for ~5 min at the adequate field. Then, the sample is transferred into the NMR magnet within ~10 s, and 15N2 signal detection is performed with a simple 90° pulse followed by data acquisition.
Fig. 2Experimental and spectral distinction between magnetization and singlet spin order.
(A) Experimental procedure. The sample is hyperpolarized by bubbling parahydrogen (p-H2) through the solution in the NMR tube for 5 min and subsequently transferred into the high-field magnet for detection. If the hyperpolarization/bubbling is performed in a magnetic shield at ~6 mG, z-magnetization is created (black). If the hyperpolarization/bubbling is performed in the laboratory field anywhere between ~0.1 and ~1 kG, singlet order is created (blue). (B) Z-magnetization and singlet spin order can easily be distinguished based on their spectral appearance. a.u., arbitrary units. Z-magnetization produces an in-phase quartet (black). Singlet order gives an anti-phase quartet (blue). The spin system parameters for the 15N2 two-spin system are JNN = 17.3 Hz and Δδ = 0.58 parts per million.
Two types of hyperpolarized states can be created: Magnetization and singlet order
We create two different types of hyperpolarization on the 15N2-diazirine. We can hyperpolarize traditional z-magnetization, which corresponds to nuclear spins aligned with the applied magnetic field and is associated with pure in-phase signal as illustrated with the black trace in Fig. 2B. Alternatively, we can hyperpolarize singlet order on the 15N2-diazirine, which corresponds to an anti-aligned spin state, with both spins pointing in opposite directions, entangled in a quantum mechanically “hidden” state. This hidden singlet order is converted into a detectable state when transferred to a high magnetic field and associated with the anti-phase signal illustrated by the blue trace in Fig. 2B (see the Supplementary Materials for details). The difference in symmetry of z-magnetization and singlet order leads to differences in signal decay rates; z-magnetization is directly exposed to all NMR relaxation mechanisms and is often associated with shorter signal lifetimes, which may impede molecular tracking on biologically relevant time scales. Singlet order, on the other hand, is protected from many relaxation mechanisms because it has no angular momentum (42–52) and can therefore exhibit much longer lifetimes, enabling hour-long molecular tracking.
The type of hyperpolarized state is selected by the magnetic field
We can control which type of hyperpolarization we create by choosing the appropriate magnetic fields for the bubbling process. Z-magnetization is created in the SABRE-SHEATH mode at low magnetic fields inside a magnetic shield (22–24, 53, 54). This behavior is explained by resonance conditions for hyperpolarizing magnetization versus singlet order that we derive in the Supplementary Materials. The condition for creating magnetization, νH − νN = |JHH ± JNN|, is field-dependent in the NMR frequencies, νH and νN, and field-independent in the J-couplings. Accordingly, hyperpolarized magnetization is created at a magnetic field where the frequency difference matches the J-couplings. This magnetic field is ~6 mG, which is obtained by using JHH = −10 Hz, JNN = −17.3 Hz, γ1H = 4.2576 kHz/G, and γ15N = −0.4316 kHz/G (see the Supplementary Materials). The theoretical prediction of 6 mG matches the experimental maximum for hyperpolarized z-magnetization illustrated by the blue data in Fig. 3A.
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