Imaging intact human organs with local resolution of cellular structures using hierarchical phase-contrast tomography.

Imaging intact human organs from the organ to the cellular scale in three dimensions is a goal of biomedical imaging. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique using the European Synchrotron Radiation Facility (ESRF)'s Extremely Brilliant Source (EBS). The spatial coherence of the ESRF-EBS combined with our beamline equipment, sample preparation and scanning developments enabled us to perform non-destructive, three-dimensional (3D) scans with hierarchically increasing resolution at any location in whole human organs. We applied HiP-CT to image five intact human organ types: brain, lung, heart, kidney and spleen. HiP-CT provided a structural overview of each whole organ followed by multiple higher-resolution volumes of interest, capturing organotypic functional units and certain individual specialized cells within intact human organs. We demonstrate the potential applications of HiP-CT through quantification and morphometry of glomeruli in an intact human kidney and identification of regional changes in the tissue architecture in a lung from a deceased donor with coronavirus disease 2019 (COVID-19).

GPU-enabled real-time optical frequency comb spectroscopy and a photonic readout.

We describe a GPU-enabled approach for real-time optical frequency comb spectroscopy in which data is recorded, Fourier transformed, normalized, and fit at data rates up to 2.2 GB/s. As an initial demonstration we have applied this approach to rapidly interrogate the motion of an optomechanical accelerometer through the use of an electro-optic frequency comb. We note that this approach is readily amenable to both self-heterodyne and dual-comb spectrometers for molecular spectroscopy as well as a photonic readout where the approach's agility, speed, and simplicity are expected to enable future improvements and applications.

Single-modulator, direct frequency comb spectroscopy via serrodyne modulation.

Traditional electro-optic frequency comb spectrometers rely upon the use of an acousto-optic modulator (AOM) to provide a differential frequency shift between probe and local oscillator (LO) legs of the interferometer. Here we show that these modulators can be replaced by an electro-optic phase modulator (EOM) which is driven by a sawtooth waveform to induce serrodyne modulation. This approach enables direct frequency comb spectroscopy to be performed with a single dual-drive Mach-Zehnder modulator (DD-MZM), allowing for lower differential phase noise. Further, this method allows for simpler production of integrated photonic comb spectrometers on the chip scale.

High dynamic range electro-optic dual-comb interrogation of optomechanical sensors.

An interleaved, chirped electro-optic dual comb system is demonstrated for rapid, high dynamic range measurements of cavity optomechanical sensors. This approach allows for the cavity displacements to be interrogated at measurement times as fast as 10 µs over ranges far larger than can be achieved with alternative methods. While the performance of this novel, to the best of our knowledge, readout approach is evaluated with an optomechanical accelerometer, this method has a wide range of applications including temperature, pressure, and humidity sensing as well as acoustics and molecular spectroscopy.

Electro-optic frequency combs for rapid interrogation in cavity optomechanics.

Electro-optic frequency combs were employed to rapidly interrogate an optomechanical sensor, demonstrating spectral resolution substantially exceeding that possible with a mode-locked frequency comb. Frequency combs were generated using an integrated-circuit-based direct digital synthesizer and utilized in a self-heterodyne configuration. Unlike approaches based upon laser locking, the present approach allows rapid, parallel measurements of full optical cavity modes, large dynamic range of sensor displacement, and acquisition across a wide frequency range between DC and 500 kHz. In addition to being well suited to measurements of acceleration, this optical frequency comb-based approach can be utilized for interrogation in a wide range of cavity optomechanical sensors.

Electromagnetically induced transparency in vacuum and buffer gas potassium cells probed via electro-optic frequency combs.

Electromagnetically induced transparency (EIT) in K39 and K41 was probed using electro-optic frequency combs generated by applying chirped waveforms to a phase modulator. The carrier tone of the frequency comb served as the pump beam and induced the necessary optical cycling. Comb tooth spacings as narrow as 20 kHz were used to probe potassium in both buffer gas and evacuated cells at elevated temperatures. Atomic absorption features as narrow as 33(5) kHz were observed, allowing for the K39 lower-state hyperfine splitting to be optically measured with a fit uncertainty of 2 kHz. Due to the ultranarrow width of the EIT features, long-lived optical free induction decays were also observed which allowed for background-free detection.

Ultra-sensitive cavity ring-down spectroscopy in the mid-infrared spectral region.

We describe an ultra-sensitive cavity ring-down spectrometer which operates in the mid-infrared spectral region near 4.5 µm. With this instrument a noise-equivalent absorption coefficient of 2.6×10-11 cm-1 Hz-1/2 was demonstrated with less than 150 nW of optical power incident on the photodetector. Quantum noise was observed in the individual ring-down decay events, leading to quantum-noise-limited short-time performance. We believe that this spectrometer's combination of high sensitivity and robustness make it well suited for measurements of ultra-trace gas species as well as applications in optics and fundamental physics.

Multiheterodyne spectroscopy with optical frequency combs generated from a continuous-wave laser.

Dual-drive Mach-Zehnder modulators were utilized to produce power-leveled optical frequency combs (OFCs) from a continuous-wave laser. The resulting OFCs contained up to 50 unique frequency components and spanned more than 200 GHz. Simple changes to the modulation frequency allowed for agile control of the comb spacing. These OFCs were then utilized for broadband, multiheterodyne measurements of CO2 using both a multipass cell and an optical cavity. This technique allows for robust measurements of trace gas species and alleviates much of the cost and complexity associated with the use of femtosecond OFCs produced with mode-locked pulsed lasers.

Comb-linked, cavity ring-down spectroscopy for measurements of molecular transition frequencies at the kHz-level.

We present a low uncertainty measurement technique for determining molecular transition frequencies. This approach is complementary to sub-Doppler saturation spectroscopies and is expected to enable new frequency measurements for a wide variety of molecular species with uncertainties at the kHz-level. The technique involves measurements of Doppler broadened lines using cavity ring-down spectroscopy whereby the probe laser is actively locked to the ring-down cavity and the spectrum frequencies are linked directly to an optical frequency comb that is referenced to an atomic frequency standard. As a demonstration we have measured the transition frequency of the (30012) ← (00001) P14e line of CO2 near 1.57 µm with a combined standard uncertainty of ~9 kHz. This technique exhibits exceptional promise for measurements of transition frequencies and pressure shifting parameters of many weak absorbers, and indicates the potential for substantially improved measurements when compared to those obtained with conventional spectroscopic methods.

Measurement of H2O broadening of O2 A-band transitions and implications for atmospheric remote sensing.

We present laboratory measurements of H(2)O-broadened (16)O(2) A-band (b(1)Σ(g)(+) ← X(3)Σ(g)(-)(0,0)) absorption spectra acquired with a laser-based photoacoustic spectroscopy method. This absorption band is widely used in a variety of high-precision atmospheric remote sensing applications. We report H(2)O broadening parameters for six of the strongest transitions in this band, and we show that these measured values are nominally 1.5-2 times greater than the corresponding air-broadening parameters. Simulations of atmospheric transmission spectra in the O(2) A-band that incorporate our measured H(2)O broadening parameters indicate that H(2)O present at concentrations typically found in the Earth's atmosphere can influence the column-integrated transmission relative to the dry air case. Further, because of spatial and seasonal variations in humidity, failure to account for the enhanced H(2)O pressure broadening effects can lead to concomitant biases in atmospheric O(2) A-band retrievals of quantities such as surface pressure and path length in greenhouse gas retrievals.

Frequency-stabilized cavity ring-down spectroscopy measurements of line mixing and collision-induced absorption in the O2 A-band.

Frequency-stabilized cavity ring-down spectroscopy measurements were performed in the P-branch of the O(2) A-band [b(1)Σ(g) (+) ← X (3)Σ(g) (-)(0,0)] near atmospheric pressure. Line mixing parameters and collision-induced absorption were quantified and reported. These measurements show qualitative differences with those taken at relatively high pressure (2 MPa-20 MPa). We also assess the implications of these measurements on atmospheric retrievals.

Multiscale three-dimensional imaging of intact human organs down to the cellular scale using hierarchical phase-contrast tomography.

Human organs are complex, three-dimensional and multiscale systems. Spatially mapping the human body down through its hierarchy, from entire organs to their individual functional units and specialised cells, is a major obstacle to fully understanding health and disease. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique utilising the European Synchrotron Radiation Facility's Extremely Brilliant Source: the world's first high-energy 4 th generation X-ray source. HiP-CT enabled three-dimensional and non-destructive imaging at near-micron resolution in soft tissues at one hundred thousand times the voxel size whilst maintaining the organ's structure. We applied HiP-CT to image five intact human parenchymal organs: brain, lung, heart, kidney and spleen. These were hierarchically assessed with HiP-CT, providing a structural overview of the whole organ alongside detail of the organ's individual functional units and cells. The potential applications of HiP-CT were demonstrated through quantification and morphometry of glomeruli in an intact human kidney, and identification of regional changes to the architecture of the air-tissue interface and alveolar morphology in the lung of a deceased COVID-19 patient. Overall, we show that HiP-CT is a powerful tool which can provide a comprehensive picture of structural information for whole intact human organs, encompassing precise details on functional units and their constituent cells to better understand human health and disease.

Multiplexed sub-Doppler spectroscopy with an optical frequency comb.

An optical frequency comb generated with an electro-optic phase modulator and a chirped radiofrequency waveform is used to perform pump-probe spectroscopy on the D1 and D2 transitions of atomic potassium at 770.1 nm and 766.7 nm, respectively. With a comb tooth spacing of 200 kHz and an optical bandwidth of 2 GHz the hyperfine transitions can be simultaneously observed. Interferograms are recorded in as little as 5 µs (a timescale corresponding to the inverse of the comb tooth spacing). Importantly, the sub-Doppler features can be measured as long as the laser carrier frequency lies within the Doppler profile, thus removing the need for slow scanning or a priori knowledge of the frequencies of the sub-Doppler features. Sub-Doppler optical frequency comb spectroscopy has the potential to dramatically reduce acquisition times and allow for rapid and accurate assignment of complex molecular and atomic spectra which are presently intractable.

Intracellular analysis of interleukin-2 induction provides direct evidence at the single cell level of differential coactivation requirements for CD45RA+ and CD45RO+ T cell subsets.

Through measurements of intracellular cytokine production, evidence is provided at the single cell level that triggering different cell surface molecules preferentially activates discrete human peripheral blood (PB) T cell subsets. T cell costimulation due to cross-linking a variety of individual molecules (beta1, beta2, and beta7 integrins, CD26, CD43, or CD44), in conjunction with the CD3/TCR complex, preferentially activated CD45RO+ PB T lymphocytes. CD28, however, costimulated interleukin-2 (IL-2) production in both CD45RO+ and CD45RA+ subpopulations. The amount of soluble IL-2 produced by CD28 coactivation was 15-30-fold higher than that due to integrin or CD26-dependent coactivation, although even the lowest amount of soluble IL-2 produced was in the range of the high-affinity IL-2 receptor. The overall proliferative responses were similar among all costimulatory settings. This was in part due to the uniform upregulation of IL-2 receptor-alpha (IL-2R alpha) (CD25) expression on the entire T cell population activated under each of the different costimulatory conditions. The data provide direct evidence on a single cell level that activation of human CD45RA+ (naive) T cells is stringently controlled and, in these studies, limited to CD28 costimulation for induction of IL-2 production. In contrast, coactivation of CD45RO+ (memory) T lymphocytes can proceed by a variety of different PB T cell surface molecules.

Deferoxamine improves spatial memory performance following experimental brain injury in rats.

Traumatic brain injury (TBI) causes impairments of both motor and spatial memory performances. Research is only beginning to reveal the biochemical mechanism(s) underlying these deficits. It has been postulated that reactive oxygen species such as the superoxide and hydroxyl radicals, as well as the peroxynitrite anion, are generated by injury and may play a critical role in the observed memory deficits. The highly reactive hydroxyl radical, which is thought to contribute to neuronal toxicity, can be generated by an iron-catalyzed reaction. The source of this iron (or iron-bound proteins) could be a compromise of the blood-brain barrier, which can occur following TBI. In this report, we investigate the ability of deferoxamine, a scavenger of free iron, the hydroxyl radical and the peroxynitrite anion, to facilitate behavioral recovery following a controlled cortical impact of rats. Intraperitoneal administration of this drug prior to the injury did not affect the rate of recovery from motor deficits in comparison to vehicle (saline)-injected animals. However, deferoxamine-treated animals showed significant improvement in spatial memory performance in a Morris water maze task. Volumetric analysis of cortical tissue loss showed no significant differences between vehicle- and drug-injected animals. Similarly, histological examination of the hippocampus did not reveal any gross differences between the two groups. These results indicate that deferoxamine improves spatial memory performance, possibly through protection from neuronal dysfunction.

High-performance liquid chromatographic assay for sodium levothyroxine in tablet formulations: content uniformity applications.

Sodium levothyroxine was quantitated in 25-300 micrograms/tablet formulations. The procedure consisted of pulverization of a suitable sample, extraction into acetonitrile-water (40:60, v/v) containing 0.05% o-phosphoric acid, and injection onto a bonded-phase cyanopropyl column; the effluent was monitored by UV detection at 225 nm. Spiked placebo recovery studies demonstrated the linearity of the method over the range of 80-120% of the label claim. Stability studies indicated that no degradation products or excipients interfered with the quantitation of the intact drug. Data demonstrating the accuracy and precision of this assay are presented, and the method was applied to the measurement of single-tablet content uniformity.

Reduced evoked release of acetylcholine in the rodent hippocampus following traumatic brain injury.

The chronic effects of traumatic brain injury on acetylcholine release were evaluated by using in vivo microdialysis. Acetylcholine release was measured in the hippocampus of anesthetized rats 2 weeks after lateral controlled cortical impact (n = 10) or sham surgery (n = 10). Prior to microdialysis, behavioral assessments of motor and spatial memory were performed. Cortical impact (6 meter/s, 2 mm deformation) produced beam balance deficits that persisted for 1 day and beam walking deficits that persisted for 3 days after injury. In addition, spatial memory, as measured by swim latencies in a Morris water maze, was compromised between 10-14 days after injury. Immediately following behavioral testing, the animals were anesthetized with halothane, and a microdialysis probe was placed into the dorsal hippocampus. After a 160 min equilibration period, extracellular levels of acetylcholine were measured prior to and after an intraperitoneal administration of scopolamine (1 mg/kg), which evokes acetylcholine release by blocking autoreceptors. Prior to scopolamine administration, there were no differences in extracellular levels of acetylcholine between injured and sham animals. However, there was a significant reduction of hippocampal acetylcholine release evoked by scopolamine in injured animals as compared to sham controls. In separate control groups, saline administration alone did not change hippocampal acetylcholine release in injured (n = 5) or sham (n = 5) animals. This study represents the first application of in vivo microdialysis to evaluate chronic neurotransmission deficits following TBI. The present study demonstrates that a magnitude of traumatic brain injury (TBI) sufficient to produce spatial memory deficits can result in a reduction in scopolamine-evoked release of acetylcholine within the hippocampus. The data further suggest that presynaptic mechanisms mediating release of acetylcholine could play a significant role in cholinergic neurotransmission deficits following TBI.

Reproducibility of intravenous linear pharmacokinetic parameters of ethanol in dogs.

Linear elimination parameters were computed after intravenous administration of the same dose (11 mmol/kg) of intravenous ethanol in anesthetized dogs twice with a period of 7-8 days (group A, n = 10) or 6 hours (group B, n = 8) between the two studies. For rate of elimination, clearance and time of disappearance of half the dose, routine statistical methods (paired t-test, correlation) showed no difference between the first and the second study of either group; however, for each of these three elimination parameters, quantitative analysis showed significant disparities between the first and second studies of each group. For Widmark ratio r, which is a measurement of ethanol distribution rather than elimination, both the routine statistical methods and the quantitative analysis showed no difference between the first and second study of either group. In dogs, the assumption that blood alcohol curve is reproducible in individuals may not be valid.

Ethanol elimination in the anesthetized dog: intragastric versus intravenous administration.

We computed by linear kinetics predicted equilibrated plasma concentrations, elimination parameters and availability of ethanol for fasting anesthetized dogs who received the same dose (11 mmol/kg) of ethanol twice, once intragastrically and once intravenously. Agreement between predicted (y) and observed (x) equilibrated plasma levels above 3 mmol/l was for intragastric ethanol y = 0.031 + 1.008x (r = 0.973) and for intravenous ethanol y = 0.2 + 0.99x (r = 0.992). Linear elimination (elimination rate, clearance, time of disappearance of half the dose) was significantly slower and Widmark's ratio r was significantly greater for intragastric than for intravenous ethanol. Apparent availability of intragastric ethanol, computed by dividing the intragastric by the intravenous plasma ethanol concentration at zero time (both values extrapolated from the linear portion of the blood alcohol curve), was 0.739 +/- 0.125. Considerable ethanol residuals were present in the stomach four hours after intragastric instillation. We conclude that retention of ethanol in the stomach, probably because of anesthesia, created the apparent differences in elimination of ethanol between intragastric and intravenous administration. Despite gastric retention, decrease of ethanol levels was linear above 3 mmol/l after intragastric instillation.

Relative lack of toxicity of transplatin compared with cisplatin in rodents.

The differential toxicity of the cis- and trans-isomers of diamminedichloroplatinum (II) (cisplatin and transplatin) was investigated in rats and guinea pigs. In both species, repeated daily administration of 1 to 2 mg per kg cisplatin produced severe histological and/or functional damage to renal and gastro-intestinal systems and resulted in death of the animals. Quantification of tissue platinum by atomic absorption spectroscopy demonstrated accumulation of large amounts of platinum in the kidney of the animals, with lesser amounts in the liver and gastro-intestinal tract. Transplatin, administered at total doses two- to four-fold that of cisplatin, was essentially non-toxic by histological and functional assessment. However, the amounts of tissue platinum measured in transplatin-treated animals were no smaller than those measured in cisplatin-treated animals; indeed, platinum concentrations in kidneys of transplatin-treated rats were more than 2.5 times those in cisplatin-treated rats. Thus tissue platinum content did not correlate with organ damage. These data suggest that mechanism(s) involving steric interactions of platinum species, perhaps with cellular macromolecules such as DNA or RNA, may be important in the differential toxicity of these two compounds.