Contrast mechanisms in pump-probe microscopy of melanin.

Pump-probe microscopy of melanin in tumors has been proposed to improve diagnosis of malignant melanoma, based on the hypothesis that aggressive cancers disaggregate melanin structure. However, measured signals of melanin are complex superpositions of multiple nonlinear processes, which makes interpretation challenging. Polarization control during measurement and data fitting are used to decompose signals of melanin into their underlying molecular mechanisms. We then identify the molecular mechanisms that are most susceptible to melanin disaggregation and derive false-coloring schemes to highlight these processes in biological tissue. We demonstrate that false-colored images of a small set of melanoma tumors correlate with clinical concern. More generally, our systematic approach of decomposing pump-probe signals can be applied to a multitude of different samples.

Nonequilibrium Control of Thermal and Mechanical Changes in a Levitated System.

Fluctuation theorems are fundamental extensions of the second law of thermodynamics for small nonequilibrium systems. While work and heat are equally important forms of energy exchange, fluctuation relations have not been experimentally assessed for the generic situation of simultaneous mechanical and thermal changes. Thermal driving is indeed generally slow and more difficult to realize than mechanical driving. Here, we use feedback cooling techniques to implement fast and controlled temperature variations of an underdamped levitated microparticle that are 1 order of magnitude faster than the equilibration time. Combining mechanical and thermal control, we verify the validity of a fluctuation theorem that accounts for both contributions, well beyond the range of linear response theory. Our results allow the investigation of general far-from-equilibrium processes in microscopic systems that involve fast mechanical and thermal changes at the same time.

Beyond intensity modulation: new approaches to pump-probe microscopy.

Pump-probe microscopy is an emerging nonlinear imaging technique based on high repetition rate lasers and fast intensity modulation. Here, we present new methods for pump-probe microscopy that keep the beam intensity constant and instead modulate the inter-pulse time delay or the relative polarization. These techniques can improve image quality for samples that have poor heat dissipation or long-lived radiative states and can selectively address nonlinear interactions in the sample. We experimentally demonstrate this approach and point out the advantages over conventional intensity modulation.

Low-cost measurement of face mask efficacy for filtering expelled droplets during speech.

Mandates for mask use in public during the recent coronavirus disease 2019 (COVID-19) pandemic, worsened by global shortage of commercial supplies, have led to widespread use of homemade masks and mask alternatives. It is assumed that wearing such masks reduces the likelihood for an infected person to spread the disease, but many of these mask designs have not been tested in practice. We have demonstrated a simple optical measurement method to evaluate the efficacy of masks to reduce the transmission of respiratory droplets during regular speech. In proof-of-principle studies, we compared a variety of commonly available mask types and observed that some mask types approach the performance of standard surgical masks, while some mask alternatives, such as neck gaiters or bandanas, offer very little protection. Our measurement setup is inexpensive and can be built and operated by nonexperts, allowing for rapid evaluation of mask performance during speech, sneezing, or coughing.

Thermodynamics of continuous non-Markovian feedback control.

Feedback control mechanisms are ubiquitous in science and technology, and play an essential role in regulating physical, biological and engineering systems. The standard second law of thermodynamics does not hold in the presence of measurement and feedback. Most studies so far have extended the second law for discrete, Markovian feedback protocols; however, non-Markovian feedback is omnipresent in processes where the control signal is applied with a non-negligible delay. Here, we experimentally investigate the thermodynamics of continuous, time-delayed feedback control using the motion of an optically levitated, underdamped microparticle. We test the validity of a generalized second law which bounds the energy extracted from the system, and study the breakdown of feedback cooling for very large time delays.

Cooling of a levitated nanoparticle to the motional quantum ground state.

Quantum control of complex objects in the regime of large size and mass provides opportunities for sensing applications and tests of fundamental physics. The realization of such extreme quantum states of matter remains a major challenge. We demonstrate a quantum interface that combines optical trapping of solids with cavity-mediated light-matter interaction. Precise control over the frequency and position of the trap laser with respect to the optical cavity allowed us to laser-cool an optically trapped nanoparticle into its quantum ground state of motion from room temperature. The particle comprises 108 atoms, similar to current Bose-Einstein condensates, with the density of a solid object. Our cooling technique, in combination with optical trap manipulation, may enable otherwise unachievable superposition states involving large masses.

Cavity Cooling of a Levitated Nanosphere by Coherent Scattering.

We report three-dimensional (3D) cooling of a levitated nanoparticle inside an optical cavity. The cooling mechanism is provided by cavity-enhanced coherent scattering off an optical tweezer. The observed 3D dynamics and cooling rates are as theoretically expected from the presence of both linear and quadratic terms in the interaction between the particle motion and the cavity field. By achieving nanometer-level control over the particle location we optimize the position-dependent coupling and demonstrate axial cooling by two orders of magnitude at background pressures of 6×10^{-2} mbar. We also estimate a significant (>40 dB) suppression of laser phase noise heating, which is a specific feature of the coherent scattering scheme. The observed performance implies that quantum ground state cavity cooling of levitated nanoparticles can be achieved for background pressures below 1×10^{-7} mbar.

Expression of type IIA secretory phospholipase A2 inhibits cholesteryl ester transfer protein activity in transgenic mice.

OBJECTIVE: High circulating levels of group IIA secretory phospholipase A2 (sPLA2-IIA) activity and mass are independent cardiovascular risk factors. Therefore, inhibition of sPLA2-IIA may be a target for the treatment of atherosclerotic cardiovascular disease. The present study evaluated the effects of sPLA2-IIA inhibition with varespladib acid in a novel mouse model, human apolipoprotein B (apoB)/human cholesteryl ester transfer protein (CETP)/human sPLA2-IIA triple transgenic mice (TTT) fed a Western-type diet. APPROACH AND RESULTS: sPLA2-IIA expression increased atherosclerotic lesion formation in TTT compared with human apoB/human CETP double transgenic mice (P<0.01). Varespladib acid effectively inhibited plasma sPLA2-IIA activity. Surprisingly, however, administration of varespladib acid to TTT had no impact on atherosclerosis, which could be attributed to a proatherogenic plasma lipoprotein profile that appears in response to sPLA2-IIA inhibition because of increased plasma CETP activity. In the TTT model, sPLA2-IIA decreased CETP activity by reducing the acceptor properties of sPLA2-IIA-modified very low-density lipoproteins specifically because of a significantly lower apoE content. Increasing very low-density lipoprotein-apoE content by means of adenovirus-mediated gene transfer in sPLA2-IIA transgenic mice restored the acceptor properties for CETP. CONCLUSIONS: These data show that in a humanized triple transgenic mouse model with hypercholesterolemia, sPLA2-IIA inhibition increases CETP activity via increasing the very low-density lipoprotein-apoE content, resulting in a proatherogenic lipoprotein profile.

Cavity cooling of an optically levitated submicron particle.

The coupling of a levitated submicron particle and an optical cavity field promises access to a unique parameter regime both for macroscopic quantum experiments and for high-precision force sensing. We report a demonstration of such controlled interactions by cavity cooling the center-of-mass motion of an optically trapped submicron particle. This paves the way for a light-matter interface that can enable room-temperature quantum experiments with mesoscopic mechanical systems.

Minimizing strain influences in a genetically modified mouse phenotyping platform.

Our approach has been to power studies to allow for detection of at least modest changes from a wild-type littermate control, include assays with overlapping physiological systems to provide cross-functional interpretive value, and to employ challenge assays.

Airborne particulate metals in the New York City subway: a pilot study to assess the potential for health impacts.

A prior study in New York City observed that airborne concentrations of three metals found in steel - iron, manganese, and chromium - are more than 100 times higher in the subway system than in aboveground air. To investigate the potential for health effects of exposure at these levels, we conducted a pilot study of subway workers comparing personal exposures to steel dust with biomarkers of metal exposure, oxidative stress, and DNA damage in blood and urine samples. Workers wore a personal air sampler operating at 4L/m for one to three work shifts with blood and urine samples collected at the end of the final shift. We found that PM(2.5) exposures varied among subway workers on the basis of job title and job activity. The subway workers' mean time-weighted PM(2.5) exposure was 52 microg/m3, with a median of 27 microg/m3, and a range of 6-469 microg/m3. The observed concentrations of PM(2.5), iron, manganese, and chromium fell well below occupational standards. Biomarker concentrations among the 39 subway workers were compared with a group of 11 bus drivers, and a group of 25 suburban office workers. Concentrations of DNA-protein crosslinks and chromium in plasma were significantly higher in subway workers than in bus drivers, but no significant difference was observed for these biomarkers between subway workers and office workers. Urinary isoprostane concentrations were significantly correlated with the number of years working in the subway system, and were detected at higher, though not significantly higher, concentrations in subway workers than in bus drivers or office workers. At the group level, there was no consistent pattern of biomarker concentrations among subway workers significantly exceeding those of the bus drivers and office workers. At the individual level, steel dust exposure was not correlated with any of the biomarkers measured.

Focal cerebral ischemia in the TNFalpha-transgenic rat.

BACKGROUND: To determine if chronic elevation of the inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), will affect infarct volume or cortical perfusion after focal cerebral ischemia. METHODS: Transgenic (TNFalpha-Tg) rats overexpressing the murine TNFalpha gene in brain were prepared by injection of mouse DNA into rat oocytes. Brain levels of TNFalpha mRNA and protein were measured and compared between TNFalpha-Tg and non-transgenic (non-Tg) littermates. Mean infarct volume was calculated 24 hours or 7 days after one hour of reversible middle cerebral artery occlusion (MCAO). Cortical perfusion was monitored by laser-Doppler flowmetry (LDF) during MCAO. Cortical vascular density was quantified by stereology. Post-ischemic cell death was assessed by immunohistochemistry and regional measurement of caspase-3 activity or DNA fragmentation. Unpaired t tests or analysis of variance with post hoc tests were used for comparison of group means. RESULTS: In TNFalpha-Tg rat brain, the aggregate mouse and rat TNFalpha mRNA level was fourfold higher than in non-Tg littermates and the corresponding TNFalpha protein level was increased fivefold (p

An extensive phenotypic characterization of the hTNFalpha transgenic mice.

BACKGROUND: Tumor necrosis factor alpha (TNFalpha) is implicated in a wide variety of pathological and physiological processes, including chronic inflammatory conditions, coronary artery disease, diabetes, obesity, and cachexia. Transgenic mice expressing human TNFalpha (hTNFalpha) have previously been described as a model for progressive rheumatoid arthritis. In this report, we describe extensive characterization of an hTNFalpha transgenic mouse line. RESULTS: In addition to arthritis, these hTNFalpha transgenic mice demonstrated major alterations in body composition, metabolic rate, leptin levels, response to a high-fat diet, bone mineral density and content, impaired fertility and male sexual function. Many phenotypes displayed an earlier onset and a higher degree of severity in males, pointing towards a significant degree of sexual dimorphism in response to deregulated expression of TNFalpha. CONCLUSION: These results highlight the potential usefulness of this transgenic model as a resource for studying the progressive effects of constitutively expressed low levels of circulating TNFalpha, a condition mimicking that observed in a number of human pathological conditions.

Noninvasive bioluminescence imaging of normal and spontaneously transformed prostate tissue in mice.

Several transgenic mouse models of prostate cancer have been developed recently that are able to recapitulate many key biological features of the human condition. It would, therefore, be desirable to employ these models to test the efficacy of new therapeutics before clinical trial; however, the variable onset and non-visible nature of prostate tumor development limit their use for such applications. We now report the generation of a transgenic reporter mouse that should obviate these limitations by enabling noninvasive in vivo bioluminescence imaging of normal and spontaneously transformed prostate tissue in the mouse. We used an 11-kb fragment of the human prostate-specific antigen (PSA) promoter to achieve specific and robust expression of firefly luciferase in the prostate glands of transgenic mice. Ex vivo bioluminescence imaging and in situ hybridization analysis confirmed that luciferase expression was restricted to the epithelium in all four lobes of the prostate. We also show that PSA-Luc mice exhibit decreased but readily detectable levels of in vivo bioluminescence over extended time periods following androgen ablation. These results suggest that this reporter should enable in vivo imaging of both androgen-dependent and androgen-independent prostate tumor models. As proof-of-principle, we show that we could noninvasively image SV40 T antigen-induced prostate tumorigenesis in mice with PSA-Luc. Furthermore, we show that our noninvasive imaging strategy can be successfully used to image tumor response to androgen ablation in transgenic mice and, as a result, that we can rapidly identify individual animals capable of sustaining tumor growth in the absence of androgen.

Steel dust in the New York City subway system as a source of manganese, chromium, and iron exposures for transit workers.

The United States Clean Air Act Amendments of 1990 reflected increasing concern about potential effects of low-level airborne metal exposure on a wide array of illnesses. Here we summarize results demonstrating that the New York City (NYC) subway system provides an important microenvironment for metal exposures for NYC commuters and subway workers and also describe an ongoing pilot study of NYC transit workers' exposure to steel dust. Results from the TEACH (Toxic Exposure Assessment, a Columbia and Harvard) study in 1999 of 41 high-school students strongly suggest that elevated levels of iron, manganese, and chromium in personal air samples were due to exposure to steel dust in the NYC subway. Airborne concentrations of these three metals associated with fine particulate matter were observed to be more than 100 times greater in the subway environment than in home indoor or outdoor settings in NYC. While there are currently no known health effects at the airborne levels observed in the subway system, the primary aim of the ongoing pilot study is to ascertain whether the levels of these metals in the subway air affect concentrations of these metals or related metabolites in the blood or urine of exposed transit workers, who due to their job activities could plausibly have appreciably higher exposures than typical commuters. The study design involves recruitment of 40 transit workers representing a large range in expected exposures to steel dust, the collection of personal air samples of fine particulate matter, and the collection of blood and urine samples from each monitored transit worker.

Experimental Helicobacter felis infection in transgenic mice expressing human group IIA phospholipase A2.

BACKGROUND: Both various virulence factors of Helicobacter pylori and host factors influence the clinical outcome of H. pylori infection. In animal experiments with Helicobacter felis, large variations in the severity of disease have been observed between different mouse strains infected with a single isolate of H. felis. C57BL/6 J mouse strain that lacks the expression of group IIA phospholipase A2 has been shown to develop more severe gastric inflammation than other mouse strains. Thus, group IIA phospholipase A2 has been suggested to play a role in regulating inflammation in gastric mucosa. The aim of this study was to examine the possible role of group IIA phospholipase A2 in experimental Helicobacter infection. MATERIALS AND METHODS: Transgenic mice expressing human group IIA phospholipase A2 and their group IIA phospholipase A2 deficient nontransgenic C57BL/6 J littermates were infected with H. felis. The mice were killed 3, 8, and 19 weeks after inoculation of bacteria to determine the histopathological changes in gastric mucosa. RESULTS: The infected mice developed chronic inflammation in gastric mucosa. We found no differences in the colonization of bacteria between transgenic and nontransgenic mice. At 3 and 8 weeks, no difference was found in the severity of inflammation between the two groups. Nineteen weeks after the administration of bacteria the inflammation was more marked in nontransgenic than transgenic mice. Group IIA phospholipase A2 was expressed by in situ hybridization in the neck cells of the glandular stomach in transgenic mice. CONCLUSIONS: The results of the present study suggest that the endogenous expression of group IIA phospholipase A2 diminishes chronic inflammation in gastric mucosa in experimental H. felis infection in mice.

Non-invasive imaging of GFAP expression after neuronal damage in mice.

Up-regulation of glial fibrillary acidic protein (GFAP) expression is often used as a surrogate marker of neuronal damage. We have created a transgenic mouse line that carries the luciferase gene under the transcriptional control of the mouse GFAP promoter. Biophotonic imaging was used to non-invasively detect the increase in GFAP expression after kainic acid induced neuronal cell death. We demonstrate that after kainic acid treatment, strong biophotonic signals were detected from the brain area. This correlated with both endogenous GFAP and luciferase RNA levels as well as with hippocampal cell death observed histologically. The transgenic mouse line will provide a powerful tool to dynamically monitor neuronal cell death in the living animal and will aid in the discovery and development of drugs to treat damage due to stroke and other neurodegenerative diseases.

Inflammation-dependent cerebral deposition of serum amyloid a protein in a mouse model of amyloidosis.

The major pathological hallmark of amyloid diseases is the presence of extracellular amyloid deposits. Serum amyloid A (SAA) is an apolipoprotein primarily produced in the liver. Serum protein levels can increase one thousandfold after inflammation. SAA is the precursor to the amyloid A protein found in deposits of systemic amyloid A amyloid (AA or reactive amyloid) in both mouse and human. To study the factors necessary for cerebral amyloid formation, we have created a transgenic mouse that expresses the amyloidogenic mouse Saa1 protein in the brain. Using the synapsin promoter to drive expression of the Saa1 gene, the brains of transgenic mice expressed both RNA and protein. Under noninflammatory conditions, transgenic mice do not develop AA amyloid deposits in the brain; however, induction of a systemic acute-phase response in transgenic mice enhanced amyloid deposition. This deposition was preceded by an increase in cytokine levels in the brain, suggesting that systemic inflammation may be a contributing factor to the development of cerebral amyloid. The nonsteroidal anti-inflammatory agent indomethacin reduced inflammation and protected against the deposition of AA amyloid in the brain. These studies indicate that inflammation plays an important role in the process of amyloid deposition, and inhibition of inflammatory cascades may attenuate amyloidogenic processes, such as Alzheimer's disease.

Human secretory phospholipase A2 mediates decreased plasma levels of HDL cholesterol and apoA-I in response to inflammation in human apoA-I transgenic mice.

OBJECTIVE: Plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein (apo)A-I are decreased in inflammatory states. Secretory phospholipase A2 (sPLA2), an acute-phase protein, may play a key role in the pathophysiology of this phenomenon. METHODS AND RESULTS: To investigate the effects of sPLA2 on human-like HDL particles in vivo, we generated transgenic mice overexpressing human apoA-I and human sPLA2 (apoA-I/sPLA2 mice). Compared with apoA-I mice, apoA-I/sPLA2 mice had significantly lower plasma levels of phospholipids, HDL cholesterol, and apoA-I (each P<0.01). HDL from apoA-I/sPLA2 mice was significantly depleted in phospholipids and cholesteryl esters (each P<0.001) but was enriched in protein and triglycerides (each P<0.001). As assessed by gel filtration and nondenaturing gel electrophoresis, sPLA2 overexpression in apoA-I mice resulted in a dramatic shift of the HDL particle size toward smaller particles. Furthermore, virtually all plasma sPLA2 in apoA-I/sPLA2 mice was found in association with the HDL fraction. The acute-phase response was induced in apoA-I/sPLA2 double-transgenic and apoA-I single-transgenic mice by intraperitoneal lipopolysaccharide (LPS) injection. Plasma sPLA2 was significantly increased after LPS injection in apoA-I/sPLA2 mice. Twelve hours after LPS administration, plasma total cholesterol, HDL cholesterol, apoA-I, and phospholipids were unchanged in apoA-I transgenic control mice but had decreased significantly in the apoA-I/sPLA2 mice (-57%, -62%, and -54%, -61%, respectively; each P<0.001). Both groups of mice had increased plasma levels of serum amyloid A (SAA) in response to LPS. To test the hypothesis that SAA may be an in vivo activator of sPLA2, we specifically overexpressed SAA in apoA-I/sPLA2 mice by means of liver-directed gene transfer. Despite high plasma levels of SAA, plasma lipid and lipoprotein profiles were not different than those in control mice. CONCLUSIONS: These results in a mouse model of human-like HDL indicate that sPLA2 expression significantly influences HDL particle size and composition and demonstrate that an induction of sPLA2 is required for the decrease in plasma HDL cholesterol in response to inflammatory stimuli in mice and that this effect is independent of SAA.