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Cancer heterogeneity and its transformation with time propels treatment resistance and confounds patient outcomes. The inability to monitor in vivo the low abundance, heterocellular phenotypes that resist treatment and ultimately lead to patient death limits the ability to design precision therapies. Here we overcome limitations in multiplexed fluorescence phenotyping to introduce real-time, cellular resolution visualization of tumor heterogeneity in vivo. This method was performed to simultaneously map for the first time 5 individual biomarkers of stemness, proliferation, metabolism, leukocytes and angiogenesis deep within the peritoneal cavities of micrometastatic cancer mouse models at 17 frames per second (fps). The newly developed imaging system revealed distinct cancer cell phenotype–immune cell spatial correlations and clearly visualized the dynamic spatial response of resistant cancer cell niches following treatment. Furthermore, wide-field datasets were generated to facilitate derivation of a mathematical framework for quantifying biomarker spatial variation and thereby overcoming the area restrictions of conventional tumor biopsy. These results pave the way for real-time identification of cancer cell phenotypes in a clinical setting, on which optimized treatment regimens can be based for personalized treatment and precision therapy e.g., tumor margin determination during surgical resection. Additionally, this modality can be used to obtain more fundamental insights into tumor heterogeneity and how treatments affect the molecular and cellular responses of patient-specific disease.

Compound prism arrays are a powerful, yet underutilized, solution for producing high transmission and customized chromatic dispersion profiles over broad bandwidths, the quality of which is unobtainable with commercially available prisms or diffraction gratings. However, the computational complexity associated with designing these prism arrays presents a barrier to the widespread adoption of their use. Here we introduce customizable prism designer software that facilitates high-speed optimization of compound arrays guided by target specifications for chromatic dispersion linearity and detector geometry. Information theory is utilized such that target parameters can be easily modified through user input to efficiently simulate a broad range of possible prism array designs. We demonstrate the capabilities of the designer software to simulate new prism array designs for multiplexed, hyperspectral microscopy that achieve chromatic dispersion linearity and a 70-90% light transmission over a significant portion of the visible wavelength range (500–820 nm). The designer software is applicable to many optical spectroscopy and spectral microscopy applications—with varying requirements for spectral resolution, light ray deviation, and physical size—that are photon-starved and for which the enhanced transmission of refraction versus diffraction warrants custom optical designs.

Mild isolated fetal ventriculomegaly (iFVM) is the most common abnormality of the fetal central nervous system. It is characterized by enlargement of one or both of the lateral ventricles (defined as ventricular width greater than 10 mm, but less than 12 mm). Despite its high prevalence, the pathophysiology of iFVM during fetal brain development and the neurobiological substrate beyond ventricular enlargement remain unexplored. In this work, we aimed to establish the relationships between the structural development of transient fetal brain zones/compartments and increased cerebrospinal fluid volume. For this purpose, we used in vivo structural T2-weighted magnetic resonance imaging of 89 fetuses (48 controls and 41 cases with iFVM). Our results indicate abnormal development of transient zones/compartments belonging to both hemispheres (i.e. on the side with and also on the contralateral side without a dilated ventricle) in fetuses with iFVM. Specifically, compared to controls, we observed enlargement of proliferative zones and overgrowth of the cortical plate in iFVM with associated reduction of volumes of central structures, subplate, and fetal white matter. These results indicate that enlarged lateral ventricles might be linked to the development of transient fetal zones and that global brain development should be taken into consideration when evaluating iFVM.

Early brain development, from the embryonic period to infancy, is characterized by rapid structural and functional changes. These changes can be studied using structural and physiological neuroimaging methods. In order to optimally acquire and accurately interpret this data, concepts from adult neuroimaging cannot be directly transferred. Instead, one must have a basic understanding of fetal and neonatal structural and physiological brain development, and the important modulators of this process. Here, we first review the major developmental milestones of transient cerebral structures and structural connectivity (axonal connectivity) followed by a summary of the contributions from ex vivo and in vivo MRI. Next, we discuss the basic biology of neuronal circuitry development (synaptic connectivity, i.e. ensemble of direct chemical and electrical connections between neurons), physiology of neurovascular coupling, baseline metabolic needs of the fetus and the infant, and functional connectivity (defined as statistical dependence of low-frequency spontaneous fluctuations seen with functional magnetic resonance imaging (fMRI)). The complementary roles of magnetic resonance imaging (MRI), electroencephalography (EEG), magnetoencephalography (MEG), and near-infrared spectroscopy (NIRS) are discussed. We include a section on modulators of brain development where we focus on the placenta and emerging placental MRI approaches. In each section we discuss key technical limitations of the imaging modalities and some of the limitations arising due to the biology of the system. Although neuroimaging approaches have contributed significantly to our understanding of early brain development, there is much yet to be done and a dire need for technical innovations and scientific discoveries to realize the future potential of early fetal and infant interventions to avert long term disease.

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression of ATP-dependent K+ transporters, particularly the Na+, K+, Cl−, and water cotransporter NKCC1. Overexpression of NKCC1 in the ChP resulted in increased CSF K+ clearance, increased cerebral compliance, and reduced circulating CSF in the brain without changes in intracranial pressure in mice. Moreover, ChP-specific NKCC1 overexpression in an obstructive hydrocephalus mouse model resulted in reduced ventriculomegaly. Collectively, our results implicate NKCC1 in regulating CSF K+ clearance through the ChP in the critical period during postnatal neurodevelopment in mice., Abnormal CSF accumulation in the brain can lead to hydrocephalus. The mechanisms regulating CSF clearance during early development are unclear. Here, the authors show that NKCC1 regulates the clearance of both CSF K+ and fluid volume through the choroid plexus during postnatal development in mice.

Tumor microenvironmental heterogeneity is a major driver of treatment resistance and variability in clinical response to therapy. This heterogeneity arises from variations in cellular phenotypes populating the tumor microenvironment (TME), their spatio-temporal localization and expression of surface markers, often associated with specific biological function – stemness, metabolism, proliferation, immune activation and others. Such features are usually studied through ex vivo immunofluorescence and cytometry to identify cellular phenotypes in TME; however, they cannot be applied in vivo for real time TME analysis. Moreover, current in vivo imaging techniques fail to provide real-time high-resolution visualization of the TME. Routinely employed biopsy tissue sampling through fine needle aspirates is limited by its invasive nature and inability to provide a global and dynamic overview of the TME, thus limiting our ability to study spatiotemporal TME dynamics and identify possible features leading to potentially resistant tumor phenotypes. In this study, we demonstrate the use of a hyperspectral fluorescence microendoscope (HFME) to monitor cellular phenotypes in TME with real-time visualization and high video imaging capability (~17 fps). Current fluorescence video microscopy is limited to simultaneous imaging of no more than 2 molecular markers with potential to be expanded to ~4 markers using dichroic mirrors and point detectors; the HFME demonstrated in this study can currently resolve 6 different molecular markers, simultaneously, using a multichannel linear array detector with potential to expand to 10 or more markers. Using a cocktail of near infra-red fluorophore-antibody conjugates targeted against key molecular (surface) markers of different cells in the TME, we are able to capture real-time TME dynamics at cellular resolution in two pre-clinical models; 1) a xenograft orthotopic mouse model of peritoneal carcinomatosis (disseminated metastases within the abdominal cavity) and imaging epidermal growth factor receptor (EGFR), CD44, CA125 (MUC16), transferrin receptor, Thomsen-Friedenreich carbohydrate antigen (T antigen), and CD45, 2) A syngeneic immunocompetent KPC cell line implanted mouse model of pancreatic ductal adenocarcinoma and imaging CD3, CD4, CD8a and CD45. Imaging on these tumor models was performed pre- and post-sub-therapeutic verteporfin (benzoporphyrin derivative monoacid A) photodynamic therapy (PDT, a cytotoxic light-based therapy). PDT treatment resulted in reduction of cancer cell burden, immune cell infiltration and alterations in their relative spatial localization. The results were confirmed by histopathological validation and ex vivo immunofluorescence staining of tumor tissue sections. Collectively, these results demonstrate the capability of HFME to image cancer cell phenotypes and the tumor microenvironment, in real time in live mice. With the ability to monitor cancer growth and treatment effects at a cellular level, HFME can potentially assist in customizing therapies in a patient-specific manner. Citation Format: Mohammad A. Saad, Bryan Q. Spring, Akilan Palanisami, Eric M. Kercher, Ryan T. Lang, Jason Sutin, Zhiming Mai, Tayyaba Hasan. Real-time visualization of tumor cell phenotype and microenvironmental heterogeneity enabled by a hyperspectral fluorescence microendoscope [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P044.

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation is deleterious for perinatal neurodevelopment, but how CSF leaves the brain during this critical period is unknown. We found in mice a postnatal neurodevelopmental transition phase featuring precipitous CSF K+ clearance, accompanied by water, through the choroid plexus (ChP). The period corresponds to a human fetal stage when canonical CSF clearance pathways have yet to form and congenital hydrocephalus begins to manifest. Unbiased ChP metabolic and ribosomal profiling highlighted this transition phase with increased ATP yield and activated energy-dependent K+ transporters, in particular the Na+-K+-Cl− and water cotransporter NKCC1. ChP-targeted NKCC1 overexpression enhanced K+-driven CSF clearance and enabled more permissive cerebral hydrodynamics. Moreover, ventriculomegaly in an obstructive hydrocephalus model was improved by ChP-targeted NKCC1 overexpression. Collectively, we identified K+-driven CSF clearance through ChP during a transient but critical neurodevelopmental phase, with translational value for pathologic conditions.

Therapeutic hypothermia decreases cerebral metabolism in neonates at risk for HIE. Here, we quantify cerebral hemodynamics in the first week of life and show significant correlations with neurodevelopmental outcomes at 18 months.

Monitoring of cerebral blood flow (CBF) and autoregulation are essential components of neurocritical care, but continuous noninvasive methods for CBF monitoring are lacking. Diffuse correlation spectroscopy (DCS) is a noninvasive diffuse optical modality that measures a CBF index ([Formula: see text]) in the cortex microvasculature by monitoring the rapid fluctuations of near-infrared light diffusing through moving red blood cells. We tested the feasibility of monitoring [Formula: see text] with DCS in at-risk patients in the Neurosciences Intensive Care Unit. DCS data were acquired continuously for up to 20 h in six patients with aneurysmal subarachnoid hemorrhage, as permitted by clinical care. Mean arterial blood pressure was recorded synchronously, allowing us to derive autoregulation curves and to compute an autoregulation index. The autoregulation curves suggest disrupted cerebral autoregulation in most patients, with the severity of disruption and the limits of preserved autoregulation varying between subjects. Our findings suggest the potential of the DCS modality for noninvasive, long-term monitoring of cerebral perfusion, and autoregulation.

Monitoring of cerebral blood flow (CBF) and autoregulation are essential components of neurocritical care, but continuous noninvasive methods for CBF monitoring are lacking. Diffuse correlation spectroscopy (DCS) is a noninvasive diffuse optical modality that measures a CBF index (

Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics.

Physiological monitoring of oxygen delivery to the brain has great significance for improving the management of patients at risk for brain injury. Diffuse correlation spectroscopy (DCS) is a rapidly growing optical technology able to non-invasively assess the blood flow index (BFi) at the bedside. The current limitations of DCS are the contamination introduced by extracerebral tissue and the need to know the tissue's optical properties to correctly quantify the BFi. To overcome these limitations, we have developed a new technology for time-resolved diffuse correlation spectroscopy. By operating DCS in the time domain (TD-DCS), we are able to simultaneously acquire the temporal point-spread function to quantify tissue optical properties and the autocorrelation function to quantify the BFi. More importantly, by applying time-gated strategies to the DCS autocorrelation functions, we are able to differentiate between short and long photon paths through the tissue and determine the BFi for different depths. Here, we present the novel device and we report the first experiments in tissue-like phantoms and in rodents. The TD-DCS method opens many possibilities for improved non-invasive monitoring of oxygen delivery in humans.

Here we describe an automated method, named serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in three dimensions, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders.

We present a novel single-molecule method for rapidly evaluating small-molecule binding to individual DNA molecules using nanopores fabricated in ultrathin silicon membranes. A measurable shift in the residual ion current through a approximately 3.5 nm pore results from threading of a dye-intercalated DNA molecule, as compared to the typical residual current of native DNA. The average level of the residual current can be used to directly quantify the fraction of bound molecules to DNA, providing a new way to determine binding isotherms. Spatial sensitivity is also demonstrated by designing a two-segment DNA molecule that contains small-molecule binding sites in one of its two segments. Translocations of such molecules exhibit two current levels upon incubation with a DNA-binding dye, caused by selectively bound dye in one of the DNA segments. Our results, as shown here with four different dyes, coincide well with bulk fluorescence measurements performed under identical conditions. The nanopore approach for "reading-out" molecular binding along a DNA molecule, combined with the miniscule amounts of DNA required and the potential for scalability using nanopore arrays, provide a novel platform for future applications in analytical drug screening.

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer-oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein-uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA-plasminogen activator inhibitor type 1-mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. © The Rockefeller University Press.

The design and prototype tests of a segmented and shielded instrumentation structure, based on tube modules connected by canoe ball type kinematic couplings, are presented with application to a high-precision microscope for biological science experiments. The segmented tube design is shown to be significantly less sensitive to deformation from uneven thermal disturbances than a single tube structure. The gaps between the tube segments relieve thermal strains and restrict axial heat conduction, and the large-radius kinematic couplings provide high thermal resistance interconnects without significantly degrading structural stiffness. Simulation results, based on two- and three-dimensional models of heat conduction, are verified with experimental measurements of prototype structures and are used to predict the dependence of the tilt error on the geometry, boundary conditions, and material properties of the tubular segments. Adding layers of low-cost foam insulation and thin metal shielding to the walls of the structure further improves performance. Kinematic couplings could allow the structure to be disassembled and reassembled without re-calibration, and thus, pre-calibrated tube modules with different components could be easily and quickly interchanged between experiments.

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.

The mechanism of burst suppression, a spontaneous neural activity arising pathologically or from anesthesia, was studied by determining the cerebral rate of metabolic oxygen during EEG bursts with near-infrared and diffuse correlation spectroscopy.

We combine a Continuous-wave NIRS (CWNIRS) system with a Diffuse Correlation Spectroscopy (DCS) system to simultaneously measure hemoglobin concentrations and blood flow to quantify cerebral metabolism changes in infants

Development of an implantable EEG and diffuse optical imaging probe for longitudinal measurements in awake rats to investigate neurovascular coupling.

Monitoring the real-time behavior of spatial arrays of single living bacteria cells is only achieved with much experimental difficulty due to the small size and mobility of the cells. To address this problem, we have designed and constructed a simple microfluidic device capable of trapping single bacteria cells in spatially well-defined locations without the use of chemical surface treatments. The device exploits hydrodynamics to slow down and trap cells flowing near a narrow aperture. We have modeled this system numerically by approximating the motion of Escherichia coli cells as rigid 3-D ellipsoids. The numerical predictions for the speed and efficiency of trapping were tested by fabricating the devices and imaging GFP expressing E. coli at a high spatio-temporal resolution. We find that our numerical simulations agree well with the actual cell flow for varying trap geometries. The trapped cells are optically accessible, and combined with our ability to predict their spatial location we demonstrate the ease of this method for monitoring multiple single cells over a time course. The simplicity of the design, inexpensive materials and straightforward fabrication make it an accessible tool for any systems biology laboratory.

Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells. Our results reveal that the target RNA is not evenly distributed within the cell and localizes laterally along the long cell axis, in a pattern suggesting the existence of ordered helical RNA structures reminiscent of known bacterial cytoskeletal cellular elements.

Capillary electrophoresis is used to separate ultrasmall (∼1 nm) carboxylate functionalized Si nanoparticles (Si-np-COO-) prepared via hydrosilylation with an ω-ester 1-alkene. The electropherograms show a monodisperse Si core size with one or two carboxylate groups added to the surface. On-column detection of their laser-induced fluorescence demonstrates that the individual Si-np-COO-have narrow emissions (full width at half maximum = 30-40 nm) with a nearly symmetric lineshape. Preparative scale electrophoresis should be a viable route for purification of the Si-np-COO-for further study and future applications. © 2006 American Institute of Physics.

Studying the properties and stability of silicon nanoparticles (Si-np) in aqueous environments may lead to novel applications in biological systems. In this work, we use absorption and photoluminescence (PL) spectroscopy to characterize ultrasmall Si-np prepared through anodic etching and ultrasonic fractionation of a crystalline Si wafer. Their behavior is studied over time in 2-propanol and during treatments with water, NaOH, HCl, and H2O2. The observed population is divided into two types of material: bright species consisting of well-etched Si-np, -1 nm in diameter, and dark species derived from partially etched or aggregated Si structures. The dark material is seen by its scattering in the 2-propanol and water solutions and is largely removed via precipitation with the NaOH or HCl treatment. The bright material includes three distinct species with their respective emissions in the UV-B, UV-A, and hard-blue regions of the spectrum. The hard-blue PL is shown to have a simple pH dependence with a pKa-3, providing an important insight into its chemical origin and signaling for possible application of Si-np as environmental probes. Our results offer some potential for tailoring the PL properties of ultrasmall Si-np through control of their surface chemistry. © 2005 American Chemical Society.

We present a new method of measurement based on fluorescence lifetime imaging that reveals molecular-scale details of the mixing process in a continuous-flow turbulent microfluidic reactor. Our data provide a glimpse of the cascade to the minimal eddy size, followed by rapid diffusion involving the smallest eddies for final mixing.

Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 mm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with onephoton absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized. © 2004 Society of Photo-Optical Instrumentation Engineers.

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses non- invasively. This paper describes the development of a micro manipulation workstation integrating two-photon, 3-D imaging with a high-force, uniform-gradient, magnetic manipulator. The uniform-gradient magnetic field applies nearly equal forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5 micrometers diameter paramagnetic particles and over 800 pN on 5.0 micrometers ferromagnetic particles. These forces vary less than 10% over an area 200 x 200 micrometers 2. The compatibility with the use of high numerical aperture (approximately equals 1.0) objectives is an integral part of the workstation design allowing sub- micron resolution 3-D two-photon imaging. Three dimensional maps of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses is not always a local deformation.© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are the enzymes responsible for the biosynthesis of the precursor to the biologically active prostaglandins, prostacyclin, and thromboxane and are the molecular targets for nonsteroidal antiinflammatory drugs (NSAIDs). Selective COX-2 inhibitors are antiinflammatory and analgesic but lack gastrointestinal toxicity, an undesirable side effect attributed to COX-1 inhibition. Crystallographic analysis of selective COX inhibitors complexed with either isoform provides some information about the molecular determinants of selectivity but does not provide information about the dynamics of inhibitor association/dissociation. We employed rapid-mixing techniques and fluorescence quenching to monitor the association and dissociation of a selective COX-2 inhibitor to COX-1 or COX-2. The association of the fluorescent diaryloxazole, SC299, with both enzymes occurs in a time-dependent fashion. Its binding to COX-2 occurs in three kinetically distinct steps whereas its binding to COX-1 occurs in two steps. In contrast to the relatively rapid association of SC299 with both enzymes, its dissociation from COX-2 is quite slow and occurs over several hours whereas the dissociation from COX-1 is complete in less than 1 min. The selectivity of SC299 as a COX-2 inhibitor correlates to its relative rates of dissociation from the two COX isoforms. A model is proposed for diarylheterocycle binding to COX's that integrates these kinetic data with available structural information.

We present the design of a magnetic tweezers microscope for cellular manipulation. Our design allows versatile and significant 3D stress application over a large sample region. For linear force application, forces up to 250 pN per 4.5 micrometers magnetic bead can be applied. Finite element analysis shows that variance in force level is around 10 percent within an area of 300 X 300 micrometers 2. Our eight-pole design potentially allows 3D liner force application and exertion of torsional stress. Furthermore, our design allows high resolution imaging using high numerical aperture objective. Both finite element analysis of magnetic field distribution and force calibration of our design are presented. As a feasibility study, we incubated fibronectin coated 4.5 micrometers polystyrene beads with Swiss 3T3 mouse fibroblast cells. Under application around 250 pN of force per magnetic particle, we observed relative movement between attached magnetic and polystyrene beads to be on the order of 1 micrometers . Elastic, viscoelastic, and creeping responses of cell surfaces were observed. Our results are consistent with previous observations using similar magnetic techniques.© (2000) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Two-photon microscopy of endogenous fluorescence has proven to be a powerful method for quantification of tissue structure and biochemistry in 3D and with micron resolution. In particular, autofluorescence of mouse brain tissue provides potentially rich signatures for identifying brain anatomical regions and structures without the need for traditional Nissl or other exogenous staining. We quantitatively investigate the identities and distributions of endogneous fluorophores in whole mouse brains using Serial Two-Photon (STP) tomography. We vary the two-photon excitation wavelength over the range of 720 to 980 nm while simultaneously performing spectrally resolved imaging from 400 nm to 650 nm and report on background autofluorescence for use in segmenting, constructing brain atlases, and identifying specific neuronal regions.View Large Image | View Hi-Res Image | Download PowerPoint Slide

A β-cyclodextrin labeled with seven naphthoyloxy chromophores was studied by steady-state and time-resolved fluorescence spectroscopy in order to get information on the dynamics of energy hopping between chromophores. The steady-state fluorescence anisotropy was recorded as a function of excitation wavelength in a mixture of methanol and ethanol at 110 K (rigid glass). The fluorescence anisotropy decay was obtained under the same conditions by the multifrequency phase-modulation technique upon excitation at 290 nm. The data were analyzed and interpreted on the basis of a theoretical model involving a unique rate constant for energy hopping between nearest neighbors. In particular, this model predicts a long-time leveling-off of the emission anisotropy at 1/7th of the fundamental anisotropy, which is confirmed by both steady-state and time-resolved data and thus indicates that there is no preferred mutual orientation between the chromophores. As regards the rate of energy hopping, an average value of 2 × 1...

We present a microfabricated, continuous-flow, turbulent mixing device that can mix two or more fluids to complete homogeneity on the molecular scale in the microsecond range. The current design is compact, portable, relatively simple to fabricate, adaptable for various measurement techniques, and consumes small sample volumes. The entire mixing process is observable and we use this feature to characterize the dependence of the progress of mixing on the flow velocity. We present details of the mixer’s construction and optical data acquisition using fluorescence. Because the mixer is constructed using microfabrication technology, it is inexpensive and alterations are easy to explore. We show that the dependence of mixing times and pressure drop on the flow velocity agree well with theoretical expectations for turbulent pipe flow. This provides useful guidelines in predicting performance of this type of mixing device.

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7–5nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150–3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.

Fluorescence lifetime images are obtained with the laser scanning microscope using two methods: the time-correlated single-photon counting method and the frequency-domain method. In the same microscope system, we implement both methods. We perform a comparison of the performance of the two approaches in terms of signal-to-noise ratio (SNR) and the speed of data acquisition. While in our practical implementation the time-correlated single-photon counting technique provides a better SNR for low-intensity images, the frequency-domain method is faster and provides less distortion for bright Samples. © 2003 Society of Photo-Optical Instrumentation Engineers.