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Microfluidics in vitro assays recapitulate a blood vessel microenvironment using surface-immobilized agonists under biofluidic flows. However, these assays do not quantify intrathrombus mass and activities of adhesive platelets at the agonist margin and use fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a label-free multimodal quantitative imaging flow assay that combines rotating optical coherent scattering microscopy and quantitative phase microscopy. The combined imaging platform enables real-time evaluation of membrane fluctuations of adhesive-only platelets and total intrathrombus mass under physiological flow rates in vitro. We call this multimodal quantitative imaging flow assay coherent optical scattering and phase interferometry (COSI). COSI records intrathrombus mass to picogram accuracy and shape changes to a platelet membrane with high spatial-temporal resolution (0.4 μm/s) under physiological and pathophysiological fluid shear stress (1800 and 7500 s−1). With COSI, we generate an axial slice of 4 μm from the coverslip surface, approximately equivalent to the thickness of a single platelet, which permits nanoscale quantification of membrane fluctuation (activity) of adhesive platelets during initial adhesion, spreading, and recruitment into a developing thrombus (mass). Under fluid shear, pretreatment with a broad range metalloproteinase inhibitor (250 μM GM6001) blocked shedding of platelet adhesion receptors that shown elevated adhesive platelet activity at average of 42.1 μm/s and minimal change in intrathrombus mass.

Moldable, transparent polydimethylsiloxane (PDMS) elastomer microdevices enable a broad range of complex studies of three-dimensional cellular networks in their microenvironment in vitro. However, the uneven distribution of refractive index change, external to PDMS devices and internally in the sample chamber, creates a significant optical path difference (OPD) that distorts the light sheet beam and so restricts diffraction limited performance. We experimentally showed that an OPD of 120 μm results in the broadening of the lateral point spread function by over 4-fold. In this paper, we demonstrate steps to adapt a commercial inverted selective plane illumination microscope (iSPIM) and remove the OPD so as to achieve sub-micrometer imaging ranging from 0.6 ± 0.04 μm to 0.91 ± 0.03 μm of a fluorescence biological sample suspended in regular saline (RI ≈1.34) enclosed in 1.2 to 2 mm thick micromolded PDMS microdevices. We have proven that the removal of the OPD from the external PDMS layer by refractive index (RI) matching with a readily accessible, inexpensive sucrose solution is critical to achieve a >3-fold imaging resolution improvement. To monitor the RI matching process, a single-mode fiber (SMF) illuminator was integrated into the iSPIM. To remove the OPD inside the PDMS channel, we used an electrically tunable lens (ETL) that par-focuses the light sheet beam with the detection objective lens and so minimised axial distortions to attain sub-micrometer imaging resolution. We termed this new light sheet imaging protocol as modified inverted selective plane illumination microscopy (m-iSPIM). Using the high spatial–temporal 3D imaging of m-iSPIM, we experimentally captured single platelet (≈2 μm) recruitment to a platelet aggregate (22.5 μm × 22.5 μm × 6 μm) under flow at a 150 μm depth within a microfluidic channel. m-iSPIM paves the way for the application of light sheet imaging to a wide range of 3D biological models in microfluidic devices which recapitulate features of the physiological microenvironment and elucidate subcellular responses.

T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of &zeta, chain in a light controlled manner. We showed that spatial clustering of the &zeta, chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of &zeta, chain, Zap70, PLC&gamma, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate &zeta, chain phosphorylation upon &zeta, chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed &zeta, chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

Intensity shot noise in digital holograms distorts the quality of the phase images after phase retrieval, limiting the usefulness of quantitative phase microscopy (QPM) systems in long term live cell imaging. In this paper, we devise a hologram-to-hologram neural network, Holo-UNet, that restores high quality digital holograms under high shot noise conditions (sub-mW/cm2 intensities) at high acquisition rates (sub-milliseconds). In comparison to current phase recovery methods, Holo-UNet denoises the recorded hologram, and so prevents shot noise from propagating through the phase retrieval step that in turn adversely affects phase and intensity images. Holo-UNet was tested on 2 independent QPM systems without any adjustment to the hardware setting. In both cases, Holo-UNet outperformed existing phase recovery and block-matching techniques by ∼ 1.8 folds in phase fidelity as measured by SSIM. Holo-UNet is immediately applicable to a wide range of other high-speed interferometric phase imaging techniques. The network paves the way towards the expansion of high-speed low light QPM biological imaging with minimal dependence on hardware constraints.

T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of CD3ζ chains in a light controlled manner. We showed that spatial clustering of the CD3ζ intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of CD3ζ, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate CD3ζ phosphorylation upon CD3ζ clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed CD3ζ clusters at the plasma membrane. Taken together, our data suggest that clustering of the TCR can initialize proximal TCR signaling and thus constitute a biophysical mechanism of TCR triggering.

In this work, we propose a label-free COSI system to quantify morphological changes and platelet activity along non-patterned collagen fibers within millisecond in microfluidics channels under flow at sub-platelet imaging resolution.

In this work, we propose a label-free COSI system to investigating morphological changes and platelet-platelet interactions within a thrombus during embolism events to interrogate prothrombotic events within a microfluidics channel under flow.

Platelets are small anucleate cells that are essential for many biological processes including hemostasis, thrombosis, inflammation, innate immunity, tumor metastasis, and wound healing. Platelets circulate in the blood and in order to perform all of their biological roles, platelets must be able to arrest their movement at an appropriate site and time. Our knowledge of how platelets achieve this has expanded as our ability to visualize and quantify discreet platelet events has improved. Platelets are exquisitely sensitive to changes in blood flow parameters and so the visualization of rapid intricate platelet processes under conditions found in flowing blood provides a substantial challenge to the platelet imaging field. The platelet's size (~2 μm), rapid activation (milliseconds), and unsuitability for genetic manipulation, means that appropriate imaging tools are limited. However, with the application of modern imaging systems to study platelet function, our understanding of molecular events mediating platelet adhesion from a single-cell perspective, to platelet recruitment and activation, leading to thrombus (clot) formation has expanded dramatically. This review will discuss current platelet imaging techniques in vitro and in vivo, describing how the advancements in imaging have helped answer/expand on platelet biology with a particular focus on hemostasis. We will focus on platelet aggregation and thrombus formation, and how platelet imaging has enhanced our understanding of key events, highlighting the knowledge gained through the application of imaging modalities to experimental models in vitro and in vivo. Furthermore, we will review the limitations of current imaging techniques, and questions in thrombosis research that remain to be addressed. Finally, we will speculate how the same imaging advancements might be applied to the imaging of other vascular cell biological functions and visualization of dynamic cell-cell interactions.

Removal of complex aberrations at millisecond time scales over millimeters in distance in multiphoton laser scanning microscopy limits the total spatiotemporal imaging throughput for deep tissue imaging. Using a single low resolution deformable mirror and time multiplexing (TM) adaptive optics, we demonstrate video rate aberration correction (5 ms update rate for a single wavefront mask) for a complex heterogeneous distribution of refractive index differences through a depth of up to 1.1 mm and an extended imaging FOV of up to 0.8 mm, with up to 167% recovery of fluorescence intensity 335 µm from the center of the FOV. The proposed approach, termed raster adaptive optics (RAO), integrates image-based aberration retrieval and video rate removal of arbitrarily defined regions of dominant, spatially varied wavefronts. The extended FOV was achieved by demonstrating rapid recovery of up to 50 distinct wavefront masks at 500 ms update rates that increased imaging throughput by 2.3-fold. Because RAO only requires a single deformable mirror with image-based aberration retrieval, it can be directly implemented on a standard laser scanning multiphoton microscope.

The heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of l-tryptophan oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Here we demonstrate that IDO1 is a mammalian nitrite reductase capable of chemically reducing nitrite to nitric oxide (NO) under hypoxia. Ultraviolet-visible absorption and resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous-IDO1 protein (FeII-IDO1) with nitrite under anaerobic conditions resulted in the time-dependent formation of an FeII-nitrosyl IDO1 species, which was inhibited by substrate l-tryptophan, dependent on the concentration of nitrite or IDO1, and independent of the concentration of the reductant, dithionite. The bimolecular rate constant for IDO1 nitrite reductase activity was determined as 5.4 M-1 s-1 (pH 7.4, 23 °C), which was comparable to that measured for myoglobin (3.6 M-1 s-1; pH 7.4, 23 °C), an efficient and biologically important mammalian heme-based nitrite reductase. IDO1 nitrite reductase activity was pH-dependent but differed with myoglobin in that it showed a reduced proton dependency at pH >7. Electron paramagnetic resonance studies measuring NO production showed that the conventional IDO1 dioxygenase reducing cofactors, ascorbate and methylene blue, enhanced IDO1's nitrite reductase activity and the time- and IDO1 concentration-dependent release of NO in a manner inhibited by l-tryptophan or the IDO inhibitor 1-methyl-l-tryptophan. These data identify IDO1 as an efficient mammalian nitrite reductase that is capable of generating NO under anaerobic conditions. IDO1's nitrite reductase activity may have important implications for the enzyme's biological actions when expressed within hypoxic tissues.

Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.

The heme enzyme indoleamine 2,3-dioxygenase (IDO) is a key regulator of immune responses through catalyzing l-tryptophan (l-Trp) oxidation. Here, we show that hydrogen peroxide (H(2)O(2)) activates the peroxidase function of IDO to induce protein oxidation and inhibit dioxygenase activity. Exposure of IDO-expressing cells or recombinant human IDO (rIDO) to H(2)O(2) inhibited dioxygenase activity in a manner abrogated by l-Trp. Dioxygenase inhibition correlated with IDO-catalyzed H(2)O(2) consumption, compound I-mediated formation of protein-centered radicals, altered protein secondary structure, and opening of the distal heme pocket to promote nonproductive substrate binding; these changes were inhibited by l-Trp, the heme ligand cyanide, or free radical scavengers. Protection by l-Trp coincided with its oxidation into oxindolylalanine and kynurenine and the formation of a compound II-type ferryl-oxo heme. Physiological peroxidase substrates, ascorbate or tyrosine, enhanced rIDO-mediated H(2)O(2) consumption and attenuated H(2)O(2)-induced protein oxidation and dioxygenase inhibition. In the presence of H(2)O(2), rIDO catalytically consumed nitric oxide (NO) and utilized nitrite to promote 3-nitrotyrosine formation on IDO. The promotion of H(2)O(2) consumption by peroxidase substrates, NO consumption, and IDO nitration was inhibited by l-Trp. This study identifies IDO as a heme peroxidase that, in the absence of substrates, self-inactivates dioxygenase activity via compound I-initiated protein oxidation. l-Trp protects against dioxygenase inactivation by reacting with compound I and retarding compound II reduction to suppress peroxidase turnover. Peroxidase-mediated dioxygenase inactivation, NO consumption, or protein nitration may modulate the biological actions of IDO expressed in inflammatory tissues where the levels of H(2)O(2) and NO are elevated and l-Trp is low.

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a critical immune regulatory enzyme capable of signalling for immune suppression in diverse pathological settings including chronic inflammation, infection and cancer. During inflammation, IDO1 is expressed within activated innate immune cells that also produce the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) by NADPH oxidase (Nox) and myeloperoxidase (MPO), respectively. We recently reported that H2O2 inhibits recombinant IDO1 dioxygenase activity by engaging the enzyme’s heme peroxidase activity resulting in oxidative self-inactivation (JBC 2013;288:1548). To test if Nox-derived H2O2 or MPO-derived HOCl also control cellular IDO1 activity, we studied IFN-γ-stimulated human monocytes that co-express IDO1, Nox and MPO. Stimulation of monocytes with PMA increased cellular production of H2O2 and HOCl that coincided with the post-translational inhibition of IDO1 activity. The ability of PMA to inhibit IDO1 was abrogated by inhibition of Nox-derived H2O2 production with the PKC inhibitor Bisindolylmaleimide or the flavoprotein oxidase inhibitor diphenyleniodonium, but not by inhibition of MPO-derived HOCl with 4-aminobenzoic acid hydrazide. IFN-γ-stimulated human monocytes not only expressed the phagocytic Nox2 isoform but also Nox4. While selective Nox4 inhibition reduced cellular H2O2 production by ~50%, it did not reverse PMA’s inhibitory action on IDO1. Also, PMA did not inhibit IDO1 activity in IFN-γ-stimulated monocytes isolated from chronic granulomatous disease (CGD) patients expressing defective Nox2. These studies establish that Nox2-derived ROS control IDO1 activity in human innate immune cells and provide important new insights into the redox control of IDO1’s immune regulatory actions.

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the initial and rate limiting step of L-tryptophan (L-Trp) oxidation along the kynurenine pathway. IDO1 functions as a central immune regulatory enzyme with important implications for inflammation, infectious disease, allergic and autoimmune disorders, neuropathology and cancer. Here we identify IDO1 is a novel mammalian nitrite reductase that is capable of reducing nitrite to nitric oxide (NO) under hypoxia. Optical and Resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous IDO1 protein (FeII-IDO) with nitrite under anaerobic conditions resulted in the time-dependent formation of a FeII-nitrosyl IDO1 species that was dependent on nitrite and IDO1 protein concentration. The bimolecular rate constant for IDO1 nitrite reductase activity was 5.1 M-1 s-1, which was comparable to that measured under identical conditions for myoglobin (3.7 M-1 s-1), an efficient and biologically important mammalian heme nitrite reductase. IDO nitrite reductase activity was also pH dependent but differed with myoglobin in that it showed a reduced proton dependency at pH>7. Electron paramagnetic resonance studies measuring NO production from nitrite showed that the conventional IDO1 dioxygenase reducing co-factors, ascorbate plus methylene blue, or ascorbate alone, supported IDO1’s nitrite reductase activity and the time-dependent release of NO in a manner modulated by substrate L-Trp and the IDO1 inhibitors 1-methyl-L-Trp and 1-methyl-D-Trp. These data identify IDO1 as a novel mammalian heme-based nitrite reductase that is capable of generating NO under hypoxia. IDO1’s nitrite reductase activity may have important implications for the immune regulatory actions of the enzyme when expressed within tumours and infected tissues that are characterized by hypoxia.

We propose a raster scanning adaptive optics method that uses digital image segmentation and a low-resolution deformable mirror with a maximum of 50 wavefront masks, which removes spatially varying aberrations (both sample and lens) across a field of view of 0.8 mm at 500 ms.