Jenny M. Tam

An imaging fiber-based optical tweezer array for microparticle array assembly

In this letter, we present a method to generate and regenerate arrays of microspheres by optically trapping through optical imaging fiber bundles. In this method, a laser beam is coupled into the proximal end of an optical fiber bundle, and the light energy is distributed across the face of the fiber. Each illuminated individual fiber in the array propagates light to the distal face of the bundle, where light focusing elements at the end of each fiber focus the laser light and form optical traps. These optical traps are capable of capturing and arraying microspheres in parallel. The number of optical traps is determined by the number of fibers in the optical fiber bundle and is capable of creating a dense array (∼5×104 traps/mm2) of optical tweezers.

Dectin-1 Activation Controls Maturation of β-1,3-Glucan-containing Phagosomes

Background: Dectin-1 is able to recognize and phagocytose the fungal carbohydrate, β-1,3-glucan, but its contribution to phagosomal maturation has not been explored. Results: Dectin-1-dependent Syk activation promotes phagolysosomal fusion and acidification. Conclusion: Dectin-1-dependent Syk-activation permits egress of early phagosomes to mature phagolysosomes. Significance: The surface recognition receptor, Dectin-1 shapes anti-fungal responses by controlling fungal phagosome maturation. Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.

Dectin-1–Dependent LC3 Recruitment to Phagosomes Enhances Fungicidal Activity in Macrophages

Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.

Toll-Like Receptor 9 Modulates Macrophage Antifungal Effector Function during Innate Recognition of Candida albicans and Saccharomyces cerevisiae†

ABSTRACT Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens. Macrophages sample the phagosomal content and orchestrate the innate immune response. Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA and is activated by fungal DNA. Here we demonstrate that specific triggering of TLR9 recruitment to the macrophage phagosomal membrane is a conserved feature of fungi of distinct phylogenetic origins, including Candida albicans, Saccharomyces cerevisiae, Malassezia furfur, and Cryptococcus neoformans. The capacity to trigger phagosomal TLR9 recruitment was not affected by a loss of fungal viability or cell wall integrity. TLR9 deficiency has been linked to increased resistance to murine candidiasis and to restriction of fungal growth in vivo. Macrophages lacking TLR9 demonstrate a comparable capacity for phagocytosis and normal phagosomal maturation compared to wild-type macrophages. We now show that TLR9 deficiency increases macrophage tumor necrosis factor alpha (TNF-α) production in response to C. albicans and S. cerevisiae, independent of yeast viability. The increase in TNF-α production was reversible by functional complementation of the TLR9 gene, confirming that TLR9 was responsible for negative modulation of the cytokine response. Consistently, TLR9 deficiency enhanced the macrophage effector response by increasing macrophage nitric oxide production. Moreover, microbicidal activity against C. albicans and S. cerevisiae was more efficient in TLR9 knockout (TLR9KO) macrophages than in wild-type macrophages. In conclusion, our data demonstrate that TLR9 is compartmentalized selectively to fungal phagosomes and negatively modulates macrophage antifungal effector functions. Our data support a model in which orchestration of antifungal innate immunity involves a complex interplay of fungal ligand combinations, host cell machinery rearrangements, and TLR cooperation and antagonism.

Parallel microparticle manipulation using an imaging fiber-bundle-based optical tweezer array and a digital micromirror device

In this letter, the authors present a method to selectively capture and release microparticles using an imaging fiber-bundle-based optical tweezer array system. By integrating a digital micromirror device into a tweezer array system, patterns of optical traps can be generated on the distal face of a fiber bundle. A 2×2 array of traps was created to capture, release, and recapture microparticles in solution. This straightforward process enables individual control of the size, shape, and position of each trap in the array.

DNA detection on ultrahigh-density optical fiber-based nanoarrays

Nanoarrays for DNA detection were fabricated on etched nanofiber bundles based on recently developed techniques for microscale arrays. Two different-sized nanoarrays were created: one with 700 nm feature sizes and a 1 microm center-to-center pitch (approximately 1x10(6) array elements/mm(2)) and one with 300 nm feature sizes and a 500 nm center-to-center pitch (4.6x10(6) array elements/mm(2)). A random, multiplexed array composed of oligonucleotide-functionalized nanospheres was constructed and used for parallel detection and analysis of fluorescently labeled DNA targets. We have used these arrays to detect a variety of target sequences including Bacillus thuringiensis kurstaki and vaccina virus sequences, two potential biowarfare agents, as well as interleukin-2 sequences, an immune system modulator that has been used for the diagnosis of HIV.

Fabrication and optical characterization of imaging fiber-based nanoarrays

In this paper, we present a technique for fabricating arrays containing a density at least 90 times higher than previously published. Specifically, we discuss the fabrication of two imaging fiber-based nanoarrays, one with 700nm features, another with 300nm features. With arrays containing up to 4.5x10(6) array elements/mm(2), these nanoarrays have an ultra-high packing density. A straightforward etching protocol is used to create nanowells into which beads can be deposited. These beads comprise the sensing elements of the nanoarray. Deposition of the nanobeads into the nanowells using two techniques is described. The surface characteristics of the etched arrays are examined with atomic force microscopy and scanning electron microscopy. Fluorescence microscopy was used to observe the arrays. The 300nm array features and the 500nm center-to-center distance approach the minimum feature sizes viewable using conventional light microscopy.

Improved In Vivo Whole-Animal Detection Limits of Green Fluorescent Protein-Expressing Tumor Lines by Spectral Fluorescence Imaging

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 × 103 cells for spectral imaging versus 1 × 106 cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.

Quantitative endovascular fluorescence-based molecular imaging through blood of arterial wall inflammation.

PURPOSE To evaluate an author-developed normalization algorithm for quantitative imaging of optical molecular probes through blood and to assess, in the rat aorta after focal aortic injury, the feasibility of measuring protease activity by using this method. MATERIALS AND METHODS This study was performed according to a protocol approved by the institutional animal care committee. A Monte Carlo simulation was used to determine the pair of near-infrared (NIR) dyes that was best suited for the normalization algorithm. The authors tested the correction method in vitro and in vivo by injecting free dye mixtures intramurally in the aortas of four rats. The potential clinical utility was then evaluated by applying the method to the endovascular measurement of protease activity in a rat model of focal aortic injury. RESULTS When the Monte Carlo simulation was used in the normalization algorithm, it was predicted that the intensities of signals from two NIR dyes would vary +/-3% across 1 mm of blood compared with the intensity of the raw fluorochrome signal, which would vary +/-60%. This result was validated in vitro. Endovascular imaging of free dye collections revealed that clinically relevant, uncontrollable differences in the amount of blood intervening between the imaging catheter and the dye collection precipitated dramatic variations in raw NIR fluorescence. However, use of the correction method resolved these variations such that the measured signal intensity correlated well with the different dye concentrations in the different animals. Moreover, endovascular imaging of the focal aortic injury model enabled successful measurement of enzyme activity in the walls of the rat aortas. CONCLUSION The authors implemented a correction method for quantitative real-time endovascular imaging of fluorescence that enables one to resolve the attenuating effects of blood on NIR signal.

Integrated genomics of crohn's disease risk variant identifies a role for CLEC12A in antibacterial autophagy

Summary The polymorphism ATG16L1 T300A, associated with increased risk of Crohn’s disease, impairs pathogen defense mechanisms including selective autophagy, but specific pathway interactions altered by the risk allele remain unknown. Here, we use perturbational profiling of human peripheral blood cells to reveal that CLEC12A is regulated in an ATG16L1-T300A-dependent manner. Antibacterial autophagy is impaired in CLEC12A-deficient cells, and this effect is exacerbated in the presence of the ATG16L1∗300A risk allele. Clec12a−/− mice are more susceptible to Salmonella infection, supporting a role for CLEC12A in antibacterial defense pathways in vivo. CLEC12A is recruited to sites of bacterial entry, bacteria-autophagosome complexes, and sites of sterile membrane damage. Integrated genomics identified a functional interaction between CLEC12A and an E3-ubiquitin ligase complex that functions in antibacterial autophagy. These data identify CLEC12A as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease.