Person: Evans, Conor
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Evans
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Conor
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Evans, Conor
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Publication Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM(Nature Publishing Group UK, 2018) Evers, Michael; Salma, Nunciada; Osseiran, Sam; Casper, Malte; Birngruber, Reginald; Evans, Conor; Manstein, DieterFluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and phasor analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest.Publication Abstract 126: Improving Post-Operative Monitoring of Autologous Breast Reconstruction: A Novel, Oxygen-Sensing Liquid Bandage First-in-Human Trial(Wolters Kluwer Health, 2018) Bucknor, Alexandra; Kamali-Sadeghian, Parisa; Maylar, Masoud; Chen, Austin; Marks, Haley; Roussakis, Emmanuel; Nowell, Nicholas H.; Evans, Conor; Lin, SamuelPublication An Integrin-Targeted, Highly Diffusive Construct for Photodynamic Therapy(Nature Publishing Group UK, 2017) Klein, Oliver J.; Yuan, Hushan; Nowell, Nicholas H.; Kaittanis, Charalambos; Josephson, Lee; Evans, ConorTargeted antineoplastic agents show great promise in the treatment of cancer, having the ability to impart cytotoxicity only to specific tumor types. However, these therapies do not experience uniform uptake throughout tumors, leading to sub-lethal cell killing that can impart treatment resistance, and cause problematic off-target effects. Here we demonstrate a photodynamic therapy construct that integrates both a cyclic RGD moiety for integrin-targeting, as well as a 5 kDa PEG chain that passivates the construct and enables its rapid diffusion throughout tumors. PEGylation of the photosensitizer construct was found to prevent photosensitizer aggregation, boost the generation of cytotoxic reactive radical species, and enable the rapid uptake of the construct into cells throughout large (>500 µm diameter) 3D tumor spheroids. Replacing the cyclic RGD with the generic RAD peptide led to the loss of cellular uptake in 3D culture, demonstrating the specificity of the construct. Photodynamic therapy with the construct was successful in inducing cytotoxicity, which could be competitively blocked by a tenfold concentration of free cyclic RGD. This construct is a first-of-its kind theranostic that may serve as a new approach in our growing therapeutic toolbox.Publication Comprehensive Evaluation of Peripheral Nerve Regeneration in the Acute Healing Phase Using Tissue Clearing and Optical Microscopy in a Rodent Model(Public Library of Science, 2014) Jung, Yookyung; Ng, Joanna H.; Keating, Cameron P.; Senthil-Kumar, Prabhu; Zhao, Jie; Randolph, Mark; Winograd, Jonathan; Evans, ConorPeripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.Publication Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning(Optical Society of America, 2012) de Groot, Mattijs; Evans, Conor; de Boer, Johannes F.We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 µm over a 500 µm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a three-dimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan.Publication Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine(Annual Reviews, 2008) Evans, Conor; Xie, XiaoliangCoherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique that is capable of real-time, nonperturbative examination of living cells and organisms based on molecular vibrational spectroscopy. Recent advances in detection schemes, understanding of contrast mechanisms, and developments of laser sources have enabled superb sensitivity and high time resolution. Emerging applications, such as metabolite and drug imaging and tumor identification, raise many exciting new possibilities for biology and medicine.Publication Longitudinal, 3D in vivo imaging of sebaceous glands by coherent anti-Stokes Raman scattering microscopy –normal function and response to cryotherapy(2014) Jung, Yookyung; Tam, Joshua; Jalian, H. Ray; Anderson, R. Rox; Evans, ConorSebaceous glands perform complex functions, and are centrally involved in the pathogenesis of acne vulgaris. Current techniques for studying sebaceous glands are mostly static in nature, whereas the gland’s main function – excretion of sebum via the holocrine mechanism – can only be evaluated over time. We present a longitudinal, real-time alternative – the in vivo, label-free imaging of sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which is used to selectively visualize lipids. In mouse ears, CARS microscopy revealed dynamic changes in sebaceous glands during the holocrine secretion process, as well as in response to damage to the glands caused by cooling. Detailed gland structure, plus the active migration of individual sebocytes and cohorts of sebocytes were measured. Cooling produced characteristic changes in sebocyte structure and migration. This study demonstrates that CARS microscopy is a promising tool for studying the sebaceous gland and its associated disorders in three-dimensions in vivo.Publication In vivo coherent Raman imaging of the melanomagenesis-associated pigment pheomelanin(Nature Publishing Group, 2016) Wang, Hequn; Osseiran, Sam; Igras, Vivien; Nichols, Alexander J.; Roider, Elisabeth; Pruessner, Joachim; Tsao, Hensin; Fisher, David; Evans, ConorMelanoma is the most deadly form of skin cancer with a yearly global incidence over 232,000 patients. Individuals with fair skin and red hair exhibit the highest risk for developing melanoma, with evidence suggesting the red/blond pigment known as pheomelanin may elevate melanoma risk through both UV radiation-dependent and -independent mechanisms. Although the ability to identify, characterize, and monitor pheomelanin within skin is vital for improving our understanding of the underlying biology of these lesions, no tools exist for real-time, in vivo detection of the pigment. Here we show that the distribution of pheomelanin in cells and tissues can be visually characterized non-destructively and noninvasively in vivo with coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. We validated our CARS imaging strategy in vitro to in vivo with synthetic pheomelanin, isolated melanocytes, and the Mc1re/e, red-haired mouse model. Nests of pheomelanotic melanocytes were observed in the red-haired animals, but not in the genetically matched Mc1re/e; Tyrc/c (“albino-red-haired”) mice. Importantly, samples from human amelanotic melanomas subjected to CARS imaging exhibited strong pheomelanotic signals. This is the first time, to our knowledge, that pheomelanin has been visualized and spatially localized in melanocytes, skin, and human amelanotic melanomas.Publication Transient alterations of cutaneous sensory nerve function by non-invasive cryolipolysis(2015) Garibyan, Lilit; Cornelissen, Laura; Sipprell, William; Pruessner, Joachim; Elmariah, Sarina; Luo, Tuan; Lerner, Ethan; Jung, Yookyung; Evans, Conor; Zurakowski, David; Berde, Charles; Rox Anderson, R.Cryolipolysis is a non-invasive, skin cooling treatment for local fat reduction that causes prolonged hypoesthesia over the treated area. We tested the hypothesis that cryolipolysis can attenuate nociception of a range of sensory stimuli, including stimuli that evoke itch. The effects of cryolipolysis on sensory phenomena were evaluated by quantitative sensory testing (QST) in 11 healthy subjects over a period of 56 days. Mechanical and thermal pain thresholds were measured on treated and contralateral untreated (control) flanks. Itch duration was evaluated following histamine iontophoresis. Unmyelinated epidermal nerve fiber and myelinated dermal nerve fiber densities were quantified in skin biopsies from six subjects. Cryolipolysis produced a marked decrease in mechanical and thermal pain sensitivity. Hyposensitivity started between two to seven days after cryolipolysis and persisted for at least thirty-five days post-treatment. Skin biopsies revealed that cryolipolysis decreased epidermal nerve fiber density as well as dermal myelinated nerve fiber density, which persisted throughout the study. In conclusion, cryolipolysis causes significant and prolonged decreases in cutaneous sensitivity. Our data suggest that controlled skin cooling to specifically target cutaneous nerve fibers has the potential to be useful for prolonged relief of cutaneous pain and might have a use as a research tool to isolate and study cutaneous itch-sensing nerves in human skin.Publication Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT(Nature Publishing Group, 2016) Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, ConorThree-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.