Soil is a highly scattering media that inhibits imaging of plant-microbial-mineral interactions that are essential to plant health and soil carbon sequestration. However, wavefront shaping can be used to focus light through or even deep inside highly scattering objects. In this work, we seek to overcome the fundamental challenges of imaging through soil minerals by developing a custom wavefront shaping method for a multiphoton microscope. We use the adaptive stochastic parallel gradient descent optimization algorithm combined with Hadamard basis to correct the aberration and the scattering in order to focus through the soil.
Soil is a highly scattering media that inhibits imaging of plant-microbial-mineral interactions that are essential to plant health and soil carbon sequestration. In this work, we seek to overcome the fundamental challenges of imaging through soil minerals by developing a custom wavefront sensor-less adaptive optics (AO) system for a multiphoton microscope. We are using a combined experimental and modeling approach, characterizing mineral optical characteristics with scatterometry, modeling the wavefront distortion and the image quality degradation after imaging through the soil medium, simulating the image quality improvement with AO correction, and experimentally testing our models with a stand-alone AO testbed.
We have developed a novel multiphoton nonlinear microscopy with a highly integrated optical imaging system that offers numerous label-free techniques including two-photon excited fluorescence, second-harmonic generation, third-harmonic generation, fluorescence lifetime imaging, and spectral focusing coherent anti-Stokes Raman scattering in one platform. We have applied our system to investigate plant-microbe-mineral interactions in the rhizosphere. The system provides time efficient monitoring of the rhizosphere, offering an array of simultaneous biomolecular information without staining, three-dimensional sub-micron resolution with deeper penetration , and less photodamage. We believe that multiphoton nonlinear optical microscopy will become a valuable imaging tool in the rhizosphere and soil mineral sciences.
Pre-clinical toxicology is a statutory requirement of drug development and plays a significant role in reducing attrition in drug discovery. Histopathology and indirect methods such as measurement of toxicity-associated systemic markers in blood or urine samples are the state-of-the-art techniques for toxicity evaluation. Further improvements over these conventional techniques are needed to detect signs of drug-induced toxicity at earlier stages with higher sensitivity and specificity. Multiphoton nonlinear imaging techniques such as two-/three-photon microscopy (2PF/3PF), fluorescence lifetime imaging microscopy (FLIM), second/third harmonic generation (SHG/THG) and coherent anti-Stokes Raman scattering (CARS) microscopy can extract complimentary structural and metabolic information of the target tissue in a label-free manner. In this study, we investigated the capability of a multimodal multiphoton microscopy technique (2PF/3PF/SHG/THG/FLIM/CARS) for detecting both functional and structural changes associated with drug-induced toxicity. Cisplatin, a platinum-based chemotherapy drug, is a cytotoxic agent used to treat many types of cancers. Common side effects of Cisplatin include nephrotoxicity and gonadal dysfunction. We obtained multimodal optical images of organs such as kidney, liver, and testis harvested from mice treated with a single dose of Cisplatin (3mg/kg) by intraperitoneal injection. A control group was treated with 0.9% saline. Structural and metabolic biomarkers related to Cisplatin-induced toxicity were identified and characterized from these multimodal optical images obtained ex vivo. The preliminary results suggest that it may be possible to develop a novel platform for drug toxicity identification and assessment based on multimodal nonlinear optical imaging techniques.
Multiphoton microscopy uses ultrafast nonlinear light-matter interactions to generate signal contrast from biological samples. The imaging of tissue from various organs plays an important role for a better understanding of cellular processes within their microenvironment and helps to reveal mechanisms of cellular changes in tissues during disease processes. Most tissue imaging studies by the pharmaceutical industry or by pathologists have typically been performed using harvested and sectioned tissue from organs to investigate drug toxicity or disease-related changes. However, immediately following biopsy, tissues begin to degrade due to cell necrosis and apoptosis, and substantial information is lost during the process. We demonstrate tissue degradation monitoring at different time points after tissue excision by using our label-free multimodal multiphoton imaging system which integrates SHG, TPEF, FLIM, and CARS in one platform. We examined whole organs and tissues harvested from mice, including kidney, liver, pancreas, and brain, and immersed each in several different media including saline, Euro-Collins solution, UW solution, HTK, and formalin. We collected time-lapse images from each sample and compared rates of cell degradation, tissue structure changes, and variations in optical properties including the intensities of NADH and FAD, the metabolic redox ratio, and FLIM of free/bound NADH. As a result, we quantified rates of degradation and metabolic changes associated with the preservation methods based on these label-free optical properties. Therefore, these results can be used as reference values for most ex vivo tissue research that relies on tissue and cell viability.
A biological sample consists of a variety of complex biomolecules, and fluorescence microscopy enables visualization of specific molecules at the sub-cellular level. However, these fluorescence techniques require certain fluorescence dyes to label the sample, and the fluorophores raise serious problems such as photo toxicity and photobleaching which could affect biological functionality in living systems. Advanced label-free optical imaging techniques based on nonlinear optical phenomena overcome these limitations of fluorescence microscopy. We have developed a novel label-free multimodal multiphoton nonlinear optical imaging system based on a near-IR femtosecond laser with photonic crystal fiber and pulse shaper. This highly integrated system offers numerous label-free techniques including third harmonic generation, three-photon excited fluorescence, second harmonic generation, two-photon excited fluorescence, fluorescence lifetime imaging, and broadband coherent anti-Stokes Raman scattering microspectroscopy in one platform. All of the nonlinear signals are spectrally separated by dichroic filters and simultaneously measured by photomultiplier tubes. Moreover, this system includes phase-variance optical coherence tomography as well to enable vascular imaging. We have applied our system to investigate processes in numerous biological samples. Our imaging technique is highly integrated and time efficient to generate big data, offering an array of biomolecular information at one time without staining, three-dimensional sub-micron resolution with deeper penetration, and less photodamage. The big data output from this system is analyzed by multivariate analysis such as principal component analysis and hierarchical cluster analysis. Therefore, this novel technology and methodology will have a great impact on fast in vivo label-free biomedical imaging as a big data generator.
Toxicology of the male reproductive system has received increased interest in recent years partly fueled by the growing reports of falling sperm counts and rising reproductive disorders in the human population. Testicular toxicity (TT) in pharmaceutical development is a challenging issue due to the lack of simple and robust screening methods. Currently, histopathologic examination and hormonal evaluation are the commonly used methods to assess TT. Improved biomarker or screening platforms that would allow identification of TT at an earlier stage can have a significant impact on the safety evaluation of pharmaceutical candidates. We investigated the potential of label-free optical nonlinear imaging technologies such as fluorescence lifetime imaging microscopy (FLIM), multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) microscopy to identify novel biomarkers for effective detection of TT. In this study, testicular damage was induced in rats by intraperitoneal injection with 3 mg/kg cisplatin, a chemotherapy drug. Multimodal optical images were obtained from the fixed, unstained testicular tissue sections of untreated and treated rats using a custom-built near-infrared multiphoton imaging system. Structural and biochemical parameters extracted from these images were compared between both groups to identify abnormal features associated with TT in the treated group. By analyzing the complimentary information obtained using these label-free optical imaging technologies, it may be possible to develop a novel platform for evaluation of TT in safety assessment of pharmaceuticals on reproduction and fertility, which reveal these changes at the molecular level and allow observation of these changes at an earlier time point than available today.
Approximately 29 million Americans have diabetes, and 86 million are living with prediabetes, increasing the risk of developing type 2 diabetes. Complications of wound healing in diabetic patients represent a significant health problem. Impaired diabetic wound healing is characterized by reduced collagen production and diminished angiogenesis. During the proliferative phase of wound healing, the injured tissue undergoes angiogenesis, re-epithelialization, and fibroplasia. Monitoring the development of new blood vessels, metabolic changes, and collagen deposition, is critical to elucidate the process of diabetic wound healing and to improve the development of therapeutic drugs. This study employs a custom-built multimodal microscope where Optical Coherence Tomography Angiography (OCTA) is used for studying neovascularization, Fluorescence Lifetime Imaging Microscopy (FLIM) for NADH/FAD assessment, Second Harmonic Generation (SHG) microscopy for analyzing collagen deposition, and Coherent anti-Stoke’s Raman Scattering (CARS) microscopy for visualizing water/lipid distribution, all together to non-invasively follow closure of a skin wound in healthy diabetic (db/db) mice treated with placebo and angiogenesis-promoting topical formulation (GlaxoSmithKline). The (db/db) mouse model presents hyperglycemia, obesity, and delayed wound healing that is pathologically similar to human type 2 diabetes mellitus. In this ongoing study, the animals are treated once daily for 14 days after wounding. Images of the wound and surrounding areas are taken at different time points for 28 days. In this experiment, the wound healing process is investigated to gain deeper understanding of the drug mechanism. The capability to non-invasively monitor wound healing mechanisms can become a valuable tool in development of new drug compounds for diabetic wound care.
We developed multimodal multiphoton microspectroscopy using a small-diameter probe with gradient-index lenses and applied it to unstained Alzheimer’s disease (AD) brain samples. Our system maintained the image quality and spatial resolution of images obtained using an objective lens of similar numerical aperture. Multicolor images of AD brain samples were obtained simultaneously by integrating two-photon excited fluorescence and second-harmonic generation on a coherent anti-Stokes Raman scattering (CARS) microendoscope platform. Measurements of two hippocampal regions, the cornus ammonis-1 and dentate gyrus, revealed more lipids, amyloid fibers, and collagen in the AD samples than in the normal samples. Normal and AD brains were clearly distinguished by a large spectral difference and quantitative analysis of the CH mode using CARS microendoscope spectroscopy. We expect this system to be an important diagnosis tool in AD research.
A multimodal multiphoton nonlinear optical (NLO) microspectroscopy imaging system was developed using a femtosecond laser and a photonic crystal fiber. Coherent anti-Stokes Raman scattering (CARS) microspectroscopy was combined with two-photon excitation fluorescence and second-harmonic generation microscopy in one platform and the system was applied to diagnose liver fibrosis. Normal and liver fibrosis tissues were clearly distinguished with the great difference from CARS spectra as well as multimodal multiphoton NLO images. We expect the system to be a rapid diagnosis tool for liver fibrosis at tissue level with label-free imaging of significant biochemical components.
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