Micro-ischemic strokes, or microinfarcts, are small blood clots that obstruct cerebral blood flow to the brain. They are commonly observed in patients with dementia and have been linked to cognitive decline. However, the precise mechanisms underlying this association remain largely unknown. To advance our understanding of micro-strokes, it is imperative to observe real-time blood flow changes during the formation of microinfarcts. In this study, we successfully achieved in vivo real-time imaging of red blood cell (RBC) and dextran flow in a microinfarction model using a video-rate intravital confocal microscope. A cortical microinfarct was induced by photo-thrombosis in the cortex of a mouse following the intravenous injection of Rose Bengal, accompanied by a 5-second exposure to 7mW, 561nm light through a cranial window. Real-time imaging of blood flow allowed us to capture dynamic changes in RBC flow, along with changes in vessel diameter and cortical tissue volume after inducing the microinfarction. Initially, the obstruction resulted in reduced blood flow in the surrounding vessels and micro-vessels with minimal change in cortical volume. However, subsequent observations revealed an increase in cerebral volume, vessel thickness, and blood flow. These changes slightly decreased again afterward. Despite these fluctuations, no angiogenesis was observed. Further in-depth analysis of these complex microvascular changes from the acute stages of microinfarction to subsequent recovery could provide valuable insights into the pathophysiology of micro-ischemic strokes and offer clues for the development of novel therapeutic strategies.
KEYWORDS: Kidney, Crystals, Second harmonic generation, Mouse models, In vivo imaging, Blood, Biological imaging, Diseases and disorders, Two photon imaging, Autofluorescence
Chronic kidney disease (CKD) is one of the most prevalent kidney disorders with no distinct symptoms and gradual loss of kidney function over time. The underlying mechanism in the pathogenesis of CKD with various causes such as high blood pressure, diabetes, high cholesterol, and kidney infection is not well understood. Following up on the pathophysiological alterations, longitudinal in vivo cellular-level imaging during CKD progression can provide unique insights and valuable information for diagnosing and managing CKD. In this study, with two-photon kidney intravital imaging, we achieved a repetitive longitudinal visualization of the kidney in an adenine diet-induced CKD mouse model over 30 days. Interestingly, based on a second-harmonics generation signal, we clearly visualized and quantified the formation of 2,8-dihydroxyadenine (DHA) crystals over time in a label-free manner. The imaging results were well correlated with CKD progression monitored by a standard blood test of blood urea nitrogen (BUN) and cystatin C levels. This result suggests the potential of the label-free second-harmonics generation crystal imaging as a novel optical technique for in vivo CKD progression monitoring.
KEYWORDS: Imaging systems, Confocal microscopy, Scanners, In vivo imaging, Endomicroscopy, Microscopes, Indocyanine green, Luminescence, Field programmable gate arrays, Active optics
This work presents a fully packaged confocal endomicroscopic system using Lissajous fiber scanner for in-vivo imaging. The confocal endomicroscopic system consists of a scanning probe part, an optical part, and an electrical part. The scanning probe uses resonant Lissajous fiber scanner based on a piezoelectric tube. The scanner successfully achieves 10 frame rate with ~ 1 kHz scanning frequencies. The probe was fully packaged for waterproofing and disinfection of medical instruments into the outer diameter of 3.4 mm. The endomicroscopic system and successfully obtained 2D reflectance imaging results, human ex-vivo imaging results and a real-time in-vivo imaging results.
KEYWORDS: Retina, Angiography, Optical coherence tomography, Visualization, Laser coagulation, Pathogens, 3D modeling, Animal model studies, Pathophysiology, Drug development
To understand the pathogenesis of ophthalmic disease, utilizing small animal models such as mouse is necessary because of their ease of maintenance and availability. For identifying pathophysiology and drug development of retinal diseases in mouse model, optical coherence tomography angiography (OCTA) is promising imaging modality visualizing not only microstructure but also microvasculature. In this study, we serially imaged 3D structure and angiography of laser-induced choroidal neovascularization (CNV) in the mouse retina with/without anti-VEGF treatment. Also, the volume changes of CNV and avascular region in choroid layer are measured for identifying effects of anti-VEGF.
A lab-built high-speed OCTA prototype using the wavelength-swept laser centered at 1040 nm with 230 kHz A-scan rate acquired 3-D volumetric data consisted of 1024 x 1024 x 3 A-scans. The OCTA scanned 1.7 mm x 1.7 mm area around ONH. For obtaining angiography, amplitude decorrelation from 3 consecutive B-scans at each position was generated. Seven days after the laser photocoagulation at mouse retina for generation of the laser-induced CNV, intravitreal administration of Fc and VEGF-Trap was given in the therapeutic arm. The OCTA were performed at 6, 14, 21 and 35 days after laser photocoagulation. Vasculatures of inner retina, outer retina and choroid layers were separately visualized after RPE flattening and layer segmentation. To investigate therapeutic effects of anti-VEGF treatment, the relative area and volume of CNV in outer retina layer is measured. Also, total volume of avascular zone surrounding the laser injury site in choroid layer is also analyzed.
Lymph nodes (LN) are major checkpoints for circulating T lymphocytes to recognize foreign antigens collected from peripheral tissue. High endothelial venule (HEV) in LN facilitates the effective transmigration of circulating T lymphocytes from the blood into LN. There have been many efforts to visualize T lymphocytes trafficking across HEV to understand the underlying mechanism. However, due to insufficient spatiotemporal resolution and the lack of an in vivo labeling method, clear visualization of dynamic behaviors of rapidly flowing T lymphocytes in HEV and their transmigration have been difficult. In this work, we adapted a custom-designed video-rate laser scanning confocal microscopy system to track individual flowing T lymphocytes in the HEV in real time in vivo. We demonstrate that the HEVs in LN can be clearly identified in vivo with its distinctive cuboidal morphology of endothelial cells fluorescently labeled by intravenously injected anti-CD31 antibody conjugated with Alexa fluorophore. By visualizing the adaptively transferred T lymphocytes, we successfully analyzed dynamic flowing behaviors of T lymphocytes and their transendothelial migration while interacting with the endothelial cells in HEV.
The ability to conduct high-resolution fluorescence imaging in internal organs of small animal models in situ and over time can make a significant impact in biomedical research. Toward this goal, we developed a real-time confocal and multiphoton endoscopic imaging system. Using 1-mm-diameter endoscopes based on gradient index lenses, we demonstrate video-rate multicolor multimodal imaging with cellular resolution in live mice.
KEYWORDS: Raman spectroscopy, Optical amplifiers, Fiber amplifiers, Single mode fibers, Signal processing, Signal attenuation, Picosecond phenomena, Signal to noise ratio, Nonlinear optics, Phase shifts
Transmission systems employing Raman amplifier technology have to put up with much higher level of design complexities, when compared to conventional transmission lines with doped fiber optical amplifier. Even for the construction of a fundamental, basic building block - a unit of a fiber Raman amplifier (FRA), the designer have to struggle with the problems associated with the interactions between pump / signal waves mediated by Raman process, have to wander within the vast degrees of freedom given the choice of pumping directions / ratios, and have to contemplate with the wavelength dependent fiber loss / noise figure profiles. The problem further evolves into steps-higher, demanding and time-consuming one when extended to that of a system design employing Raman amplifiers. Optimizing OSNR and designing ultra-long haul links with best Q performances, while adjusting variables in the span length, Rayleigh penalty, pump noise, nonlinear penalty, dispersion and gain distribution is a problem which can be easily stated, but in reality is not a process which can be easily achieved. We present efficient, optimal design methods for Raman amplified WDM transmission links: 1. for the multi-channel gain flatness, 2. transient control under signal reconfigurations, and 3. for the estimation of optimum system Q value and corresponding link design parameters.
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