Jinhyo Ahn

Optical clearing based cellular-level 3D visualization of intact lymph node cortex.

Lymph node (LN) is an important immune organ that controls adaptive immune responses against foreign pathogens and abnormal cells. To facilitate efficient immune function, LN has highly organized 3D cellular structures, vascular and lymphatic system. Unfortunately, conventional histological analysis relying on thin-sliced tissue has limitations in 3D cellular analysis due to structural disruption and tissue loss in the processes of fixation and tissue slicing. Optical sectioning confocal microscopy has been utilized to analyze 3D structure of intact LN tissue without physical tissue slicing. However, light scattering within biological tissues limits the imaging depth only to superficial portion of LN cortex. Recently, optical clearing techniques have shown enhancement of imaging depth in various biological tissues, but their efficacy for LN are remained to be investigated. In this work, we established optical clearing procedure for LN and achieved 3D volumetric visualization of the whole cortex of LN. More than 4 times improvement in imaging depth was confirmed by using LN obtained from H2B-GFP/actin-DsRed double reporter transgenic mouse. With adoptive transfer of GFP expressing B cells and DsRed expressing T cells and fluorescent vascular labeling by anti-CD31 and anti-LYVE-1 antibody conjugates, we successfully visualized major cellular-level structures such as T-cell zone, B-cell follicle and germinal center. Further, we visualized the GFP expressing metastatic melanoma cell colony, vasculature and lymphatic vessels in the LN cortex.

Gradient index lens based combined two-photon microscopy and optical coherence tomography.

We report a miniaturized probe-based combined two-photon microscopy (TPM) and optical coherence tomography (OCT) system. This system is to study the colorectal cancer in mouse models by visualizing both cellular and structural information of the colon in 3D with TPM and OCT respectively. The probe consisted of gradient index (GRIN) lenses and a 90° reflecting prism at its distal end for side-viewing, and it was added onto an objective lens-based TPM and OCT system. The probe was 2.2 mm in diameter and 60 mm in length. TPM imaging was performed by raster scanning of the excitation focus at the imaging speed of 15.4 frames/s. OCT imaging was performed by combining the linear sample translation and probe rotation along its axis. This miniaturized probe based dual-modal system was characterized with tissue phantoms containing fluorescent microspheres, and applied to image mouse colonic tissues ex vivo as a demonstration. As OCT and TPM provided structural and cellular information of the tissues respectively, this probe based multi-modal imaging system can be helpful for in vivo studies of preclinical animal models such as mouse colonic tumorigenesis.

In vivo longitudinal cellular imaging of small intestine by side-view endomicroscopy

Visualization of cellular dynamics in the gastrointestinal tract of living mouse model to investigate the pathophysiology has been a long-pursuing goal. Especially, for chronic disease such as Crohns disease, a longitudinal observation of the luminal surface of the small intestine in the single mouse is highly desirable to investigate the complex pathogenesis in sequential time points. In this work, by utilizing a micro-GRIN lens based side-view endomicroscope integrated into a video-rate confocal microscopy system, we successfully performed minimally-invasive in vivo cellular-level visualization of various fluorescent cells and microvasculature in the small intestinal villi. Also, with a transgenic mouse universally expressing photoconvertible protein, Kaede, we demonstrated repetitive cellular-level confocal endoscopic visualization of same area in the small intestinal lumen of a single mouse, which revealed the continuous homeostatic renewal of the small intestinal epithelium.

Frequency selection rule for high definition and high frame rate Lissajous scanning

Lissajous microscanners are very attractive in compact laser scanning applications such as endomicroscopy or pro-projection display owing to high mechanical stability and low operating voltages. The scanning frequency serves as a critical factor for determining the scanning imaging quality. Here we report the selection rule of scanning frequencies that can realize high definition and high frame-rate (HDHF) full-repeated Lissajous scanning imaging. The fill factor (FF) monotonically increases with the total lobe number of a Lissajous curve, i.e., the sum of scanning frequencies divided by the great common divisor (GCD) of bi-axial scanning frequencies. The frames per second (FPS), called the pattern repeated rate or the frame rate, linearly increases with GCD. HDHF Lissajous scanning is achieved at the bi-axial scanning frequencies, where the GCD has the maximum value among various sets of the scanning frequencies satisfying the total lobe number for a target FF. Based on this selection rule, the experimental results clearly demonstrate that conventional Lissajous scanners substantially increase both FF and FPS by slightly modulating the scanning frequencies at near the resonance within the resonance bandwidth of a Lissajous scanner. This selection rule provides a new guideline for HDHF Lissajous scanning in compact laser scanning systems.

Fully packaged confocal endomicroscopic system using Lissajous fiber scanner for indocyanine green in-vivo imaging

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.

1457: CAPILLARY ENTRAPMENT OF MAC-1+ NEUTROPHIL DISTURBS PULMONARY MICROCIRCULATION IN SEPSIS-INDUCED ARDS

www.ccmjournal.org Critical Care Medicine • Volume 46 • Number 1 (Supplement) Learning Objectives: Ascorbic acid appears to be important for endothelial function and immune function. Ascorbid acid levels fall during critical illness. We hypothesized that considerable disruption of metabolic homeostasis would occur in critical illness relative to plasma ascorbic acid levels. Methods: We performed a metabolomics study on biorepository plasma samples collected from a single academic medical center on 90 adults with systemic inflammatory response syndrome or sepsis. We first generated metabolomic data using gas and liquid chromatography mass spectroscopy. We then utilized multivariable logistic regression to determine the association between plasma ascorbic acid and 28-day mortality. We performed fold change analysis based on false discovery rate adjusted p values to evaluate the distribution of individual metabolite concentrations relative to ascorbic acid levels. We followed this by partial least squaresdiscriminant analysis to identify individual metabolites that discriminated ascorbic acid levels. We then interrogated the entire metabolomics profile using pathway over-representation analysis to identify groups of metabolite pathways that were differential relative to ascorbic acid levels. Results: Ascorbic acid plasma levels were significantly lower in patients who died by 28 days (False Discovery Rate adjusted P = 0.043). Following adjustment for APACHE II score, renal function and sepsis, higher plasma ascorbic acid levels early in the ICU stay were associated with lower 28-day mortality [OR = 0.72 (95%CI 0.52–0.98);P = 0.039]. Metabolomic profiles significantly differed in critically ill patients relative to plasma ascorbic acid levels. In particular, decreased erythrulose (a metabolite of ascorbic acid) was the only strong predictor of low ascorbic acid levels. No metabolite pathways were significantly altered with regard to Ascorbic Acid levels. Conclusions: Low Ascorbic Acid levels early in severe critical illness are a robust predictor of 28day mortality. Differential metabolic profiles during critical illness exist according to Ascorbic Acid levels but limited to direct metabolites of Ascorbic Acid. Metabololite pathyways did not appear to be disrupted relative to differential Ascorbic Acid levels.

Nanoparticle-Assisted Transcutaneous Delivery of a Signal Transducer and Activator of Transcription 3-Inhibiting Peptide Ameliorates Psoriasis-like Skin Inflammation

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in psoriatic skin inflammation and acts as a key player in the pathogenesis and progression of this autoimmune disease. Although numerous inhibitors that intervene in STAT3-associated pathways have been tested, an effective, highly specific inhibitor of STAT3 has yet to be identified. Here, we evaluated the in vitro and in vivo biological activity and therapeutic efficacy of a high-affinity peptide specific for STAT3 (APTstat3) after topical treatment via intradermal and transcutaneous delivery. Using a preclinical model of psoriasis, we show that intradermal injection of APTstat3 tagged with a 9-arginine cell-penetrating peptide (APTstat3-9R) reduced disease progression and modulated psoriasis-related cytokine signaling through inhibition of STAT3 phosphorylation. Furthermore, by complexing APTstat3-9R with specific lipid formulations led to formation of discoidal lipid nanoparticles (DLNPs), we were able to achieve efficient skin penetration of the STAT3-inhibiting peptide after transcutaneous administration, thereby effectively inhibiting psoriatic skin inflammation. Collectively, these findings suggest that DLNP-assisted transcutaneous delivery of a STAT3-inhibiting peptide could be a promising strategy for treating psoriatic skin inflammation without causing adverse systemic events. Moreover, the DLNP system could be used for transdermal delivery of other therapeutic peptides.

Fully packaged video-rate confocal laser scanning endomicroscope using Lissajous fiber scanner

This paper reports a fully packaged confocal endomicroscope high resolution and high frame-rate (HRHF) Lissajous fiber scanning. The confocal endomicroscope features a resonant scanning fiber with ∼1kHz actuated by a piezoelectric tube (PZT). The Lissajous scanning with high resolution and high frame rate has been successfully achieved by using the selection rule of scanning frequency, i.e., strong correlation between the total lobe number of Lissajous images and the greatest common divisor (GCD) between two scanning frequencies. Our main results clearly demonstrate exceptional fill factor of 85 % at 10 Hz in frame rate. Besides, this fully packaged endomicroscopic catheter was further combined with a portable confocal microscopic system to obtain video-rate 2D reflectance as well as in-vivo mouse vascular imaging.

Mouse tissue imaging using real-time Lissajous confocal endomicroscopic system

We present mouse tissue imaging using real-time Lissajous confocal endomicroscopic system. The system consists of endomicroscopic catheter, confocal imaging system, and image processing module. The system obtained 2D fluorescence ex-vivo imaging results of mouse tissue.

In Vivo Cellular-level Real-time Pharmacokinetic Imaging of Free-form and Liposomal Indocyanine Green in Liver

Indocyanine green (ICG) is a near-infrared fluorophore approved for human use which has been widely used for various clinical applications. Despite the well-established clinical usage, our understanding about the microscopic in vivo pharmacokinetics of systemically administered ICG has been relatively limited. In this work, we successfully visualized real-time in vivo pharmacokinetic dynamics of the intravenously injected free-form and liposomal ICG in cellular resolution by utilizing a custom-built video-rate near infrared laser-scanning confocal microscopy system. Initial perfusion and clearance from blood stream, diffusion into perisinusoidal space, and subsequent absorption into hepatocyte were directly visualized in vivo. The quantification analysis utilizing the real-time image sequences revealed distinct dynamic in vivo pharmacokinetic behavior of free-form and liposomal ICG.