Micro-capillary and individual MHC-Class II:GFP+ cells are visualized by novel side-view endomicroscopy, Nature Methods, 7, pp. 303-305, Apr. 2010
In vivo visualization of B16 melanoma in skin
Highly curled angiogeneic vasculature and infiltrated MHC-Class II:GFP+ cells including macrophages around B16 melanoma are visualized by custom-built in vivo microscopy setup.
Advances in these novel visualization technologies have allowed us to have a glimpse of numerous exciting processes those never have directly seen in vivo such as gene expression, regulation, protein activity, drug delivery, systemic cell trafficking / interaction, physiological response to external stimuli in the natural in vivo micro-environment. We're on the verge of a new era of micro-nano-scale in vivo visualization technologies as a novel tool for basic and translational biomedical research as well as a novel clinical imaging modality for diagnosis and monitoring for human care. Only with extensive interactions between multiple disciplines of physics, chemistry, biology, medicine and engineering, these visions can be accomplished.
Laser scanning microscopy
Customized video-rate laser scanning system with multiple laser lines.
Advanced In Vivo Cellular Imaging System
♦ Multi-modality in vivo laser scanning endomicroscopy
♦ In situ and in vivo endomicroscopic visualization of viscera and thoracic organs in animal
♦ In vivo nano-scale visualization / In vivo deep-tissue 3D visualization
♦ Label-free molecular profile visualization / High-throughput 3D visualization
Systemic Cellular Visualization of Animal Model for Human Disease
♦ Visualization of dynamic lymphocyte trafficking in secondary lymphoid organs
♦ Monitoring of neural signaling at the cortex and hippocampus in brain
♦ Cellular visualization of the metastasized cancer micronodules at brain and liver
High-speed, Nano-scale Visualization of Organic and Inorganic Materials
♦ Ultrafast time-resolved visualization of nano-scale phenomena in advanced photonic material
In Vivo Visualization of Circulating T Lymphocytes in Lymph Node
Lymph nodes (LNs) distributed over whole body are major checkpoints for circulating T lymphocytes to recognize foreign antigens. High endothelial venules (HEVs) in LN facilitate effective recruitment of circulating T lymphocytes from the blood into the LN. Highly dynamic behaviors of rapidly flowing lymphocytes in HEV and their transendothelial/perivascular migration have not been clearly visualized due to an insufficient spatiotemporal imaging resolution and a lack of appropriate in vivo labeling method of HEV-endothelial cells and perivascular region.
In this work, we adapted a custom-design video-rate triple-color laser scanning confocal microscopy system to track rapidly flowing T lymphocytes in HEV in real time in vivo. HEVs in LN were clearly identified in vivo with its distinctive cuboidal morphology of endothelial cells fluorescently labeled by intravenous injection of Alexa488-conjugated anti-CD31 antibody (green). By visualizing the adaptively transferred T lymphocytes and red blood cells (RBCs) labeled with CMTMR (red) and DiD (NIR) respectively, we successfully analyzed flowing behaviors of T lymphocytes in comparison with RBCs in HEVs. In addition, for the first time to our knowledge, the paracellular transendothelial migration of T lymphocytes squeezing in between cuboidal-shaped endothelial cells of HEV was clearly visualized in vivo. After the transenothelial migration, the T lymphocytes searched appropriate exit site of perivascular channels surrounded by fibro-reticular cells labeled with Alexa647-conjugated anti-ER-TR7 antibody (NIR).
In vivo high-spatiotemporal resolution tracking of flowing RBCs and T cells. Colormap visualization of velocity
• K. Choe, et. al., Journal of Biomedical Optics 18(3):036005 (2013)
• K. Choe, et. al., SPIE Photonics West BiOS '2014, San Francisco, USA (2014)
In Vivo Analysis of Cellular Effects by THz Wave Radiation
The recent development of THz sources in a wide range of THz frequencies and power levels has led to greatly increased interest in potential biomedical applications such as cancer and burn wound diagnosis. However, despite its importance in realizing THz wave based applications, our knowledge of how THz wave irradiation can affect a live tissue at the cellular level is very limited. In this study, an acute inflammatory response caused by pulsed THz wave irradiation on the skin of a live mouse was analyzed at the cellular level using intravital laser-scanning confocal microscopy. Pulsed THz wave (2.7 THz, 4 μs pulsewidth, 61.4 μJ per pulse, 3Hz repetition), generated using compact FEL, was used to irradiate an anesthetized mouse’s ear skin with an average power of 260 mW/cm² for 30 minutes using a high-precision focused THz wave irradiation setup. In contrast to in vitro analysis using cultured cells at similar power levels of CW THz wave irradiation, no temperature change at the surface of the ear skin was observed when skin was examined with an IR camera. To monitor any potential inflammatory response, resident neutrophils in the same area of ear skin were repeatedly visualized before and after THz wave irradiation using a custom-built laser-scanning confocal microscopy system optimized for in vivo visualization. While non-irradiated control skin area showed no changes in the number of resident neutrophils, a massive recruitment of newly infiltrated neutrophils was observed in the THz wave irradiated skin area after 6 hours, which suggests an induction of acute inflammatory response by the pulsed THz wave irradiation on the skin via a non-thermal process.
In Vivo Quantification of Circulating Tumor Cell (CTC)
The number of circulating tumor cells (CTCs) in blood of cancer patients is a potentially useful indicator for precise monitoring of cancer metastasis, evaluation of cancer treatment and even early detection of recurrent cancer. Currently, the major strategy for CTC quantitation is using microfluidic chip isolating CTCs from drawn blood sample. However, this ex vivo quantitation of rare CTCs, one CTC per one billion hematologic cells, from the limited volume of blood sample is fundamentally challenging and suffers extremely low sensitivity. Direct in vivo quantitation of CTCs in blood circulation, potentially from whole body blood, is an attractive approach to increase sensitivity. In this study, we implemented custom-design video-rate confocal microscopy system based on fast-rotating polygonal mirror. The system could acquire images of 512x512 pixels at 30 frames/sec, which allowed us direct imaging of fast-flowing individual cells in great saphenous vein (GSV) of mouse model in vivo. After intravenous injection of CT26 colorectal cancer cells and RBC labeled with CFSE (green) and DiD (NIR) respectively, we clearly imaged fast circulating CT26 and RBC at GSV. The number of flowing CT26 rapidly decreased below 10% of initially detected number at 3 minutes after the injection, while the number of flowing RBC remained at initially detected number over 60 minutes. From the pre-determined total number of injected RBCs, we could calculate a relative calibration factor and estimated circulating CTC in whole body blood in vivo.
Comprehensive 3D Visualization of Biological Tissue with Optical Clearing
Lymph node is an important immune organ where foreign pathogens are recognized and adaptive immune respons is initiated. Complex 3D cellular network of immune cells such as T, B cells and dendritic cells in conjunction with specialized lymphatic and vascular network are established. Unfortunately, the conventional histological analysis has critical limitations in 3D cellular network analysis due to structural disruption by chemical fixation and substantial tissue loss in slicing. However, light scattering within biological samples limits the imaging depth to only superficial portion of LN cortex. Herein, we applied optical clearing technique and custom-built high-power laser-scanning confocal microscope to visualize cellular network in whole cortex of intact LN. To identify immune cellular network inside LN, we adaptively transferred many subtypes of immune cells expressing various fluorescent proteins. Also, we utilized anti-CD31- or anti-LYVE1-antibody conjugated with NIR fluorophore to label vascular or lymphatic network. From the optically cleared LN, we successfully achieved 3D volumetric visualization of whole cortex of LN, which revealed major cellular structures such as T-cell zone, B-cell follicle, HEV, lymphatic sinus and germinal center.
Intravital images of whole PLN (a) and HEVs (b, c). T cells (red), Vascular endothelial cells (green) in HEVs
Circulating tumor cell imaged in great saphenous vein.
3D visualization of lymph node after optical clearing.
B cells (green, GFP), T cells (red, DsRed).
• Y. Hwang, et. al., Optics Express 22(10):11465-11475 (2014)
• Y. Hwang, et. al., SPIE Photonics West BiOS '2014, San Francisco, USA (2014)
High precision THz wave irradiation setup for live mouse model.
Infiltration of neutrophil at the THz wave irradiated skin.
• H. Seo, et. al. Biomedical Optics Express,6(6):2158-2167 (2015)
• H. Seo, et. al. SPIE Photonics West BiOS '2014, San Francisco, USA (2014)
Animal model, particularly mouse, has been an important test bed for basic and translational biomedical study preceding clinical application. Recent advances in genomic technology have allowed a creation of the animal model for human disease with a genetically encoded biomarker, notably green fluorescent protein (GFP) awarded Nobel Prize at 2008. Combined with a rapid development of fluorescent probes, including newly emerging nano-material, and mature molecular biology tools, it has opened up a new avenue to the observation of complex pathophysiology of human disease in the animal model with much greater detail at cellular and molecular level in high contrast and specificity. Accordingly, novel fluorescence imaging methods that can visualize anatomical structure with functional and molecular information provided by fluorescent probes in an animal model in vivo have drawn great attentions. While all of the major clinical imaging modalities such as ultrasound, CT, MRI and PET has been modified and adapted, optical imaging technologies, especiallylaser scanning confocal and multiphoton fluorescence microscopy, are the only one readily providing cellular resolution of sub-micrometer in a live animal. Over the recent years, these technologies enabled dynamic 3D visualization of the living specimen as various biological processes unfold in real-time, providing unprecedented insights those were impossible to obtain by traditional static 2D snapshots (i.e. histopathology). With the development of highly specific targeting agents and new endogenous contrast, potentially to be empowered by nano-technology, these new imaging methods have advanced to be directly applied to live animal in vivo, beyond conventional ex vivo thin tissue sections or in vitro cell cultures on Petri dish.
In Vivo Micro-Visualization Laboratory
Graduate School of Nanoscience and Technology (GSNT)
Graduate School of Medical Science and Engineering (GSMSE)
Korea Advanced Institute of Science and Technology (KAIST)