MD1 Utilizing nonlinear optical microscopy to investigate the development of early cancer in nude mice in vivo

Chun-Chin Wang1, Feng-Chieh Li1, Sung-Jan Lin2, Wen Lo1, Chen-Yuan Dong1

1Department of Physics, National Taiwan University.
2 Department of Dermatology, National Taiwan University Hospital and College of Medicine.

 

In this investigation, we used nonlinear optical microscopy to image and analyze normal and carcinogen DMBA treated skin tissues of nude mice in vivo. Using the results obtained from two-photon autofluroescence and second harmonic generation (SHG) images and the application of ASI (Autofluorescence versus SHG Index), we can visualize the interaction between mouse skin cells and connective tissue.

We found that as the imaging depth increases, ASI has different distribution in normal and treated skin tissues. Since the DMBA treated skin eventually became squamous cell carcinoma (SCC), our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy. We envision this approach can be effective in studying intravital tumor biology, leading to improved tumor treatment procedures.

 

 

MD2 Multiphoton Fluorescence Diagnosis of Hepatocellular Carcinoma

Tzu-Lin Sun1,Yuan Liu1, Ming-Chin Sung1, Chun-Hui Yang1,
Hsuan-Shu Lee2,3,Chen-Yuan Dong1

1Department of Physics National Taiwan University,
2National Taiwan University College of Medicine,
3National Taiwan University Hospital

 

Hepatic disease has always been a major health problem in Taiwan, and hepatocelular carcinoma (HCC) is among the leading causes of death. Although doctors diagnose the degree of liver carcinoma according to their clinical experience, there is a need for an objective diagnosis of the extent of this cancer. In this work, we attempt to develop an objective criterion to help improving the accuracy of HCC classification which will be a useful aid for clinical investigations.

To achieve the goal, we make use of two-photon fluorescence (TPF) and second harmonic generation (SHG) microscopy in imaging hepatocellular carcinoma (HCC) specimens. By scanning the slices of different HCC specimens, we attempt to correlate the change in autofluorescence with other parameters such as tumor size. We hope that this approach can lead to a more exact classification of HCC.

Our preliminary results show that the autoflurescence intensity of non-tumor is larger than tumor. Our approach demonstrates the possibility of using tissue autofluroescence for tumor classification and that this methodology may be a useful too for the clinical diagnosis of diseases.

 

 

MD3 The effects of depilatory agents as a penetration enhancer on human stratum corneum structures

Jin-Ning Lee 1, Shiou-Hwa Jee 2, Chih-Chieh Chan 2, Wen Lo 1, Sung-Jan Lin 1,3 , Chen-Yuan Dong 1

1 Department of Physics, National Taiwan University, Taipei, Taiwan;
2 Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan;
3 Institute of Biomedical Engineering, College of Engineering and College of Medicine, National Taiwan University, Taipei, Taiwan

 

The aim of this research is to characterize the penetration enhancing effect of depilatory, and to investigate the change of stratum corneum(SC). Human foreskin was treated by a depilatory for 10 minutes and then using fluorescent probe, hydrophilic sulforhodamine (SRB), to model drugs penetration that was quantified by twophoton microscopy. The structural alternations of SC are assessed by routine histology, transmission electron microscopy and Nile red staining. The results showed that the penetration of fluorescent probe can be enhanced. Nile red staining revealed, instead of a regular lipid layers around the brick of corneocytes, a disorganized and homogenized pattern of lipid distribution. Ultrastructural analysis showed that the protein envelope of coenocytes disintegrated, especially intercellular lipid of SC. We concluded that depilatory enhance drug penetration by disrupting both the cellular integrity of corneocytes and the regular packing of intercellular lipid of SC.

 

 

MD4 Intravital multiphoton microscopy for imaging hepatobiliary function

Feng-Chien Li1 , Tzu-Lin Sun 1, Shu-Mei Yang 2, Hsuan-Shu Lee 2, Chen-Yuan Dong 1

1 Department of Physics, National Taiwan University, Taipei 106, Taiwan;
,2 Department of Internal Medicine, National Taiwan University Hospital, Taipei100, Taiwan

 

Liver is the chemical factory in body responsible for important functions such as metabolism and detoxification. When liver can not be regenerated in time to amend damages that has occurred, failure of hepatic functions such as liver failure and metabolic disease can result. Traditionally, the study of liver pathology has depended on histological techniques, but such methods are limited to ex-vivo observation. In order to study hepatic metabolism in vivo, we have designed a hepatic imaging chamber made of biocompatible titanium alloy (6V4Al-Ti, ELI grade). In combination with multiphoton and second harmonic generation microscopy, our approach allows the intravital observation of hepatic intravital activities to be achieved. Processes such as hepatic metabolism and disease progression can be studied using this methodology.

 

 

MD5 The Observation of Interaction between Pseudomonas aeruginosa and Cornea by the Use of Two-Photon Fluorescence and Second Harmonic Generation Microscopy

Yu Lin Chang1 Wen Lo1 Hsin Yuan Tan2 Chen Yuan Dong1

1Department Of Physics, National Taiwan University , Taipei 106, Taiwan,
2Department Of Ophthalmology, Chung Gun Memorial Hospital , Linko 333, Taiwan

 

As the contact lens becomes popular, the issue of associated corneal infection becomes increasingly important in ophthalmology. Pseudomonas aeruginosa is a bacteria species which occurs frequently in infected corneas. Using minimally invasive, two-photon fluorescence and second harmonic generation microscopy, the structural alteration to collagen fibrils of corneal stroma can be investigated. We acquired multi-photon images on infected corneal button specimens at different times following the introduction of pathogens. Our results show that collagen destruction becomes increasingly more significant with time. We hope that this approach can eventually be used for the clinical diagnosis of the infectious corneas without destructive biopsy procedures.

 

 

MD6 Observation of the influence of force on collagen production by chondrocytes using two-photon and second harmonic generation.

Chia-Cheng Chang1,Hsuan-Shu Lee2.3,Ling-Ling Chiou3,Chi-Hsiu Huang1,Wen Lo1,Chen-Yan Dong1.

1National Taiwan University Department of Physics Taipei 106 Taiwan
2National Taiwan University College of Bio-Resources and Agricultural Institute of Biotechnology Taipei 106 Taiwan
3National Taiwan University Hospital and National Taiwan University college of Medicine Department of Internal Medicine Taipei 100 Tsaiwan

 

We cultivate mesenchymal stem cells (MSCs) in a medium with TGF-£]3 added. TGF-£]3 induce the MSCs to differentiate into chomdrocytes and the chondrocytes subsequently produces collagen. During culture, we impart force on the cells by using a centrifuge. We choose three forces-0G , 10000G , 20000G and exerted the force on the cells for 15 minutes a day for 4 weeks. We observed that the cells exerted with greater force produce collagen that tend to accumulate in lumps at early stage, and generate stronger second harmonic signal.

 

 

MD7 Imaging Human Bone Marrow Mesenchymal Stem Cell Morphogenesis in Chitosan Scaffold by Multiphoton Microscopy

Chi-Hsiu Huang 1, Hsuan-Shu Lee 2,3, Ling-Ling Chiou 3, Chia-Cheng Chang 1, Wen Lo 1, Chen-Yuan Dong 1

1 National Taiwan University Department of Physics Taipei 106 Taiwan;
2 National Taiwan University College of Bio-Resources and Agricultural Institute of Biotechnology Taipei 106 Taiwan;
3 National Taiwan University Hospital and National Taiwan University College of Medicine Department of Internal Medicine Taipei 100 Taiwan

 

One important goal in tissue engineering is cultiviating cartilage tissue for transplantation. The most common method to cultivate cartilage is to seed human bone marrow mesenchymal stem cells on three dimensional biocompatiable and biodegradable scaffold materials,then add the signal factor TGF£]-3 to induce the cells to produce collagen that would grow into cartilage tissue.In this study,we seeded mesenchymal stem cells on a chitosan scaffold and added TGF£]-3 to induce cartilage tissue producing and image the process using multiphoton and second harmonic generation microscopy.

 

 

MD8 Structural Alteration of Corneal Scar Tissue Revealed by Multiphoton Microscopy

Wen Lo 1,Chiu-Mei Hsueh 1, Shu-Wen Teng 1,Yen Sun 1, Sung-Jan Lin 2,3, Ching-Hsi Hsiao 4,5, Wei-Chou Lin 6, Hsin-Yuan Tan 2,4,5,
Chen-Yuan Dong 1

1 Department of Physics, National Taiwan University, Taipei 106, Taiwan,
2 Institute of Biomedical Engineering, College and Medicine and Engineering, Taipei 100, Taiwan,
3 Department of Dermatology, National Taiwan University Hospital, Taipei 100, Taiwan,
4 Department of Ophthalmology, Chang Gung Memorial Hospital, Linko 333, Taiwan,
5 College of Medicine, Chang Gung University, Linko 333, Taiwan
6 Department of Pathology, National Taiwan University Hospital, Taipei 100, Taiwan

 

The special arrangement of collagen fibers in corneal stroma provides extraordinary high transparency of cornea. However, wound healing process after clinical treatments, such as refractive surgeries, can disrupt collagen alignment and hinder corneal transparency. While works have been focused on the mechanism, the availability of a non-invasive in vivo imaging technique may be of valuable importance for investigating this complicated physiological response. Here we demonstrate the multiphoton fluorescence and second harmonic generation (SHG) ex vivo imaging of a full-thickness corneal scar tissue ten years following wounding. We intend to demonstrate the structural alterations to the cornea following wound healing process and the potential of application of this novel optical technique as a clinical diagnostic and monitoring tool for corneal pathologies.

 

 

MD9 High resolution multiphoton imaging of acute liver inflammation using hepatic imaging chamber

Tzu-Lin Sun1,Feng-Chieh Li1,Shu-Mei Yang3,Wen-Pu Hong1,
Hsuan-Shu Lee2,3,Chen-Yuan Dong1

1Department of Physics National Taiwan University,
2National Taiwan University College of Medicine,
3National Taiwan University Hospital

 

Conventional hepatic research relies heavily on histological images for morphological information of liver tissue. Static histological images, however, can not provide real-time dynamic information of in vivo physiological processes such as cell motion. In this work, we developed an intravital imaging system to study the effects of acute inflammation of liver on immune system reaction. We hope that this methodology can be used to reveal in vivo details of acute inflammation of liver and allow us to better understand hepatic processes.

In our approach, we used two-photon fluorescence (TPF) microscopy to observe in vivo liver acute inflammation in a mouse and determined the dynamics of leukocyte due to immune system reaction. To obtain real-time images of liver, we developed a high resolution intravital hepatic imaging chamber. This device allows us to make short term in vivo examination.

To demonstrate the usefulness of our approach, we used the effect of immune system reaction in response to acute inflammation as a liver disease model. We injected lipopolysaccharide (LPS) to induce acute inflammation and imaged leukocytes motion by using the liver chamber. The blood vessels and leukocytes were labeled by staining with Fluorescein Isothiocyanate-Dextran (FITC-dextran) and Rhodamine 6G respectively. Analyzing the change in the number of leukocyte after inflammation, we can determine how leukocytes react as part of the immune mechanism. Our preliminary results show that leukocyte number increased and the velocity of blood flow decreased due to inflammation. These changes are consistent with leukocytes aggregation to protect the body against bacteria invasion.

 

 

MD10 Ex vivo optical biopsy of eyes by using mulitphoton microscopy

Wen Lo1, Shu-Wen Teng1, Hsin-Yuan Tan2,3, Hsiao-Ching Chen9, Hsuan-Shu Lee9, Yang-Fan Chen1, Ju-Li Peng4, Huei-Hsing Lin4, Ki Hean Kim5, Yen Sun1, Wei-Chou Lin6, Sung-Jan Lin3,7, Shiou-Hwa Jee7,8, Peter T. C. So5, Chen-Yuan Dong1

1Microscopic Biophysics Laboratory, Department of Physics, National Taiwan University, Taipei 106, Taiwan
2 Department of Ophthalmology, Chang Gung Memorial Hospital, Linko 333, Taiwan
3 Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan
4 Department of Life Science, National Taiwan University, Taipei 106, Taiwan
5Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
6 Department of Pathology, National Taiwan University Hospital, Taipei 100, Taiwan
7 Department of Dermatology, National Taiwan University Hospital, Taipei 100, Taiwan
8 Department of Dermatology, College of Medicine, National Taiwan University Hospital, Taipei 100, Taiwan
9 Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei 100, Taiwan

 

The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to obtain spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in ex vivo eyes without slicing and staining. At the micron resolution, multiphoton imaging can provide both large histological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes.
The structural information of cornea, limbus and sclera were revealed by multiphoton imaging of intact porcine eye balls. In order to highlight the quiescent keratocytes in corneal stroma, a large area multiphoton cross-section across an intact eye of a GFP mouse was obtained by a homebuilt multiphoton microscope. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling improved the fluorescent contrast of cellular area and facilitated visualizing of inactive keratocytes.
Our results show that multiphoton imaging provides highly resolved structural information of cornea, limbus, sclera and conjunctiva. The multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.