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Hepatobiliary metabolism is one of the major functions of the liver. However, little is known of the relationship between the physiological location of the hepatocytes and their metabolic potential. By the combination of time-lapse multiphoton microscopy and first order kinetic constant image analysis, the hepatocellular metabolic rate of the model compound 6-carboxyfluorescein diacetate (6-CFDA) is quantified at the single cell level. We found that the mouse liver can be divided into three zones, each with distinct metabolic rate constants. The sinusoidal uptake coefficients k1 of Zones 1, 2, and 3 are respectively 0.239 ± 0.077, 0.295 ± 0.087, and 0.338 ± 0.133 min−1, the apical excreting coefficients k2 of Zones 1, 2, and 3 are 0.0117 ± 0.0052, 0.0175 ± 0.0052, and 0.0332 ± 0.0195 min−1, respectively. Our results show not only the existence of heterogeneities in hepatobiliary metabolism, but they also show that Zone 3 is the main area of metabolism.

We tried to image obstructive cholestasis by using a newly developed imaging system to measure the alterations of hepatobiliary function in living mice with their bile ducts ligated. A hepatic imaging window was installed on the upper abdomen soon after the mice underwent ligation of the common bile duct. On the next day, the mice received intravenous injection of rhodamine B isothiocyanate-dextran and carboxyfluorescein diacetate. The later would be transformed into fluorogenic carboxyfluorescein (detected at approximately 500-550 nm) by hepatocytes and then excreted into bile canaliculi. The images were acquired by multiphoton microscopy. The fluorescence intensities at approximately 500-550 nm within hepatocytes or sinusoids were measured in time series. In mice with bile duct ligation, bile canaliculi failed to appear during the whole observation period over 100 min following carboxyfluorescein diacetate injection, whereas the fluorescence was retained much longer within sinusoids. Furthermore, the fluorescence intensities in sinusoids were persistently higher than in hepatocytes during the course. Bile duct ligation impedes hepatocytes to excrete carboxyfluorescein into bile canaliculi. The kinetics of fluorescence intensities in hepatocytes and sinusoids indicated there is an active machinery operating backflow of this fluorogenic bile solute from hepatocytes into sinusoids in the liver with obstructive cholestasis.

The advantages offered by multiphoton microscopy enabled this technique to be applied for in vivo understanding of physiological processes. However, multiphoton intravital microscopy also requires associated technologies to be developed. In this work, we detailed the design of a dorsal skin fold chamber made of titanium alloy that allow high resolution multiphoton fluorescence and second harmonic generation (SHG) microscopy to be achieved. We demonstrate that our apparatus is capable of obtaining high resolution, images of blood vessels and collagen matrix in nude mice. Such an imaging chamber will allow physiological processes to be investigated at high resolution

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). In combination with multiphoton and a method of quantification by using optical signal, our approach allows the intravital observation of hepatic activities to be achieved. Processes such as hepatic metabolism and disease progression can be studied using this methodology.

In this investigation, we used in vivo nonlinear optical microscopy to image normal and carcinogen DMBA treated skin tissues of nude mice. We acquired two-photon autofluroescence and second harmonic generation (SHG) images of the skin tissue, and applied the ASI (Autofluorescence versus SHG Index) to the resulting image. This allows us to visualize and quantify the interaction between mouse skin cells and the surrounding connective tissue. We found that as the imaging depth increases, ASI has a different distribution in the normal and the 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 to be effective in studying tumor biology and tumor treatment procedures.

Atomic layer deposition (ALD) has been used to grow zinc oxide (ZnO) into a template of anodic aluminum oxide with quasi-dendritic nanochannels to form quasi-dendritic nanostructures. The characteristic photoluminescence (PL) emission from the inner region of the quasi-dendritic ZnO nanostructure peaks at 397 nm while that from its outer region at 424 nm. In between the two regions, the PL peak shows monotonic shift. In other words, the different layers of the single quasi-dendritic ZnO nanostructure emit PL with graded wavelengths spontaneously. The red shift in the PL peak positions is likely to be caused by the change in local stoichiometry between Zn and O, which are resulted from the limited supply of materials through the quasi-dendritic nanochannels during the ALD. The process to fabricate such quasi-dendritic ZnO nanostructures with spontaneously graded emission could help expand applications of ZnO-based devices.

Lung tumor is a major disease in Taiwan and the development of minimally invasive imaging to detect and diagnose lung tumor in vivo is of biomedical value. However, prior to in vivo investigation, it is important to obtain the multiphoton signatures associated with lung tumor ex vivo. In this investigation, we used multiphoton microscopy to image normal and cancerous lung tissues (adenocarcinoma). Our results show that a combination of harmonic generation and multiphoton autofluorescence imaging may be used to acquire structural information in discerning lung carcinoma from normal lung tissues.

For many years, the dorsal skinfold chamber has been used for intravital observation in animal models. We have developed a chamber built using medical-grade titanium. Our design allows long-term, high resolution, and large-area observation in nude mice in vivo. Combined with two-photon fluorescence and second-harmonic generation microscopy, our chamber can be used to obtain qualitative and quantitative information for in vivo analysis of diseases such as tumors.

Morphological changes of hepatocyte death have so far only been described on cells in culture or in tissue sections. Using a high-resolution and high-magnification multiphoton microscopic system, we recorded in living mice serial changes of acetaminophen (APAP)-induced hepatocyte necrosis in relevance to metabolism of a fluorogenic bile solute. Initial changes of hepatocyte injury included basal membrane disruption and loss of mitochondrial membrane potential. An overwhelming event of rupture at adjacent apical membrane resulting in flooding of bile into these hepatocytes might ensue. Belbs formed on basal membrane and then dislodged into the sinusoid circulation. Transmission electron microscopy disclosed a necrotic hepatocyte depicting well the changes after apical membrane rupture and bile flooding. Administration of the antidote N-acetylcysteine dramatically reduced the occurrence of apical membrane rupture. The present results demonstrated a hidden but critical step of apical membrane rupture leading to irreversible APAP-induced hepatocyte injury.

We performed intravital multiphoton microscopy to image and analyze normal and carcinogen treated skintissues of nude mice in vivo. Using intravital images and the quantitative pixel to pixel ratiometric processing of multiphotonautofluorescence to second harmonic generation index (MAFSI), we can visualize the interaction between epithelial cells and extracellular matrix. We found that as the imaging depth increases, MAFSI has different distribution in normal and treated cutaneous specimens. Since the treated skin eventually became squamous cellcarcinoma, our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy.

We utilize multiphoton microscopy for the label-free diagnosis of noncancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from humans. Our results show that the combination of second-harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire morphological and quantitative information in discriminating cancerous from noncancerous lung tissues. Specifically, noncancerous lung tissues are largely fibrotic in structure, while cancerous specimens are composed primarily of tumor masses. Quantitative ratiometric analysis using MAF to SHG index (MAFSI) shows that the average MAFSI for noncancerous and LAC lung tissue pairs are 0.55+/-0.23 and 0.87+/-0.15, respectively. In comparison, the MAFSIs for the noncancerous and SCC tissue pairs are 0.50+/-0.12 and 0.72+/-0.13, respectively. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from noncancerous tissues.