In vivo multiphoton imaging of bile duct ligation

Invited Paper

In vivo multiphoton imaging of bile duct ligation Yuan Liua, Feng-Chieh Lia, Hsiao-Chin Chenb, Po-shou Changa,Shu-Mei Yangb, Hsuan-Shu Leeb, Chen-Yuan Donga a Department of Physics, National Taiwan University, Taipei 106, Taiwan b Department of Internal Medicine, National Taiwan University Hospital, Taipei100, Taiwan ABSTRACT Bile is the exocrine secretion of liver and synthesized by hepatocytes. It is drained into duodenum for the function of digestion or drained into gallbladder for of storage. Bile duct obstruction is a blockage in the tubes that carry bile to the gallbladder and small intestine. However, Bile duct ligation results in the changes of bile acids in serum, liver, urine, and feces1, 2. In this work, we demonstrate a novel technique to image this pathological condition by using a newly developed in vivo imaging system, which includes multiphoton microscopy and intravital hepatic imaging chamber. The images we acquired demonstrate the uptake, processing of 6-CFDA in hepatocytes and excretion of CF in the bile canaliculi. In addition to imaging, we can also measure kinetics of the green fluorescence intensity. Keywords: multiphoton, bile duct ligation, in vivo, liver

1. MATERIALS AND METHODS 1.1 Animal C57BL/6 mice (about 5-6 weeks of age, weighing 20-25g) are the animals used in this study. 1.2 Rhodamine B isothiocyanate-Dextran (R9379, Sigma) Dextran labeled with Rhodamine B is used in perfusion or microcirculation studies in animals. It is injected into the mice (50 mg/ml, 50µl) to indicate the sinusoids in liver. The average molecular weight of this dye is 70,000 and the size is large enough to keep in the blood circulation for observation. The peak emission wavelength is 573 nm. 1.3 6-Carboxyfluorescein diacetate, 6-CFDA (C5041, Sigma) 6-CFDA is used in monitoring the hepatic metabolic activities in observation of the sequential uptake and processing from hepatocytes. 6-CFDA is a non-fluorescent material, but can be observed after being absorbed by hepatocytes and hydrolyzed by esterase into CF with peak emission wavelength of 517 nm3, 4. The subsequent excretion of CF can help to identify bile canaliculi as well. Signals of CF can be observed in the detection channel with 525±25 nm filter and we prefer jugular vein injection (10 mg/ml, 5µl) over tail vein injection. 1.4 Two-photon Microscope The two-photon microscope we used in this study is illustrated in Fig. 1. The titanium-sapphire laser with 780-nm output (Tsunami, Spectra Physics, Mountain View, California) was energized by a diode-pumped solid-state laser (Millennia X, Spectra Physics). The pulse width is about 75 femtosecond with a repetition rate of 80 MHz. After passing through the beam control optics the laser light was scanned by an x-y mirror scanning system (Model 6220, Combridge Technology, Cambridge, Massachusetts). The laser was then guided toward the modified inverted microscope (TE2000, Nikon, Japan), and was beam-expanded into the back aperture of an objective (S Fluor 40Xoil, NA 1.3, Nikon) by a primary dichroic mirror (700DCSPXRUV-3p, Chroma Technology,Rockingham, Vermont). The laser beam power that reached the liver of the mice was approximately 10 mW and the luminescence coming from the sample was collected in the epi-illuminated geometry. After passing through the primary dichroic mirror, sample luminescence was separated into four detection channels by secondary dichroic mirrors (435DCXR, 495DCXR, 555DCLP, Chroma Technology) and additional bandpass filters (HQ390/20, HQ460/50, HQ525/50, HQ590/80, Chroma Technology). In

Multiphoton Microscopy in the Biomedical Sciences VIII, edited by Ammasi Periasamy, Peter T. C. So, Proc. of SPIE Vol. 6860, 68600G, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.764264

Proc. of SPIE Vol. 6860 68600G-1 2008 SPIE Digital Library -- Subscriber Archive Copy

this manner, second harmonic generation and blue, green and red fluorescence signals were collected by the detections with bandwidth of 390±10, 460±25, 525±25 and 590±40nm, respectively. Specimen

Objective 40Xoil

XY Scanner Dichroic 700nm

PMT Box

Emission Light

Computer Readout

Beam Control Optics

Ti-Sapphire Laser 780nm

PMT box Dichroic 495nm

Dichroic 555nm

Filter 525±25nm Green 500~550nm

Filter 590±40nm

Blue 435~485nm

Dichroic 435nm Filter 460±25nm Filter 300±10nm

Red 550~630nm

SHG 380~400nm Fig. 1. Schematic of a two-photon microscope

Proc. of SPIE Vol. 6860 68600G-2

1.5 Installation of Hepatic Image Chamber Installation of the intravital hepatic imaging chamber was accomplished in the following manners:4 1. Anesthetize the mouse. 2. Shave the hair on the abdomen of the mouse. 3. Operate a vertical incision of the skin and peritoneum on the skin at top of liver. 4. Make a circular wound with similar size of hepatic window device. 5. Mount the hepatic window device onto the skin. 6. Cover and adhere the round glass. 7. Move the device into the U-shape track on the steel plate, and fix it by another rectangular metal plate. After fixing it on a specific stage, the chamber apparatus is placed on the inverted microscope.

4

Fig. 2. Installation of Hepatic Imaging Chamber

Figure 2 Installation of Hepatic Image Chamber


2. RESULTS AND DISCUSSION The representative multiphoton micrographs in Figs. 3 and 4 show the hepatobiliary excretory function in normal and bile duct ligation mice, respectively, and the images were taken at about 30 µm below the capsule, which is the collagen fiber (capsule) at the liver surface. Fig. 3 shows the time course observation of normal mouse liver. Micrograph (a) was at the moment of 6-CFDA injection, sinusoids are visible due to the red fluorescence of rhodamine dextran. Few leukocytes moving and some erythrocytes flowing can be visualized. Micrograph (b) was taken 2 minutes later, cords of hepatocytes and bile canaliculi emerged immediately. It was because the 6-CFDA was being taken up by hepatocytes and hydrolyzed into carboxyfluorescein (CF) and eventually drained into bile canaliculi. Micrographs took within ten minutes (as shown in (c), (d) and (e)) show presence of canaliculi. However, the fluorescence intensity of distributed bile canaliculi reached the peak at about 10min point and started to decrease. As shown in micrograph (g), cords of hepatocytes and bile canaliculi gradually faded out, which took approximately 30 minutes after 6-CFDA injection. The last micrograph (h) was taken at the 50 minute time point and the green fluorescence found in the bile canaliculi at earlier time points has become almost invisible. Fig. 4 shows the time course observation of bile duct ligation mouse liver. To compare with normal one we demonstrate micrographs at identical time points. As shown in (a), which was taken at the moment of 6-CFDA injection, sinusoids were labeled with rhodamine dextran and few hepatocytes in this micrographic field were green. Therefore most of the hepatic cells in mouse with obstructive cholestasis 24 hours appear to have lost metabolic function. In the series micrographs at 2, 4, 6, 8 and 10-min point, we did not find appreciable change in green fluorescence intensity. Moreover, we did not observe bile canaliculi which was widespread and obvious in normal mouse at the same time points. In the last two images, which were taken at 30 and 60-min time points, respectively, we observed the green fluorescence in the sinusoidal veins was more intense than previous time points.

3. CONCLUSION Our results demonstrate that the kinetics of hepatic metabolism with bile duct ligation can be studied using intravital multiphoton microscopy. From high resolution imaging, we also have confidence that our unique technique, which combines multiphoton microscopy and intravital hepatic imaging chamber, has the potential in the investigation of the hepatobiliary excretory function in vivo.

4. ACKNOWLEDGMENT This work was supported by the National Science Council (Taiwan) and was completed in the Optical Molecular Imaging Microscopy Core Facility (A5) of Taiwan’s National Research Program for Genomic Medicine (NRPGM)


Fig.3. Representative multiphoton hepatic micrographs (normal mouse)


Fig.4. Representative multiphoton hepatic micrographs (bile duct ligation)


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