Feng-Chieh Li

Visualizing dynamics of sub-hepatic distribution of nanoparticles using intravital multiphoton fluorescence microscopy.

Nanoparticles that do not undergo renal excretion or in vivo degradation into biocompatible debris often accumulate in the reticuloendothelial system, also know as the mononuclear phagocyte system, with undesired consequences that limit their clinical utility. In this work, we report the first application of intravital multiphoton fluorescence microscopy to dynamically track the hepatic metabolism of nanoparticles with subcellular resolution in real time. Using fluorescently labeled mesoporous silica nanoparticles (MSNs) in mice as a prototypical model, we observed significant hepatocyte uptake of positively charged, but not negatively charged, moieties. Conversely, in vivo imaging of negatively charged, but not positively charged, MSNs reveals an overwhelming propensity for the formers rapid uptake by Kupffer cells in liver sinusoids. Since the only prerequisite for these studies was that nanoparticles are fluorescently labeled and not of a specific composition or structure, the techniques we present can readily be extended to a wide variety of nanoparticle structures and surface modifications (e.g., shape, charge, hydrophobicity, PEGylation) in the preclinical assessment and tailoring of their hepatotoxicities and clearances.

In vivo dynamic metabolic imaging of obstructive cholestasis in mice

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.

Differentiation of normal and cancerous lung tissues by multiphoton imaging

In this work, we utilized multiphoton microscopy for the label-free diagnosis of non-cancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from human. 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 non-cancerous lung tissues. Specifically, non-cancerous 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 or SAAID) 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. Intrinsic fluorescence ratio (FAD/NADH) of SCC and non-cancerous tissues are 0.40±0.05 and 0.53±0.05 respectively, the redox ratio of SCC diminishes significantly, indicating that increased cellular metabolic activity. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from non-cancerous tissues. With additional development, multiphoton microscopy may be used for the clinical diagnosis of lung cancers.

The modulation effect of transverse, antibonding, and higher-order longitudinal modes on the two-photon photoluminescence of gold plasmonic nanoantennas.

Plasmonic nanoantennas exhibit various resonant modes with distinct properties. Upon resonant excitation, plasmonic gold nanoantennas can generate strong two-photon photoluminescence (TPPL). The TPPL from gold is broadband and depolarized, and may serve as an ideal local source for the investigation of antenna eigenmodes. In this work, TPPL spectra of three arrays of single-crystalline gold nanoantennas are comprehensively investigated. We carefully compare the TPPL spectra with dark-field scattering spectra and numerically simulated spectra. We show the modulation effect of the transverse resonant mode and the nonfundamental longitudinal mode on the TPPL spectrum. We also demonstrate suppression of TPPL due to the subradiant antibonding modes and study the influence of antenna resonant modes on the overall TPPL yield. Our work provides direct experimental evidence on nanoantenna-mediated near-to-far-field energy coupling and gains insight into the emission spectrum of the TPPL from gold nanoantennas.

Apical membrane rupture and backward bile flooding in acetaminophen-induced hepatocyte necrosis

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.

Direct visualization of functional heterogeneity in hepatobiliary metabolism using 6-CFDA as model compound

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.

In vivo multiphoton imaging of bile duct ligation

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.

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

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.

Intravital multiphoton microscopy for imaging hepatobiliary function

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 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.

Dorsal skinfold titanium chamber for noninvasive imaging in nude mice using multiphoton and harmonic generation microscopy

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.