Dynamic visualization of the recovery of mouse hepatobiliary metabolism to acetaminophen-overdose damage.

Acetaminophen (APAP) overdose is one of the world's leading causes of drug-induced hepatotoxicity. Although traditional methods such as histological imaging and biochemical assays have been successfully applied to evaluate the extent of APAP-induced liver damage, detailed effect of how APAP overdose affect the recovery of hepatobiliary metabolism and is not completely understood. In this work, we used intravital multiphoton microscopy to image and quantify hepatobiliary metabolism of the probe 6-carboxyfluorescein diacetate in APAP-overdose mice. We analyzed hepatobiliary metabolism for up to 7 days following the overdose and found that the excretion of the probe molecule was the most rapid on Day 1 following APAP overdose and slowed down on Days 2 and 3. On Day 7, probe excretion capability has exceeded that of the normal mice, suggesting that newly regenerated hepatocytes have higher metabolic capabilities. Our approach may be further developed applied to studying drug-induced hepatotoxicity in vivo.

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.

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.

Interactions between fluorescence of atomically layered graphene oxide and metallic nanoparticles.

Graphene oxide (GO) demonstrates interesting photoluminescence (PL) because of its unique heterogeneous atomic structure, which consists of variable sp(2)- and sp(3)-bonded carbons. In this study, we report the interaction between the luminescence of GO ranging from a single atomic layer to few-layered thin films and localized surface plasmon resonance (LSPR) from silver nanoparticles (Ag NPs). The photoluminescence of GO in the vicinity of the Ag NPs is enhanced significantly due to the near-field plasmonic effect by coupling electron-hole pairs of GO with oscillating electrons in Ag NPs, leading to an increased PL intensity and a decreased PL decay lifetime. The maxima 30-fold enhancement in PL intensity is obtained with an optimized film thickness of GO, and the luminescence image from a single atomic layer GO sheet is successfully observed with the assistance of the LSPR effect. The results provide an ideal platform for exploring the interactions between the fluorescence of two-dimensional layered materials and the LSPR effect.

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 former's 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.

Differentiation of normal and cancerous lung tissues by multiphoton imaging.

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.

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.