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Cancer initiating cells (CICs) have been the focus of recent anti-cancer therapies, exhibiting strong invasion capability via potentially enhanced ability to remodel extracellular matrices (ECM). We have identified CICs in a human breast cancer cell line, MX-1, and developed a xenograft model in SCID mice. We investigated the CICs’ matrix-remodeling effects using Second Harmonic Generation (SHG) microscopy to identify potential phenotypic signatures of the CIC-rich tumors. The isolated CICs exhibit higher proliferation, drug efflux and drug resistant properties in vitro; were more tumorigenic than non-CICs, resulting in more and larger tumors in the xenograft model. The CIC-rich tumors have less collagen in the tumor interior than in the CIC-poor tumors supporting the idea that the CICs can remodel the collagen more effectively. The collagen fibers were preferentially aligned perpendicular to the CIC-rich tumor boundary while parallel to the CIC-poor tumor boundary suggesting more invasive behavior of the CIC-rich tumors. These findings would provide potential translational values in quantifying and monitoring CIC-rich tumors in future anti-cancer therapies. CIC-rich tumors remodel the collagen matrix more than CIC-poor tumors.

Background & Aims: There is increasing need for accurate assessment of liver fibrosis/cirrhosis. We aimed to develop qFibrosis, a fully-automated assessment method combining quantification of histopathological architectural features, to address unmet needs in core biopsy evaluation of fibrosis in chronic hepatitis B (CHB) patients.Methods: qFibrosis was established as a combined index based on 87 parameters of architectural features. Images acquired from 25 Thioacetamide-treated rat samples and 162 CHB core biopsies were used to train and test qFibrosis and to demonstrate its reproducibility. qFibrosis scoring was analyzed employing Metavir and Ishak fibrosis staging as standard references, and collagen proportionate area (CPA) measurement for comparison. Results: qFibrosis faithfully and reliably recapitulates Metavir fibrosis scores, as it can identify differences between all stages in both animal samples (p

Significant tissue structures exist in cardiac ventricular tissue that are of supracellular dimension. It is hypothesized that these tissue structures contribute to the discontinuous spread of electrical activation, may contribute to arrhymogenesis and also provide a substrate for effective cardioversion. However, the influences of these mesoscale tissue structures in intact ventricular tissue are difficult to understand solely on the basis of experimental measurement. Current measurement technology is able to record at both the macroscale tissue level and the microscale cellular or subcellular level, but to date it has not been possible to obtain large volume, direct measurements at the mesoscales. To bridge this scale gap in experimental measurements, we use tissue-specific structure and mathematical modelling. Our models have enabled us to consider key hypotheses regarding discontinuous activation. We also consider the future developments of our intact tissue experimental programme.

Fast voltage-sensitive dyes are widely used to image cardiac electrical activity. Typically, the emission spectrum of these fluorochromes is wavelength shifted with altered membrane potential, but the optical signals obtained also decay with time and are affected by contraction. Ratiometry reduces, but may not fully remove, these artifacts. An alternate approach has been developed in which the time decay in simultaneously acquired short- and long-wavelength signals is characterized nonparametrically and removed. Motion artifact is then identified as the time-varying signal component common to both decay-corrected signals and subtracted. Performance of this subtraction technique was compared with ratiometry for intramural optical signals acquired with a fiber-optic probe in an isolated, Langendorff-perfused pig heart preparation ( n = 4) stained with di-4-ANEPPS. Perfusate concentration of 2,3-butanedione monoxime was adjusted (7.5–12.5 mM) to alter contractile activity. Short-wavelength (520–600 nm) and long-wavelength (>600 nm) signals were recorded over 8–16 cardiac cycles at 6 sites across the left ventricular free wall in sinus rhythm and during pacing. A total of 451 such data sets were acquired. Appreciable wall motion was observed in 225 cases, with motion artifact classed as moderate (less than modulation due to action potential) in 187 and substantial (more than modulation due to action potential) in 38. In all cases, subtraction performed as well as, or better than, ratiometry in removing motion artifact and decay. Action potential morphology was recovered more faithfully by subtraction than by ratiometry in 58 of 187 and 31 of 38 cases with moderate and substantial motion artifact, respectively. This novel subtraction approach may therefore provide a means of reducing the concentration of uncoupling agents used in cardiac optical mapping studies.

We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging tech- nique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling. C 2011 Society of Photo-Optical Instrumentation Engineers

Spectroscopic measurements of cardiac action potential using a high resolution spectrometer offers more insights into the complex process of spectral modulation associated with membrane potential and mechanical activity. This shows the dependence of the ratiometric behavior of di-4-ANEPPS for different excitation wavelengths.

Liver biopsy remains the gold standard in monitoring progression of liver fibrosis associated with an abnormal increase in collagen. Descriptive scoring systems are still being widely used to grade biopsy samples. In this study, we propose a new set of features by clustering collagen fibers into three groups first based on their localization and connectivity properties, and then by extracting morphological features of collagen fibers. The new feature set is compared to the earlier features used in classification of liver fibrosis, which were based on the total amount of collagen fibers. Our results show that new features lead to more accurate grading of liver fibrosis.

Liver steatosis/fibrosis represents the major conditions and symptoms for many liver diseases. Nonlinear optical microscopy has emerged as a powerful tool for label-free tissue imaging with high sensitivity and chemical specificity for several typical biochemical compounds. Three nonlinear microscopy imaging modalities are implemented on the sectioned tissues from diseased livers induced by high fat diet (HFD). Coherent anti-Stokes Raman scattering (CARS) imaging visualizes and quantifies the lipid droplets accumulated in the liver, Second harmonic generation (SHG) is used to map the distribution of aggregated collagen fibers, and two-photon excitation fluorescence (TPEF) reveals the morphology of hepatic cells based on the autofluorescence signals from NADH and flavins within the hepatocytes. Our results demonstrate that obesity induces liver steatosis in the beginning stage, which may progress into liver fibrosis with high risk. There is a certain correlation between liver steatosis and fibrosis. This study may provide new insights into the understanding of the mechanisms of steatosis/fibrosis transformations at the cellular and molecular levels.

Monitoring liver fibrosis progression by liver biopsy is important for certain treatment decisions, but repeated biopsy is invasive. We envision redefinition or elimination of liver biopsy with surface scanning of the liver with minimally invasive optical methods. This would be possible only if the information contained on or near liver surfaces accurately reflects the liver fibrosis progression in the liver interior. In our study, we acquired the second-harmonic generation and two-photon excitation fluorescence microscopy images of liver tissues from bile duct-ligated rat model of liver fibrosis. We extracted morphology-based features, such as total collagen, collagen in bile duct areas, bile duct proliferation, and areas occupied by remnant hepatocytes, and defined the capsule and subcapsular regions on the liver surface based on image analysis of features. We discovered a strong correlation between the liver fibrosis progression on the anterior surface and interior in both liver lobes, where biopsy is typically obtained. The posterior surface exhibits less correlation with the rest of the liver. Therefore, scanning the anterior liver surface would obtain similar information to that obtained from biopsy for monitoring liver fibrosis progression.

Pulse-modulated second harmonic imaging microscopes (PM-SHIMs) exhibit improved signal-to-noise ratio (SNR) over conventional SHIMs on sensitive imaging and quantification of weak collagen signals inside tissues. We quantify the spatial distribution of sparse collagen inside a xenograft model of human acute myeloid leukemia (AML) tumor specimens treated with a new drug against receptor tyrosine kinase (ABT-869), and observe a significant increase in collagen area percentage, collagen fiber length, fiber width, and fiber number after chemotherapy. This finding reveals new insights into tumor responses to chemotherapy and suggests caution in developing new drugs and therapeutic regimens against cancers.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins such as collagens, which may result in cirrhosis, liver failure, and portal hypertension. In this study, we apply a multimodal nonlinear optical microscopy platform developed to investigate the fibrotic liver diseases in rat models established by performing bile duct ligation (BDL) surgery. The three nonlinear microscopy imaging modalities are implemented on the same sectioned tissues of diseased model sequentially: i.e., second harmonic generation (SHG) imaging quantifies the contents of the collagens, the two-photon excitation fluorescence (TPEF) imaging reveals the morphology of hepatic cells, while coherent anti-Stokes Raman scattering (CARS) imaging maps the distributions of fats or lipids quantitatively across the tissue. Our imaging results show that during the development of liver fibrosis (collagens) in BDL model, fatty liver disease also occurs. The aggregated concentrations of collagen and fat constituents in liver fibrosis model show a certain correlationship between each other.

We have developed a microfluidic 3D hepatocyte chip (3D HepaTox Chip) for in vitro drug toxicity testing to predict in vivo drug hepatotoxicity. The 3D HepaTox Chip is based on multiplexed microfluidic channels where a 3D microenvironment is engineered in each channel to maintain the hepatocytes' synthetic and metabolic functions. The multiplexed channels allow for simultaneous administration of multiple drug doses to functional primary hepatocytes while an incorporated concentration gradient generator enables the in vitro dose-dependent drug responses to predict in vivo hepatotoxicity. The IC(50) values of 5 model drugs derived from the dose-dependent on-chip testing correlate well with the reported in vivo LD(50) values. The 3D HepaTox Chip can be integrated with on-chip sensors and actuators as the next generation cell-based on-chip drug testing platform.

Determining the extent of liver fibrosis has clinically been difficult due to the lack of a simple, objective method that can accurately quantify the amount of collagen in the diseased tissue. Second harmonic generation (SHG) microscopy has been shown to produce bright and robust signals from non-centrosymmetric fibrillar collagen. We designed a SHG system that can objectively measure collagen presence in livers. In addition, we have developed a fully automated algorithm to quantify liver fibrosis in an efficient, standardized and reproducible manner. SHG microscopy was performed on livers harvested from bile duct ligated Wistar rats using a confocal microscope with a mode-locked Ti:Sapphire laser. Images acquired were later analyzed with Otsu method and a custom-developed, fully automated algorithm to measure area of fibers. Both the qualitative and quantitative progression of collagen over time was observed using SHG microscopy. We have compared the modified quantification algorithm with Otsu segmentation algorithm. With the modified algorithm, we have maintained greater amount of collagen after segmentation. We have also shown modified algorithm was able to identify the presence of finer collagen which was completely removed with the normal Otsu method. We have built an imaging system using state-of-the-art SHG microscopy. In addition, a fully-automated algorithm was developed to quantify collagen content in tissue to measure the presence of collagen in high accuracy. By combining SHG microscopy and our quantification algorithm, we can provide sensitive measurement for liver fibrosis which accurately reflects the progression of liver fibrosis, especially in early stages.

We describe a simple fiber optic fluorescence spectrometry system with a wide variety of biomedical applications. This low-cost, all-fiber system is portable, robust and has the capacity to acquire fluorescence spectra at rates up to 1 kHz. We demonstrate the capabilities of the system by presenting experimental measurements of action potentials in the di-4-ANEPPS stained heart and the concentration of GFP tagged bacteria.

We have developed a standardized quantification technique for assessing the progression of liver fibrosis by combining second harmonic generation microscopy and automated quantification algorithms. We have also validated the technique using animal models.

Illumination and fluorescence collection volumes of different optical fibres in tissue were characterized using 2-photon techniques. A 3D illumination model was also developed and validated. This study provides important information for optical probe design in biomedical applications.

Introduction: Transmembrane potentials can be recorded at multiple intramural sites in the intact heart using fiber optic probes or optrodes. The technique has considerable potential utility for studies of arrhythmia and defibrillation, but has not been validated in large mammalian hearts. Methods and Results: An optrode was used to acquire intramural transmembrane potentials in six isolated Langendorff-perfused pig hearts. Mechanical activity was suppressed with 2,3-butanedione monoxime (BDM). Excitation light (488 nm) was delivered to and fluorescence collected from six sites, each spaced 1.4 mm apart across the left ventricle (LV) free wall that was stained with di-4 ANEPPS. Intramural membrane potentials were compared with extracellular potentials recorded at adjacent locations in sinus rhythm, and during atrial and subepicardial ventricular pacing (1–3 Hz). In three hearts, epicardial intracellular potentials were also measured close to the optrode. Optical action potentials were reproducible, with no significant transmural variation in morphology. There was close correspondence between subepicardial optical and intracellular potentials (R2= 0.948, n = 23). The onset of activation at and its progression across adjacent optical and extracellular recording sites were consistent, as was the variation of action potential duration (APD) with cycle length. However, there was greater variability in absolute APD estimated from optical and extracellular records (R2= 0.773, n = 258). Comparison of extracellular potentials at the same intramural sites in vivo confirms that heart isolation and BDM slow electrical propagation and depress restitution relationships, but otherwise preserve intramural patterns of electrical activation. Conclusions: We have demonstrated that our optrode provides reliable intramural transmembrane potential recordings in the isolated pig heart preparation.

Fluorescence imaging techniques have been central to much biomedical science research over the past two decades. In particular, functional imaging has provided important new information about processes that occur at cellular and sub-cellular levels. With this approach, living tissues are stained with dyes whose emission is modulated by changes in the environment to which the dye is exposed. The fluorescence imaging systems used within this context typically incorporate relatively complex free space optical assemblies and a stable platform is necessary to maintain appropriate alignment of their components. Because of the poor efficiency of these systems, it is necessary to use powerful light sources and sensitive photo-detectors. We have developed a novel fluorescence imaging system in which free-space optics are replaced by optical fibers, passive optical splitters and associated components. Solid state lasers are used as the excitation light source. A variety of detection systems have been utilized including a spectrometer. The feasibility of the approach has been established using a rat heart preparation stained with the membrane potential-sensitive dye, di-4-ANEPPS. Detailed emission spectra for this dye, at different levels of resting membrane potential, are presented here for 532 nm and 488 nm excitation. Cardiac action potentials obtained with the modular fiber optic system correspond closely to intracellular potentials acquired at adjacent sites in the isolated rat heart preparation. Our modular fiber optic system is cheaper, more efficient, more flexible and more robust than conventional fluorescence imaging systems. Using a high-speed spectrometer for photodetection, it is possible to implement the signal processing required for multi-line or ratiometric imaging in software, which further enhances the efficiency and flexibility of the system. We believe that this approach has wide potential applications for biomedical fluorescence imaging.

We develop a standardized, fully automated, quantification system for liver fibrosis assessment using second harmonic generation microscopy and a morphology-based quantification algorithm. Liver fibrosis is associated with an abnormal increase in collagen as a result of chronic liver diseases. Histopathological scoring is the most commonly used method for liver fibrosis assessment, where a liver biopsy is stained and scored by experienced pathologists. Due to the intrinsic limited sensitivity and operator-dependent variations, there exist high inter- and intraobserver discrepancies. We validate our quantification system, Fibro-C-Index, with a comprehensive animal study and demonstrate its potential application in clinical diagnosis to reduce inter- and intraobserver discrepancies.

Liver fibrosis is associated with an abnormal increase in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second-harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

Optical fibers can deliver light to, and collect it from, regions deep in tissue. However, reported illumination and fluorescence collection volumes adjacent to the fiber tip have been inconsistent, and systematic data on this topic are not available. Illumination and fluorescence collection profiles were characterized with high spatial resolution for different optical fibers in tissue and various fluids using two-photon flash photolysis and excitation. We confirm that illumination and fluorescence collection volumes for optical fibers are near identical. Collection volume is determined by the core dimensions and numerical aperture (NA) of the fiber and the scattering properties of the medium. For a multimode optical fiber with 100 microm core diam and NA=0.22, 80% of the total fluorescence is collected from a depth of 170 microm in tissue and 465 microm in nonscattering fluid. A semiempirical mathematical description of photon flux adjacent to the fiber tip was also developed and validated. This was used to quantify the extent of temporal blurring associated with propagation of a wavefront of altered fluorescence emission across the region addressed by fiber optic probes. We provide information that will facilitate the design of optical probes for tissue imaging or therapeutic applications.