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Bruce J Tromberg

Publications and source records attributed to Bruce J Tromberg.

At least 19 recordsLinked to original sources
PubMed2006

Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy.

Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth ( approximately 1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4-37 degrees C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37-4 degrees C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G', from 23 +/- 3 Pa to 0.28 +/- 0.16 Pa, respectively, mean +/- SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 +/- 3.5 Pa before to 138 +/- 40 Pa after cross-linking, mean +/- SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics.

Biomechanical Phenomena↗
PubMed2006

Epithelial-derived TGF-beta2 modulates basal and wound-healing subepithelial matrix homeostasis.

The epithelium influences the mesenchyme during dynamic processes such as embryogenesis, wound healing, fibrosis, and carcinogenesis. Since transforming growth factor-beta (TGF-beta) modulates these processes, we hypothesized that epithelial-derived TGF-beta also plays a critical role in maintaining the extracellular matrix at basal conditions. We utilized an in vitro model of the epithelial-mesenchymal trophic unit in the human airways to determine the role of epithelial-derived TGF-beta in modulating the extracellular matrix under basal and wound-healing conditions. When differentiated at an air-liquid interface, the human bronchial epithelium produces active TGF-beta2 at a concentration of 50-70 pg/ml, whereas TGF-beta1 is undetectable. TGF-beta2 increases two- to threefold following scrape injury in a dose-dependent fashion and significantly enhances both alpha-smooth muscle actin expression in the underlying collagen-embedded fibroblasts and secretion of tenascin-C into the matrix. Multiphoton microscopy demonstrates substantially enhanced second harmonic generation from fibrillar collagen in the matrix. Pretreatment of the matrix with either sirolimus (2.5 nM) or paclitaxel (10 nM) abolishes the increases in both TGF-beta2 and second harmonic generation in response to epithelial injury. In the absence of the epithelium, exogenous active TGF-beta2 (0-400 pg/ml) produces a biphasic response in the second harmonic signal with a minimum occurring at the epithelial-derived basal level. We conclude that epithelial-derived TGF-beta2 is secreted in response to injury, significantly alters the bulk optical properties of the extracellular matrix, and its tight regulation may be required for normal collagen homeostasis.

Cells, Cultured↗
PubMed2006

Multicell tumor spheroids in photodynamic therapy.

BACKGROUND AND OBJECTIVES: Multicell spheroids (MCSs) represent a simple in vitro system ideally suited for studying the effects of a wide variety of investigational treatments including photodynamic therapy (PDT). STUDY DESIGN/MATERIALS AND METHODS: In the first section of this review study, an overview of the current literature on MCS in PDT will be presented. Knowledge of basic PDT parameters has been gained from numerous MCS studies, in particular, the mechanisms of sensitizer photobleaching have been elucidated. MCSs have also proven useful for the study of complex PDT treatment regimens including multiple treatments and combined therapies involving PDT and ionizing radiation or hyperthermia. The purpose of the second part of this review is to present results from recent studies in our laboratory aimed at developing MCS models suitable for investigating tumor cell invasion and angiogenesis-processes characteristic of high-grade gliomas. RESULTS AND CONCLUSION: To that end, progress has recently been made to develop a more accurate in vivo brain tumor model consisting of biopsy-derived human tumor spheroids implanted into the brains of immunodeficient rats. Finally, recent work suggests that computer simulations may prove useful to describe the growth of MCS and predict the effects of investigational therapies including PDT. Such in silico models have made a number of counterintuitive predictions that have been verified in vitro and, as such, could guide the development of improved therapeutics.

Animals↗
PubMed2005

Quantitative spectroscopy of superficial turbid media.

We report a novel diffuse optical spectroscopy probe design for determining optical properties of superficial volumes of turbid samples. The fiber-based probe employs a highly scattering layer placed in contact with the sample of interest. This layer diffuses photons from a collimated light source before they enter the sample and provides a basis for describing light transported in superficial media by the diffusion approximation. We compare the performance of this modified two-layer diffusion model with Monte Carlo simulations. A set of experiments that demonstrate the feasibility of this method in turbid tissue phantoms is also presented. Optical properties deduced by this approach are in good agreement with those derived by use of a benchmark method for determining optical properties. The average interrogation depth of the probe design investigated here is estimated to be less than 1 mm.

Equipment Design↗
PubMed2005

Imaging in breast cancer: diffuse optics in breast cancer: detecting tumors in pre-menopausal women and monitoring neoadjuvant chemotherapy.

Diffuse optical spectroscopy (DOS) and diffuse optical imaging (DOI) are non-invasive diagnostic techniques that employ near-infrared (NIR) light to quantitatively characterize the optical properties of centimeter-thick, multiple-scattering tissues. Although NIR was first applied to breast diaphanography more than 70 years ago, quantitative optical methods employing time- or frequency-domain 'photon migration' technologies have only recently been used for breast imaging. Because their performance is not limited by mammographic density, optical methods can provide new insight regarding tissue functional changes associated with the appearance, progression, and treatment of breast cancer, particularly for younger women and high-risk subjects who may not benefit from conventional imaging methods. This paper reviews the principles of diffuse optics and describes the development of broadband DOS for quantitatively measuring the optical and physiological properties of thick tissues. Clinical results are shown highlighting the sensitivity of diffuse optics to malignant breast tumors in 12 pre-menopausal subjects ranging in age from 30 to 39 years and a patient undergoing neoadjuvant chemotherapy for locally advanced breast cancer. Significant contrast was observed between normal and tumor regions of tissue for deoxy-hemoglobin (p = 0.005), oxy-hemoglobin (p = 0.002), water (p = 0.014), and lipids (p = 0.0003). Tissue hemoglobin saturation was not found to be a reliable parameter for distinguishing between tumor and normal tissues. Optical data were converted into a tissue optical index that decreased 50% within 1 week in response to neoadjuvant chemotherapy. These results suggest a potential role for diffuse optics as a bedside monitoring tool that could aid the development of new strategies for individualized patient care.

Adult↗
PubMed2005

Noninvasive in vivo monitoring of methemoglobin formation and reduction with broadband diffuse optical spectroscopy.

We present noninvasive, quantitative in vivo measurements of methemoglobin formation and reduction in a rabbit model using broadband diffuse optical spectroscopy (DOS). Broadband DOS combines multifrequency frequency-domain photon migration (FDPM) with time-independent near infrared (NIR) spectroscopy to quantitatively measure bulk tissue absorption and scattering spectra between 600 nm and 1,000 nm. Tissue concentrations (denoted by brackets) of methemoglobin ([MetHb]), deoxyhemoglobin ([Hb-R]), and oxyhemoglobin ([HbO2]) were determined from absorption spectra acquired in "real time" during nitrite infusions in nine pathogen-free New Zealand White rabbits. As little as 30 nM [MetHb] changes were detected for levels of [MetHb] that ranged from 0.80 to 5.72 microM, representing 2.2 to 14.9% of the total hemoglobin content (%MetHb). These values agreed well with on-site ex vivo cooximetry data (r2= 0.902, P < 0.0001, n = 4). The reduction of MetHb to functional hemoglobins was also carried out with intravenous injections of methylene blue (MB). As little as 10 nM changes in [MB] were detectable at levels of up to 150 nM in tissue. Our results demonstrate, for the first time, the ability of broadband DOS to noninvasively quantify real-time changes in [MetHb] and four additional chromophore concentrations ([Hb-R], [HbO2], [H2O], and [MB]) despite significant overlapping spectral features. These techniques are expected to be useful in evaluating dynamics of drug delivery and therapeutic efficacy in blood chemistry, human, and preclinical animal models.

Animals↗
PubMed2005

Nonlinear optical microscopy of articular cartilage.

OBJECTIVE: To assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage. METHOD: Fresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination. RESULTS: NLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes. CONCLUSION: Thin images of living articular cartilage using NLOM may be obtained with (sub-)cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.

Animals↗
PubMed2004

Enhanced cytotoxic effects of 5-aminolevulinic acid-mediated photodynamic therapy by concurrent hyperthermia in glioma spheroids.

During photodynamic therapy (PDT) both normal and pathological brain tissue, in close proximity to the light source, can experience significant temperature increases. The purpose of this study was to investigate the anti-tumor effects of concurrent 5-aminolevulinic acid (ALA)-mediated PDT and hyperthermia (HT) in human and rat glioma spheroids. Human or rat glioma spheroids were subjected to PDT, HT, or a combination of the two treatments. Therapies were given concurrently to simulate the conditions that will occur during patient PDT. Predictions of diffusion theory suggest that brain tissue immediately adjacent to a spherical light applicator may experience temperature increases approaching 8 degrees C for laser input powers of 2 W. In the in vitro model employed here, HT had no effect on spheroid survival at temperatures below 49 degrees C, while sub-threshold fluence PDT results in only modest decrease in survival. HT (40-46 degrees C) and PDT interact in a synergistic manner if the two treatments are given concurrently. The degree of synergism increases with increasing temperature and light fluence. Apoptosis is the primary mode of cell death following both low-fluence rate PDT and combined HT + PDT.

Aminolevulinic Acid↗
PubMed2004

Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy.

The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the blood vessel wall.

Animals↗
PubMed2003

In vivo quantification of optical contrast agent dynamics in rat tumors by use of diffuse optical spectroscopy with magnetic resonance imaging coregistration.

We present a study of the dynamics of optical contrast agents indocyanine green (ICG) and methylene blue (MB) in an adenocarcinoma rat tumor model. Measurements are conducted with a combined frequency-domain and steady-state optical technique that facilitates rapid measurement of tissue absorption in the 650-1000-nm spectral region. Tumors were also imaged by use of contrast-enhanced magnetic resonance imaging (MRI) and coregistered with the location of the optical probe. The absolute concentrations of contrast agent, oxyhemoglobin, deoxyhemoglobin, and water are measured simultaneously each second for approximately 10 min. The differing tissue uptake kinetics of ICG and MB in these late-stage tumors arise from differences in their effective molecular weights. ICG, because of its binding to plasma proteins, behaves as a macromolecular contrast agent with a low vascular permeability. A compartmental model describing ICG dynamics is used to quantify physiologic parameters related to capillary permeability. In contrast, MB behaves as a small-molecular-weight contrast agent that leaks rapidly from the vasculature into the extravascular, extracellular space, and is sensitive to blood flow and the arterial input function.

Adenocarcinoma↗
PubMed2003

Coregistration of diffuse optical spectroscopy and magnetic resonance imaging in a rat tumor model.

We report coregistration of near-infrared diffuse optical spectroscopy (DOS) and magnetic resonance imaging (MRI) for the study of animal model tumors. A combined broadband steady-state and frequency-domain apparatus was used to determine tissue oxyhemoglobin, deoxyhemoglobin, and water concentration locally in tumors. Simultaneous MRI coregistration provided structural (T2-weighted) and contrast-enhanced images of the tumor that were correlated with the optical measurements. By use of Monte Carlo simulations, the optically sampled volume was superimposed on the MR images, showing precisely which tissue structure was probed optically. DOS and MRI coregistration measurements were performed on seven rats over 20 days and were separated into three tumor tissue classifications: viable, edematous, and necrotic. A ratio of water concentration to total hemoglobin concentration, as measured optically, was performed for each tissue type and showed values for edematous tissue to be greater than viable tissue (1.2 +/- 0.49 M/microM versus 0.48 +/- 0.15 M/microM). Tissue hemoglobin oxygen saturation (StO2) also showed a large variation between tissue types: viable tissue had an optically measured StO2 value of 61 +/- 5%, whereas StO2 determined for necrotic tissue was 43 +/- 6%.

Adenocarcinoma↗
PubMed2003

Farnesyltransferase inhibition: a novel method of immunomodulation.

Farnesyltransferase inhibitors (FTIs) are anticancer compounds that inhibit Ras GTPases. Since Ras GTPases play key roles in T cell activation and function, we hypothesized that FTIs have immunomodulatory properties and are potential antirejection agents. An investigation was performed on a potent FTI to evaluate this hypothesis in the in vitro setting. The in vitro effects of the FTI A-228839 were evaluated. Lectin- or antigen presenting cell (APC)-induced lymphocyte proliferation in the presence of A-228839 was measured. The effects of A-228839 on 1E5 T cell polarity were assessed by microscopy. Intracellular calcium ([Ca(2+)](i)) kinetics of lectin-activated lymphocytes was monitored by flow cytometry. The effects of A-228839 on peripheral blood mononuclear cell (PBMC) cytokine production was assessed by a cytometric bead array method. Activation-induced apoptosis was measured with an annexin V staining assay.A-228839 inhibited lectin-induced proliferation (IC(50)=0.24+/-0.11 microM). The inhibitory effects of A-228839 on lectin induced lymphocyte proliferation were additive to those of CsA. A-228839 was more effective in inhibiting APC-induced T cell proliferation (IC(50)=0.10+/-0.09 microM). A-228839 significantly disrupted the polarized shape of 1E5 T cells at physiologic concentrations. A-228839 altered PBMC baseline [Ca(2+)](i) but did not affect [Ca(2+)](i) kinetics during lectin-induced lymphocyte activation. A-228839 inhibited lymphocyte Th1 cytokine production at submicromolar levels and promoted apoptosis in lectin-activated lymphocytes.A-228839 potently inhibits lymphocyte activation and function. Our results suggest that FTIs may represent a new class of clinically useful immunomodulatory agents. A-228839 has potent in vitro immunomodulatory properties that warrant in vivo evaluation as an antirejection agent.

Adjuvants, Immunologic↗
PubMed2003

Repetitive 5-aminolevulinic acid-mediated photodynamic therapy on human glioma spheroids.

The response of human glioma spheroids to repetitive 5-aminolevulinic acid-mediated photodynamic therapy (PDT) was investigated. In all cases, light fluences were kept below toxic thresholds to simulate conditions typically found at 1-2 cm depths in brain adjacent to tumor. Significant inhibition of spheroid growth was observed following multiple PDT treatments at sub-threshold light fluences. The effect appears to be insensitive to the treatment intervals investigated (weekly or bi-monthly). In all cases, suppression of growth was observed for the duration of treatment. Low fluence rates (< or = 5 mW cm(-2)) appear to be more effective than high fluence rates (25 mW cm(-2)). No evidence of PDT resistance was found in this investigation.

Aminolevulinic Acid↗
PubMed2003

Reversible dissociation of collagen in tissues.

The turbidity of most biologic tissues hinders the use of lasers for diagnostic and therapeutic purposes. Hyperosmotic agents such as glycerol have been used to alter the optical scattering properties of turbid tissues. The mechanism of this effect, "optical clearing", however, remains incompletely understood. Multiphoton microscopy utilizing second harmonic generation can be used to monitor collagen structural changes in the presence of glycerol. This study suggests that the use of glycerol for tissue "optical clearing" screens noncovalent intermolecular interactions between low-order collagen structures, resulting in fiber disassembly. Dissociation of collagen fibers in native and engineered tissues in the presence of glycerol and reassociation with the application of saline are imaged dynamically. Collagen fiber reassembly is governed by the displacement of glycerol by saline in native and engineered skin. These results reveal the assembly process of high-order collagen structures and propose a molecular mechanism for the increase in tissue transparency observed after glycerol application.

Animals↗
PubMed2003

Spectroscopic approach for monitoring two-photon excited fluorescence resonance energy transfer from homodimers at the subcellular level.

We have employed a spectroscopic approach for monitoring fluorescence resonance energy transfer (FRET) in living cells. This method provides excellent spectral separation of green fluorescent protein (GFP) mutant signals within a subcellular imaging volume using two-photon excited fluorescence imaging and spectroscopy (TPIS-FRET). In contrast to current FRET-based methodologies, TPIS-FRET does not rely on the selection of optical filters, ratiometric image analysis, or bleedthrough correction algorithms. Utilizing the intrinsic optical sectioning capabilities of TPIS-FRET, we have identified protein-protein interactions within discrete subcellular domains. To illustrate the applicability of this technique to the detection of homodimer formation, we demonstrated the in vivo association of promyleocyte (PML) homodimers within their corresponding nuclear body.

Cell Line, Tumor↗
PubMed2002

Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.

Multiphoton microscopy relies on nonlinear light-matter interactions to provide contrast and optical sectioning capability for high-resolution imaging. Most multiphoton microscopy studies in biological systems have relied on two-photon excited fluorescence (TPEF) to produce images. With increasing applications of multiphoton microscopy to thick-tissue "intravital" imaging, second-harmonic generation (SHG) from structural proteins has emerged as a potentially important new contrast mechanism. However, SHG is typically detected in transmission mode, thus limiting TPEF/SHG coregistration and its practical utility for in vivo thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730-880 nm) to demonstrate that TPEF/SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF/SHG technique was applied to imaging a three-dimensional organotypic tissue model (RAFT). The structural and molecular origin of the image-forming signal from the various tissue constituents was determined by simultaneous spectroscopic measurements and confirming immunofluorescence staining. Our results show that at shorter excitation wavelengths (<800 nm), the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. Endogenous cellular signals are consistent with TPEF spectra of cofactors NAD(P)H and FAD at all excitation wavelengths. The reflected SHG intensity follows a quadratic dependence on the excitation power, decays exponentially with depth, and exhibits a spectral dependence in accordance with previous theoretical studies. The use of SHG and TPEF in combination provides complementary information that allows noninvasive, spatially localized in vivo characterization of cell-ECM interactions in unstained thick tissues.

Animals↗