Ramez Saroufeem

Dynamic tissue analysis using time- and wavelength-resolved fluorescence spectroscopy for atherosclerosis diagnosis

Simultaneous time- and wavelength-resolved fluorescence spectroscopy (STWRFS) was developed and tested for the dynamic characterization of atherosclerotic tissue ex vivo and arterial vessels in vivo. Autofluorescence, induced by a 337 nm, 700 ps pulsed laser, was split to three wavelength sub-bands using dichroic filters, with each sub-band coupled into a different length of optical fiber for temporal separation. STWRFS allows for fast recording/analysis (few microseconds) of time-resolved fluorescence emission in these sub-bands and rapid scanning. Distinct compositions of excised human atherosclerotic aorta were clearly discriminated over scanning lengths of several centimeters based on fluorescence lifetime and the intensity ratio between 390 and 452 nm. Operation of STWRFS blood flow was further validated in pig femoral arteries in vivo using a single-fiber probe integrated with an ultrasound imaging catheter. Current results demonstrate the potential of STWRFS as a tool for real-time optical characterization of arterial tissue composition and for atherosclerosis research and diagnosis.

Fluorescence lifetime imaging for the characterization of the biochemical composition of atherosclerotic plaques

This study investigates the ability of a flexible fiberoptic-based fluorescence lifetime imaging microscopy (FLIM) technique to resolve biochemical features in plaque fibrotic cap associated with plaque instability and based solely on fluorescence decay characteristics. Autofluorescence of atherosclerotic human aorta (11 autopsy samples) was measured at 48 locations through two filters, F377: 377∕50 and F460: 460∕60 nm (center wavelength∕bandwidth). The fluorescence decay dynamic was described by average lifetime (τ) and four Laguerre coefficients (LECs) retrieved through a Laguerre deconvolution technique. FLIM-derived parameters discriminated between four groups [elastin-rich (ER), elastin and macrophage-rich (E+M), collagen-rich (CR), and lipid-rich (LR)]. For example, τ(F377) discriminated ER from CR (R = 0.84); τ(F460) discriminated E+M from CR and ER (R = 0.60 and 0.54, respectively); LEC-1(F377) discriminated CR from LR and E+M (R = 0.69 and 0.77, respectively); P < 0.05 for all correlations. Linear discriminant analysis was used to classify this data set with specificity >87% (all cases) and sensitivity as high as 86%. Current results demonstrate for the first time that clinically relevant features (e.g., ratios of lipid versus collagen versus elastin) can be evaluated with a flexible-fiber based FLIM technique without the need for fluorescence intensity information or contrast agents.

Multimodal near infrared spectral imaging as an exploratory tool for dysplastic esophageal lesion identification

We explore nine different combinations of fluorescence, light scattering, and polarization spectral imaging approaches in the near-infrare spectral region toward the diagnosis of pathologic and normal esophageal lesions. The combinations of all the imaging techniques were evaluated for maximal sensitivity and specificity. The results suggest that this multimodal approach is capable of highly accurate detection of the presence of pathologic tissue.

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 microm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque.

Characterizing the origin of autofluorescence in human esophageal epithelium under ultraviolet excitation

The autofluorescence under ultraviolet excitation arising from normal squamous and columnar esophageal mucosa is investigated using multispectral microscopy. The results suggest that the autofluorescence signal arises from the superficial tissue layer due to the short penetration depth of the ultraviolet excitation. As a result, visualization of esophageal epithelial cells and their organization can be attained using wide-field autofluorescence microscopy. Our results show tryptophan to be the dominant source of emission under 266 nm excitation, while emission from NADH and collagen are dominant under 355 nm excitation. The analysis of multispectral microscopy images reveals that tryptophan offers the highest image contrast due to its non-uniform distribution in the sub-cellular matrix. This technique can simultaneously provide functional and structural imaging of the microstructure using only the intrinsic tissue fluorophores.

Real-time microscopic imaging of esophageal epithelial disease with autofluorescence under ultraviolet excitation

Detection of esophageal disease in current clinical practice is limited to visualization of macroscopic epithelial morphology. In this work, we investigate high resolution autofluorescence imaging under ultra violet excitation to visualize microscopic epithelial changes related to disease progression using a bench top prototype microscope. The approach is based on the hypothesis that UV excitation light can only penetrate the superficial layer of cells resulting in autofluorescence images of the epithelial layer without using an additional image sectioning approach. The experiments were performed using ex vivo human esophagus biopsy specimens. The results indicate that cellular morphology information related to disease progression is attainable without tissue preparation.

Fluorescence lifetime imaging microscopy for the characterization of atherosclerotic plaques

Atherosclerotic plaque composition has been associated with plaque instability and rupture. This study investigates the use of fluorescence lifetime imaging microscopy (FLIM) for mapping plaque composition and assessing features of vulnerability. Measurements were conducted in atherosclerotic human aortic samples using an endoscopic FLIM system (spatial resolution of 35 µm; temporal resolution 200 ps) developed in our lab which allows mapping in one measurement the composition within a volume of 4 mm diameter x 250 µm depth. Each pixel in the image represents a corresponding fluorescence lifetime value; images are formed through a flexible 0.6 mm side-viewing imaging bundle which allows for further intravascular applications. Based on previously recorded spectra of human atherosclerotic plaque, fluorescence emission was collected through two filters: f1: 377/50 and f2: 460/60 (center wavelength/bandwidth), which together provides the greatest discrimination between intrinsic fluorophores related to plaque vulnerability. We have imaged nine aortas and lifetime images were retrieved using a Laguerre expansion deconvolution technique and correlated with histopathology. Early results demonstrate discrimination using fluorescence lifetime between early, lipid-rich, and collagen-rich lesions which are consistent with previously reported time-resolved atherosclerotic plaque measurements.

Role of Intestinal Myofibroblasts in HIV-Associated Intestinal Collagen Deposition and Immune Reconstitution following Combination Antiretroviral Therapy

Objective:To investigate the potential role of mucosal intestinal myofibroblasts (IMFs) in HIV and associated fibrosis in gut-associated lymphoid tissue. Design:Profibrotic changes within the secondary lymphoid organs and mucosa have been implicated in failed immune reconstitution following effective combination antiretroviral therapy (cART). Microbial translocation is believed to be sustaining these systemic inflammatory pathways. IMFs are nonprofessional antigen-presenting cells with both immunoregulatory and mesenchymal functions that are ideally positioned to respond to translocating microbial antigen. Methods:Duodenal biopsies, obtained from patients naive to cART, underwent trichrome staining and were examined for tissue growth factor-beta (TGF-&bgr;) expression. Combined immunostaining and second harmonic generation analysis were used to determine IMF activation and collagen deposition. Confocal microscopy was performed to examine IMF activation and Toll-like receptor (TLR)4 expression. Finally, primary IMF cultures were stimulated with lipopolysaccharide to demonstrate the expression of the inflammatory biomarkers. Results:The expression of the fibrosis-promoting molecule, TGF-&bgr;1, is significantly increased in duodenal biopsies from HIV patients naïve to cART, and negatively correlated with subsequent peripheral CD4+ recovery. The increase in TGF-&bgr;1 coincided with an increase in collagen deposition in the duodenal mucosa in the tissue area adjacent to the IMFs. We also observed that IMFs expressed TLR4 and had an activated phenotype since they were positive for fibroblast activation protein. Finally, stimulation of IMFs from HIV patients with TLR4 resulted in significantly increased expression of profibrotic molecules, TGF-&bgr;1, and interleukin-6. Conclusion:Our data support the hypothesis that activated IMFs may be among the major cells contributing to the profibrotic changes, and thus, the establishment and maintenance of systemic inflammation interfering with immune reconstitution in HIV patients.

Establishment of rules for interpreting ultraviolet autofluorescence microscopy images for noninvasive detection of Barrett’s esophagus and dysplasia

The diagnostic potential of autofluorescence (AF) microscopy under ultraviolet (UV) excitation is explored using ex vivo human specimens. The aim is to establish optical patterns (the rules for interpretation) that correspond to normal and abnormal histologies of the esophagus, spanning from early benign modifications (Barretts esophagus) to subsequent dysplastic change and progression toward carcinoma. This was achieved by developing an image library categorized by disease progression. We considered morphological changes of disease as they are compared with histological diagnosis of the pathological specimen, as well as control samples of normal esophagus, proximal stomach, and small intestine tissue. Our experimental results indicate that UV AF microscopy could provide real-time histological information for visualizing changes in tissue microstructure that are currently undetectable using conventional endoscopic methods.

Endomicroscopy imaging of epithelial structures using tissue autofluorescence

We explore autofluorescence endomicroscopy as a potential tool for real-time visualization of epithelial tissue microstructure and organization in a clinical setting. The design parameters are explored using two experimental systems--an Olympus Medical Systems Corp. stand-alone clinical prototype probe, and a custom built bench-top rigid fiber conduit prototype. Both systems entail ultraviolet excitation at 266 nm and/or 325 nm using compact laser sources. Preliminary results using ex vivo animal and human tissue specimens suggest that this technology can be translated toward in vivo application to address the need for real-time histology.