Thanassis Papaioannou

Time-domain laser-induced fluorescence spectroscopy apparatus for clinical diagnostics

We report the design and development of a compact optical fiber-based apparatus for in situ time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) of biological systems. The apparatus is modular, optically robust, and compatible with the clinical environment. It incorporates a dual output imaging spectrograph, a gated multichannel plate photomultiplier (MCP-PMT), an intensified charge-coupled-device (ICCD) camera, and a fast digitizer. It can accommodate various types of light sources and optical fiber probes for selective excitation and remote light delivery/collection as required by different applications. The apparatus allows direct recording of the entire fluorescence decay with high sensitivity (nM range fluorescein dye concentration with signal-to-noise ratio of 46) and with four decades dynamic range. It is capable of resolving a broad range of fluorescence lifetimes from hundreds of picoseconds (as low as 300 ps) using the MCP-PMT coupled to the digitizer to milliseconds using the ICCD. T...

Fast model-free deconvolution of fluorescence decay for analysis of biological systems

For complex biological systems, conventional analysis of fluorescence intensity decay in terms of discrete exponential components cannot readily provide a true representation of the underlying fluorescence dynamics. We investigate an alternative nonparametric method for the analysis of time-resolved fluorescence data from biochemical and biological systems based on the expansion of fluorescence decay in a discrete Laguerre basis. We report that a unique Laguerre expansion can be found for fluorescence intensity decays of arbitrary form with convergence to a correct solution significantly faster than conventional multiexponential approximation methods. The Laguerre expansion coefficients are shown to be highly correlated with intrinsic fluorescence lifetimes and allow direct characterization of the fluorescence dynamics. A novel method for prediction of concentrations in mixtures of biochemical components using these coefficients is developed and successfully tested (prediction error <2%) using data from different mixtures of fluorescence lifetime standards. These findings suggest that the use of Laguerre expansion coefficients is a fast approach for the characterization and discrimination of complex biological systems such as tissues and cells, and that the method has potential for applications of fluorescence lifetime techniques to tissue diagnostics and imaging microscopy of living cells.

Detection of rupture-prone atherosclerotic plaques by time-resolved laser-induced fluorescence spectroscopy

OBJECTIVE Plaque with dense inflammatory cells, including macrophages, thin fibrous cap and superficial necrotic/lipid core is thought to be prone-to-rupture. We report a time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) technique for detection of such markers of plaque vulnerability in human plaques. METHODS The autofluorescence of carotid plaques (65 endarterectomy patients) induced by a pulsed laser (337 nm, 0.7 ns) was measured from 831 distinct areas. The emission was resolved spectrally (360-550 nm range) and temporally (0.3 ns resolution) using a prototype fiber-optic TR-LIFS apparatus. Lesions were evaluated microscopically and quantified as to the % of different components (fibrous cap, necrotic core, inflammatory cells, foam cells, mature and degraded collagen, elastic fibers, calcification, and smooth muscle cell of the vessel wall). RESULTS We determined that the spectral intensities and time-dependent parameters at discrete emission wavelengths (1) allow for discrimination (sensitivity >81%, specificity >94%) of various compositional and pathological features associated with plaque vulnerability including infiltration of macrophages into intima and necrotic/lipid core under a thin fibrous cap, and (2) show a linear correlation with plaque biochemical content: elastin (P<0.008), collagen (P<0.02), inflammatory cells (P<0.003), necrosis (P<0.004). CONCLUSION Our results demonstrate the feasibility of TR-LIFS as a method for the identification of markers of plaque vulnerability. Current findings enable future development of TR-LIFS-based clinical devices for rapid investigation of atherosclerotic plaques and detection of those at high-risk.

Effects of fiber-optic probe design and probe-to-target distance on diffuse reflectance measurements of turbid media: an experimental and computational study at 337 nm

Fiber-optic probes are widely used in optical spectroscopy of biological tissues and other turbid media. Only limited information exists, however, on the ways in which the illumination-collection geometry and the overall probe design influence the interrogation of media. We have investigated both experimentally and computationally the effect of probe-to-target distance (PTD) on the diffuse reflectance collected from an isotropically (Lambertian) scattering target and an agar-based tissue phantom. Studies were conducted with three probes characterized by either common (single-fiber) or separate (two bifurcated multifiber probes) illumination and collection channels. This study demonstrates that PTD, probe design, and tissue scattering anisotropy influence the extent of the transport of light into the medium, the light-collection efficiency, and the sampling volume of collected light. The findings can be applied toward optimization of fiber-optic probe designs for quantitative optical spectroscopy of turbid media including biological tissues.

Laguerre-based method for analysis of time-resolved fluorescence data: application to in-vivo characterization and diagnosis of atherosclerotic lesions

We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability.

Role of laser and thermal ablation devices in the treatment of vascular diseases

Since the first coronary angioplasty in 1977, both the number and complexity of interventional procedures have grown dramatically. Continuous-wave and pulsed lasers may further extend the capabilities of balloon angioplasty. Fiberoptic catheters may be used to transmit continuous-wave laser energy to ablate plaque via thermal mechanisms. Pulsed laser systems (such as the excimer) are technologically more complex than the continuous-wave systems, but may prove superior in small vessels given their ability to ablate plaque with minimal associated effects. On the other hand, modifications of the fiber-optic tip, such as the placement of a metal cap, have yielded even better results than current bare fiber systems. Such laser thermal techniques have proved a useful adjunct to balloon dilatation in peripheral vessels, but further research is necessary to determine their effect on coronary arteries. New, nonlaser technologies, however, may provide simpler power sources for thermal angioplasty. Although balloon angioplasty remains the cornerstone of interventional vascular therapy, new technologies should help to further expand the indications for nonsurgical interventions.

One-year results of excimer laser photorefractive keratectomy for myopia

BACKGROUND Excimer laser photorefractive keratectomy for the correction of myopia is presently under investigation in the United States by the Food and Drug Administration (FDA). The Phase II-B FDA study is being conducted on 75 normally sighted myopic eyes utilizing three currently available excimer lasers. This report presents the 1-year results on 12 myopic eyes treated with the VISX excimer laser system at the Ellis Eye Center at Cedars-Sinai Medical Center in Los Angeles under the Phase II-B FDA protocol. METHODS Twelve eyes of 12 patients with myopia between -1.75 and -5.00 diopters underwent 193 nm argon/fluoride excimer laser photorefractive keratectomy. The epithelium was mechanically removed, and fixation was accomplished with a suction ring which provided nitrogen flow across the corneal surface. The computer controlled corneal ablations were 5.00 mm in diameter and were accomplished with an iris diaphragm closing from large to small. RESULTS The preoperative spherical equivalent myopia was -3.50 D (SD = 1.02) and the postoperative myopia was -0.25 (SD = 0.48). Eleven of the 12 patients achieved an uncorrected visual acuity of 20/30 or better and were corrected to within +/- 0.50 D of emmetropia. All corneas demonstrated a mild reticular subepithelial haze which was barely visible at 1 year. There were no vision-threatening complications and none of the eyes experienced a loss of best corrected visual acuity. CONCLUSIONS In this small trial, the excimer laser appears to be capable of accurately changing the refractive power of the cornea for the correction of myopia with minimal side effects. Only when larger numbers of patients undergo the procedure will we be able to determine the safety and efficacy of photorefractive keratectomy as a refractive surgical procedure.

Excimer laser ablation of the human lens at 308 nm with a fiber delivery system

ABSTRACT A 308 nm excimer laser has been used with a fiber delivery system to perform ablation of the human lens. Preliminary results demonstrate the systems ability to ablate lens nucleus and cortex selectively, preserving the anterior and posterior capsules. The total delivered energy necessary to ablate a human lens ranged from 35 to 63 joules. Laser tissue interaction and ablation rates were computed for the different components of the human lens (capsule, cortex, nucleus) for the operatively useful energy densities (fluences). Operative experience suggests that cortex and nucleus can be ablated while preserving the capsule if an adequate irrigation and aspiration system is developed. These results also suggest that this modality may be adequate for performing endocapsular cataract extraction. Laser tissue interactions were also computed at variable distances between the fiber tip and tissue. As this distance increased, the spread of the beam increased and a significant increase in energy was necessary to induce tissue ablation. This was due to the decrease in fluence with increasing distance to the target tissue and/or the absorption and scattering of the delivered energy within a short distance from the fiber tip by the ablated material. Evidence of a sonic effect was also present.

Intraluminal fluorescence spectroscopy catheter with ultrasound guidance

We demonstrate the feasibility of a time-resolved fluorescence spectroscopy (TRFS) technique for intraluminal investigation of arterial vessel composition under intravascular ultrasound (IVUS) guidance. A prototype 1.8-mm (5.4 Fr) catheter combining a side-viewing optical fiber (SVOF) and an IVUS catheter was constructed and tested with in vitro vessel phantoms. The prototype catheter can locate a fluorophore in the phantom vessel wall, steer the SVOF in place, perform blood flushing under flow conditions, and acquire high-quality TRFS data using 337-nm wavelength excitation. The catheter steering capability used for the coregistration of the IVUS image plane and the SVOF beam produce a guiding precision to an arterial phantom wall site location of 0.53+/-0.16 mm. This new intravascular multimodal catheter enables the potential for in vivo arterial plaque composition identification using TRFS.

Performance evaluation of fiber optic probes for tissue lifetime fluorescence spectroscopy

The design of fiber-optic probes plays an important role in optical spectroscopic studies, including fluorescence spectroscopy of biological tissues. It can affect the light delivery and propagation into the tissue, the collection efficiency (total number of photons collected vs. total number of photons launched) and the origin of collected light. This in turn affects the signal to noise ratio (SNR) and the extend of tissue interrogation, thus influencing the diagnostic value of such techniques. Three specific fiber-optic probe designs were tested both experimentally and computationally via Monte Carlo simulations. In particular, the effects of probe architecture (single-fiber vs. two bifurcated multifiber probes), probe-to-target distance (PTD), and source-to-detector separation (SDS) were investigated on the collected diffuse reflectance of a Lambertian target and an agar-based tissue phantom. This study demonstrated that probe architecture, PTD, and SDS are closely intertwined and considerably affect the light collection efficiency, the extend of target illumination, and the origin of the collected reflected light. Our findings can be applied towards optimization of fiber-optic probe designs for quantitative fluorescence spectroscopy of diseased tissues.