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.

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 laserinduced 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.

Articles you may be interested inDesign and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging Rev. Sci. Instrum. 85, 034303 (2014); 10.1063/1.4869037 A multichannel time-domain scanning fluorescence mammograph: Performance assessment and first in vivo results Rev. Sci. Instrum. 82, 024302 (2011); 10.1063/1.3543820 Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy Rev. Sci. Instrum. 80, 065104 (2009); 10.1063/1.3142478 Design and development of a rapid acquisition laser-based fluorometer with simultaneous spectral and temporal resolution Rev. Sci. Instrum. 72, 3061 (2001); 10.1063/1.1379957Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications Rev.We report the design and development of a compact optical fiber-based apparatus for in situ time-resolved laser-induced fluorescence spectroscopy ͑tr-LIFS͒ 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-PMT͒, an intensified charge-coupled-device ͑ICCD͒ 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 46͒ 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 ps͒ using the MCP-PMT coupled to the digitizer to milliseconds using the ICCD. The data acquisition and analysis process is fully automated, enabling fast recording of fluorescence intensity decay across the entire emission spectrum ͑0.8 s per wavelength or ϳ40 s for a 200 nm wavelength range at 5 nm increments͒. The spectral and temporal responses of the apparatus were calibrated and its performance was validated using fluorescence lifetime standard dyes ͑Rhodamin B, 9-cyanoanthracene, and rose Bengal͒ and tissue endogenous fluorophores ͑elastin, collagen, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide͒. Fluorescence decay lifetimes and emission spectra of all tested compounds measured with the current tr-LIFS apparatus were found in good agreement with the values reported in the literature. The design and performance of tr-LIFS apparatus have enabled in vivo studies of atherosclerotic plaques and brain tumors.

We report the application of the Laguerre deconvolution technique (LDT) to the analysis of invivo 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.

Accumulation of numerous macrophages in the fibrous cap is a key identifying feature of plaque inflammation and vulnerability. This study investigates the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a potential tool for detection of macrophage foam cells in the intima of atherosclerotic plaques. Experiments were conducted in vivo on 14 New Zealand rabbits (6 control, 8 hypercholesterolemic) following aortotomy to expose the intimal luminal surface of the aorta. Tissue autofluorescence was induced with a nitrogen pulse laser (337 nm, 1 ns). Lesions were histologically classified by the percent of collagen or macrophage foam cells as well as thickness of the intima. Using parameters derived from the time-resolved fluorescence emission of plaques, we determined that intima rich in macrophage foam cells can be distinguished from intima rich in collagen with high sensitivity (>85%) and specificity (>95%). This study demonstrates, for the first time, that a time-resolved fluorescence-based technique can differentiate and demark macrophage content versus collagen content in vivo. Our results suggest that TR-LIFS technique can be used in clinical applications for identification of inflammatory cells important in plaque formation and rupture.