Biochemical characterization of atherosclerotic plaques by endogenous multispectral fluorescence lifetime imaging microscopy

Abstract

Objective: To investigate the potential of endogenous multispectral fluorescence lifetime imaging microscopy (FLIM) for biochemical characterization of human coronary atherosclerotic plaques.

Methods: Endogenous multispectral FLIM imaging was performed on the lumen of 58 segments of postmortem human coronary artery. The fluorescence was separated into three emission bands targeting the three main arterial endogenous fluorophores (390±20 nm for collagen, 452±22.5 nm for elastin, and 550±20 for lipids). The fluorescence normalized intensity and average lifetime from each emission band was used to classify each pixel of an image as either "High-Collagen", "High-Lipids" or "Low-Collagen/Lipids" via multiclass Fisher's linear discriminant analysis.

Results: Classification of plaques as either "High-Collagen", "High-Lipids" or "Low-Collagen/Lipids" based on the endogenous multispectral FLIM was achieved with a sensitivity/specificity of 96/98%, 89/99%, and 99/99%, respectively, where histopathology served as the gold standard.

Conclusion: The endogenous multispectral FLIM approach we have taken, which can readily be adapted for in vivo intravascular catheter based imaging, is capable of reliably identifying plaques with high content of either collagen or lipids.

Figure 1

Summary of the experimental methods. A) Schematic diagram of FLIM instrumentation, B) Multispectral FLIM data acquisition, C) Multispectral FLIM data processing, D) Statistical analysis and classification, and E) Generation of biochemical map.

Figure 2

Sample multispectral FLIM maps (left panels) and representative histopathology (right panels) corresponding to A) High-Collagen (HC), B) High-Lipids (HL), and C) Low-Collagen/Lipids (LCL) plaques selected from the training set. (IT: intimal thickening, PIT: pathological IT, FA: fibroatheroma, FC-FA: FA with foam-cell infiltration, TCFA: thin-cap FA, CA: calcification).

Figure 3

Results of the statistical analysis on the multispectral FLIM features. The values of the normalized fluorescence intensity at the three spectral channels from the HC, HL and LCL groups (A) resemble the fluorescence emission spectra from collagen, LDL and elastin, respectively (B). Similarly, the values of the fluorescence lifetime from the HC, HL and LCL groups (C) resemble the fluorescence lifetime from collagen, LDL and elastin, respectively (D).

Figure 4

Results of the statistical M-FLDA classification based on the six multispectral FLIM features. (A) 2-D Linear discriminant score space, and (B) Classification performance table assessed by 10-fold cross-validation.

Figure 5

Sample biochemical maps of plaques showing heterogeneous histopathology. A) Plaque showing regions of LCL and HL. B) Plaque showing regions of HL and HC. C) Plaque showing regions of HL and LCL. D) Plaque showing regions of LCL, HC, and HL.