Multispectral analog-mean-delay fluorescence lifetime imaging combined with optical coherence tomography

Abstract

The pathophysiological progression of chronic diseases, including atherosclerosis and cancer, is closely related to compositional changes in biological tissues containing endogenous fluorophores such as collagen, elastin, and NADH, which exhibit strong autofluorescence under ultraviolet excitation. Fluorescence lifetime imaging (FLIm) provides robust detection of the compositional changes by measuring fluorescence lifetime, which is an inherent property of a fluorophore. In this paper, we present a dual-modality system combining a multispectral analog-mean-delay (AMD) FLIm and a high-speed swept-source optical coherence tomography (OCT) to simultaneously visualize the cross-sectional morphology and biochemical compositional information of a biological tissue. Experiments using standard fluorescent solutions showed that the fluorescence lifetime could be measured with a precision of less than 40 psec using the multispectral AMD-FLIm without averaging. In addition, we performed ex vivo imaging on rabbit iliac normal-looking and atherosclerotic specimens to demonstrate the feasibility of the combined FLIm-OCT system for atherosclerosis imaging. We expect that the combined FLIm-OCT will be a promising next-generation imaging technique for diagnosing atherosclerosis and cancer due to the advantages of the proposed label-free high-precision multispectral lifetime measurement.

Keywords: (170.2520) Fluorescence microscopy; (170.3880) Medical and biological imaging; (170.4500) Optical coherence tomography; (170.6510) Time-resolved imaging; (170.6935) Tissue characterization.

Conflict of interest statement

The authors declare that there are no conflicts of interest related to this article.

Figures

Fig. 1

A schematic diagram of the combined FLIm-OCT system with the OCT subsystem (orange), the FLIm subsystem (sky-blue), the combining optics of FLIm and OCT (green), and the workstation and data acquisition system (navy). Gray and black lines are electronic wires and optical fibers, respectively. DM: dichroic mirror; L: lens; F: filter; GVM: galvano mirror; DC: dispersion compensator; FS: acousto-optic frequency shifter; PC: polarization controller.

Fig. 2

Workflow of signal and image processing for the combined FLIm-OCT image visualization, which is split into six steps: two-channel raw data acquisition, raw data deinterleaving, OCT processing, FLIm processing, data conversion, and combined image visualization. Each box represents a task.

Fig. 3

Monte-Carlo simulation results for the multispectral AMD-FLIm subsystem. (a) Simulated pulse sequence waveforms for the different fluorescence lifetimes (0, 2, 4, 6 nsec) with the optical delay-line lengths of 1 m (IRF), 16 m (Ch 1), 31 m (Ch 2), and 46 m (Ch 3). (b) Results of the simulation for fluorescence lifetime measurements according to the length difference between the two optical delay-lines for the fluorescence emission pulse and the IRF from 0 m to 50 m. (c) Compensation results for the increase in measured fluorescence lifetime due to the use of optical delay-lines. Solid lines and dashed lines in (b) and (c) denote the expected value and the standard deviation (precision) of each simulation with the different fluorescence lifetimes.

Fig. 4

A combined FLIm-OCT imaging result for a normal-looking rabbit iliac artery tissue specimen. (a) An OCT image of the normal-looking specimen combined with the co-registered fluorescence lifetime distribution of the three spectral channels. (b-d) Pseudocolor en face maps of fluorescence lifetime distribution of channels (b) 1, (c) 2, and (d) 3, which are represented in HSV color space. Each red dashed line indicates the corresponding position to the image in (a). (e-h) The corresponding histopathological sections: (e) H&E staining, (f) ORO staining, (g) PSR staining, and (h) RAM11 immunostaining. Red arrowheads in (a) and (e-h) show the exact match between the combined FLIm-OCT image and the histological sections. Scale bars are 1 mm.

Fig. 5

A combined FLIm-OCT imaging result for an atherosclerotic rabbit iliac artery tissue specimen. (a) An OCT image of the atherosclerotic specimen combined with the co-registered fluorescence lifetime distribution of the three spectral channels. (b-d) Pseudocolor en face maps of fluorescence lifetime distribution of channels (b) 1, (c) 2 and (d) 3, which are represented in HSV color space. Each red dashed line indicates the corresponding position to the image in (a). (e-h) The corresponding histopathological sections: (e) H&E staining, (f) ORO staining, (g) PSR staining, and (h) RAM11 immunostaining. Red arrowheads in (a) and (e-h) show the exact match between the combined FLIm-OCT image and the histological sections. Scale bars are 1 mm.