Nonlinear optical microscopy in decoding arterial diseases

Abstract

Pathological understanding of arterial diseases is mainly attributable to histological observations based on conventional tissue staining protocols. The emerging development of nonlinear optical microscopy (NLOM), particularly in second-harmonic generation, two-photon excited fluorescence and coherent Raman scattering, provides a new venue to visualize pathological changes in the extracellular matrix caused by atherosclerosis progression. These techniques in general require minimal tissue preparation and offer rapid three-dimensional imaging. The capability of label-free microscopic imaging enables disease impact to be studied directly on the bulk artery tissue, thus minimally perturbing the sample. In this review, we look at recent progress in applications related to arterial disease imaging using various forms of NLOM.

Keywords: Artery; Atherosclerosis; Coherent anti-Stokes Raman; Nonlinear optical microscopy; Second-harmonic generation; Two-photon excited fluorescence.

Fig. 1

a Typical anatomy of a healthy artery wall showing three distinct pathological layers, i.e. intima, media and adventitia, each separated by internal elastic lamina (IEL) and external elastic lamina (EEL). b Atherosclerotic plaque, IEL is partially broken with no clear boundary between the lesion body and the media layer. SMC, Smooth muscle cells, ECM extracellular matrix

Fig. 2

An energy diagram illustrating three best known nonlinear optical (NLO) processes used in biomedical imaging: two-photon excited fluorescence (TPEF), second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS)

Fig. 3

Schematic illustration of two common configurations of multimodal NLO microscopes. Each can provide contrast from SHG, TPEF and CARS. a Picosecond system with optical parametric oscillator (OPO). b Femtosecond system where the Stokes beam is generated in a photonic crystal fiber (PCF)

Fig. 4

TPEF image of a 95 % ethanol-fixed, unstained, rabbit’s artery cryosection (a) and an unfixed, unstained rabbit’s artery cryosection (b). Marked differences are observed in the structure of the elastic fibers located within the tunica media layer

Fig. 5

Representative two-photon emission spectra of a healthy and atherosclerotic artery lumen, respectively, at an excitation at 800 nm. The smaller/sharper band centered at 400 nm is SHG (collagen type-I) and the broader emission band centered around 500 nm is TPEF (elastin/other macromolecules, such as foam cells and macrophages). Note the dramatic different SHG/TPEF ratios between healthy and plaque-laden lumen

Fig. 6

En face NLOM images of the healthy artery lumen showing the IEL (a) and the elastic fiber network underneath the IEL (b). The imaging plane is approx. 50 μm below lumen surface (within tunica media)

Fig. 7

En face NLOM images of atherosclerotic lesions. Red/orange CARS illustrating lipid-laden structures, green TPEF illustrating fluorescent macromolecules (e.g. oxidized low-density lipoprotein), blue SHG illustrating fibrillar collagen type-I

Fig. 8

Epi-NLO images of an advanced plaque obtained at approx. depths of 10 μm (a) and 60 μm (b) from the lumen surface. Blue SHG fibrillar collagen type-I, red/orange CARS lipid-rich structure buried under a fibrous cap