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. 2016 Aug 4;4(3):102-111.
doi: 10.1016/j.pacs.2016.07.001. eCollection 2016 Sep.

Multimodal optoacoustic and multiphoton microscopy of human carotid atheroma

Affiliations

Multimodal optoacoustic and multiphoton microscopy of human carotid atheroma

Markus Seeger et al. Photoacoustics. .

Abstract

Carotid artery atherosclerosis is a main cause of stroke. Understanding atherosclerosis biology is critical in the development of targeted prevention and treatment strategies. Consequently, there is demand for advanced tools investigating atheroma pathology. We consider hybrid optoacoustic and multiphoton microscopy for the integrated and complementary interrogation of plaque tissue constituents and their mutual interactions. Herein, we visualize human carotid plaque using a hybrid multimodal imaging system that combines optical resolution optoacoustic (photoacoustic) microscopy, second and third harmonic generation microscopy, and two-photon excitation fluorescence microscopy. Our data suggest more comprehensive insights in the pathophysiology of atheroma formation and destabilization, by enabling congruent visualization of structural and biological features critical for the atherosclerotic process and its acute complications, such as red blood cells and collagen.

Keywords: BF, Brightfield; CAE, Carotid thrombendarterectomy; CMR, Continuous multirecord; Collagen; DAQ, Data acquisition; FOV, Field of view; GM, Galvanometric mirrors; HE, Hemalaun-Eosin; Human carotid atheroma; IPH, Intraplaque hemorrhage; LDL, Low density lipoprotein; MAP, Maximum amplitude projection; MPM, Multiphoton microscopy; MPOM, Multiphoton and optoacoustic microscopy; Multimodal microscopy; NLO, Non-linear optical; Non-linear optical microscopy; OAM, Optoacoustic microscopy; Optoacoustic microscopy; PMT, Photo multiplier tube; PSR, Picro-Sirius Red; Photoacoustic microscopy; RBC, Red blood cell; ROI, Region of interest; Red blood cells; SHG, Second harmonic generation; SMC, Smooth muscle cell; THG, Third harmonic generation; TPEF, Two-photon excitation fluorescence.

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Figures

Fig. 1
Fig. 1
Schematic depiction of the MPOM system consisting of two interchangeable microscopy systems, namely OAM and MPM. Abbreviations: AFG, arbitrary function generator; AMP, amplifier; BS, beamsplitter; DAQ, data acquisition card; F, optical filter; FM, flippable mirror; GM, galvanometric mirrors; GMC, GM control; I, iris diaphragm; L, lens; LP-DM, longpass dichroic mirror; M, mirror; ND, neutral density filter; OA, optoacoustic signal; OL, microscope objective lens; P, prism; PD, photodiode; PMT, photomultiplier tube; S, xyz stage; SHG, second harmonic generation signal; SP-DM, shortpass dichroic mirror; UT, ultrasound transducer; THG, third harmonic generation signal; TPEF, two-photon excitation fluorescence signal.
Fig. 2
Fig. 2
Coarse imaging and ROI selection of human carotid atheroma. (a) Schematic depiction of a typical atherosclerotic vascular cross-section. (b) Widefield BF observation of the unstained atheroma sample used for MPOM imaging and identification of lumen, cap, and shoulders. (c) Coarse OAM scan indicating a progressed intraplaque RBC embedding. Final ROI selection for subsequent MPOM imaging is indicated by the red and white boxes.
Fig. 3
Fig. 3
Hybrid microscopy imaging of human carotid atheroma at the shoulder region (ROI 1). (a) Overlay of OA, SHG, THG, and TPEF demonstrates an unscathed condition of the connective tissue (e.g. collagen and elastin), no large inclusions or fissures, and a negligible amount of embedded blood residues. Separate depiction of (b) RBC embeddings (OAM), (c) collagen (SHG), (d) tissue appearance (BF), (e) tissue morphology (THG), and (f) mainly elastin (TPEF). Profile for subsequent analysis is indicated by the white line in (a).
Fig. 4
Fig. 4
Hybrid microscopy imaging of human carotid atheroma at the cap region (ROI 2). (a) Overlay of OAM, SHG, THG, and TPEF indicates coarse interleaving structure of embedded blood residues and connective tissue bands of collagen and elastin. Separate depiction of (b) RBC embeddings (OAM), (c) collagen (SHG), (d) tissue appearance (BF), (e) tissue morphology (THG), and (f) mainly elastin and RBCs (TPEF). Profile for subsequent analysis is indicated by the white line in (a).
Fig. 5
Fig. 5
Hybrid microscopy imaging of human carotid atheroma at the lipid core (ROI 3). (a) Overlay of OAM, SHG, THG, and TPEF reveals fine interleaving structure of embedded blood residues and collagen bands. Separate depiction of (b) RBC embeddings (OAM), (c) collagen (SHG), (d) tissue appearance (BF), (e) tissue morphology (THG), and (f) elastin, LDL, foam cells, and RBCs (TPEF). Profile for subsequent analysis is indicated by the white line in (a).
Fig. 6
Fig. 6
Profiles of embedded blood residues, collagen, and elastin along the white lines indicated in Figs. 3 –5 a. The interleaving interaction between intraplaque coagulated RBCs and collagen occurs on different size levels leading to undisturbed collagen bands (a; ROI 1), coarse interrupted collagen stripes (b; ROI 2), and fine expanded structures (c; ROI 3). Partially shared signatures among OAM and TPEF reveal blood distribution whereas signals occurring only in TPEF present elastin within the sample. Colored areas represent the dominant compound in the respective region (red: RBC (OAM); green: collagen (SHG)).
Fig. 7
Fig. 7
Histological images of human carotid atheroma validating MPOM performance. (a) HE staining of cell nuclei (blue), cytoplasm (pink), and RBCs (dark red), (b) PSR staining of collagen (dark red), and (c) Orcein staining of elastin of the sample regions at the shoulder (ROI 1). Analogous depiction of (d,g) HE, (e,h) PSR, and (f,i) Orcein stainings for ROI 2 and ROI 3, respectively.

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