2.1.

SHG Microscopy

The first biological imaging experiment using SHG microscopy to study the orientation of collagen fibers was done by Freund ¹³ in 1986. Recently, SHG has been applied in different biotissues consisting of tendon,¹⁴ bone,¹⁵ tubulin,^{16, 17} muscle fibers,^{18, 19, 20, 21, 22, 23} zona pellucida,²⁴ strain in enamel rods,²⁵ and polyhedral inclusion bodies of viruses.²⁶ Collagen fiber has a highly crystalline triple-helix structure that is not centrosymmetric. Thus, SHG microscopy is an ideal tool to observe collagen fiber structure.^{16, 18, 27, 28, 29, 30, 31} The heart structure is also an interesting subject for SHG imaging to increase understanding of heart disease, including heart valves,^{32, 33} and cardiac myocytes.^{34, 35, 36} In the human atrial myocardium, the major components are cardiac muscles and collagen fibers; hence, we utilized SHG microscopy to image the human atrial myocardium. Figure 1 shows the experimental setup of the SHG microscope. The excitation light source was a home-built Cr:forsterite laser that operates at $1230\mathrm{nm}$ with a pulse width of $140\mathrm{fs}$ , a repetition rate of $110\mathrm{MHz}$ , and $450\mathrm{mW}$ of average output. The Cr:forsterite laser was pumped by $10\mathrm{W}$ of $1064\mathrm{nm}$ light from a diode-pumped $\mathrm{Nd}:\mathrm{Y}\mathrm{V}{\mathrm{O}}_4$ laser. All optics were modified to enable the passage of the excitation source light. We adapted the high-speed galvanometer mirrors (GMs) inside the FV300 scanning system with a BX51 upright microscope and a high-numerical-aperture (NA) objective (LUMPlanFl/IR $60\times$ /water/NA 0.90), all from Olympus. Real-time SHG images can be obtained by using a photomultiplier tube (PMT). CF is color filter to filter the excitation light, DM is the dichroic mirror, and S is the sample, which is mounted on the translation stage to form 2-D sectioned images.

Fig. 1

Schematic diagram of an SHG microscope: PMT, photomultiplier tube; CF, color filter; DM, dichroic mirror; S, sample.

2.2.

Fourier Transform Image Analysis

The Fourier transform is an important image processing tool that has also been used to determine the orientation and anisotropy of the microstructure, such as collagen fibers.^{37, 38, 39} Many studies have combined SHG microscopy and a Fourier transform to analyze the orientation or periodicity of the studied biological structures, such as skeletal muscle,⁴⁰ corneal tissues,⁴¹ and collagen gels.⁴² We Fourier transformed the SHG images to obtain the spatial distribution characteristics of collagen fibers in the human atrial myocardium. The acquired SHG image is composed of $512\times 512\text{pixels}$ and each pixel can be considered as a spatial function $f(x,y)$ , representing the image intensity at a point $(x,y)$ , while its spatial Fourier transform is defined by

2.3.

Materials

The study group consists of 10 patients with AF and 10 patients with NSR. All patients received open heart surgery with valvular heart disease and their age was more than $18\text{years}$ . Exclusion criteria include patients with cardiogenic shock, patients receiving major surgery within past $6\text{weeks}$ , patients with concomitant infection, patients with abnormal liver function, and pregnant women. Atrial tissues from all patients were obtained from the right atrium and were cut into sections with about $5\mathrm{mm}$ thicknesses. The specimens were incubated directly into optimal cutting temperature (OCT) compound at temperature $-20°\mathrm{C}$ until we observed these samples by using the SHG microscope at room temperature. The Ethics Committee of National Taiwan University Hospital approved the study and all patients provided written informed consent.

3.1.

Epi-SHG Images of Human Atrial Myocardium

The human atrial myocardium samples for SHG imaging study were received by open heart surgery and were cut into sections with approximately $5\mathrm{mm}$ thicknesses. The heart consists of three layers: epicardium, myocardium, and endocardium, and the cardiac muscles exist only in the myocardium. Thus, by observing the collagen fibers and cardiac muscles simultaneously, we can make sure that we observed the collagen fibers in the myocardium, not in the epicardium nor in the endocardium. Because SHG is sensitive to collagen fibers and cardiac muscle fibers, the arrangement of collagen fibers in the atrial myocardium can be revealed by SHG images. Figures 2, 2, 2, 2, 2, 2 show the SHG images taken in the atrial myocardia from different patients with NSR. The orderly arranged collagen fibers can be found to be parallel to the cardiac muscles in the same layer. We also can observe the different epi-SHG intensities between collagen fibers and muscle fibers. The epi-SHG intensity from collagen fibers is observed to be about 3 to 10 times that from muscle fibers and a similar result in skeletal muscles were revealed due to phase matching and the thickness of the tissues.⁴⁴ In contrast, the SHG images of the atrial myocardia from different AF patients are shown in Figs. 3, 3, 3, 3, 3, 3 . The collagen fibers are found to be entangled and the arrangements in AF tissues are less orderly than in NSR tissues. Clear differences in collagen fiber arrangements between NSR and AF tissues can be revealed by comparing Fig. 2 with Fig. 3.

Fig. 2

(a) to (f) SHG images of the atrial myocadia from patients with NSR. The arrangements of the collagen fibers (dashed arrows) are regular and directional to a specific angle. The cardiac muscles (solid arrows) can also be observed. (Scale bar: $50\mu \mathrm{m}$ )

Fig. 3

(a) to (f) SHG images of the atrial myocadia from patients with AF. The collagen fibers are found to be entangled and the arrangements in AF tissues are less orderly than in NSR tissues. Collagen fibrosis can be observed. (Scale bar: $50\mu \mathrm{m}$ )

3.2.

Histological Results of the Human Atrial Myocardium

After obtaining the SHG images of the atrial myocardium, the specimens were fixed in formalin and were stained with Masson’s trichrome. The sections of the atrial myocardium with the Masson’s trichrome stain by using the bright-field microscope are shown in Fig. 4 for NSR and in Fig. 4 for AF tissues. The dashed and solid arrows indicate the positions of the collagen fibers and the cardiac muscles, respectively. From Fig. 4, the collagen fibers in NSR tissues reveal orderly arrangement and are parallel to the cardiac muscles. Figure 4 shows the entangled and disoriented arrangement of the collagen fibers in AF tissues. The conclusion from the histological results is consistent with the SHG images as shown in Figs. 2 and 3 and shows that the structures of the human myocardium, including collagen fibers and cardiac muscles, can be revealed with SHG microscopy.

Fig. 4

Sections of the atrial myocardium with the Masson’s trichrome stain by using the bright-field microscope for (a) NSR and (b) AF tissues. The collagen fibers (dashed arrow) in NSR tissues show an orderly arrangement and are parallel to cardiac muscles (solid arrow). The entangled and disoriented arrangement of the collagen fibers is revealed in AF tissues and the results are consistent with SHG images.

3.3.

Fourier Transform Analysis

A Fourier transform is an ideal tool to determine the orientation and anisotropy of the collagen fibers. Utilizing a Fourier transform to analyze SHG images, the arrangement of collagen fibers can be quantified without depending on the intensities of the SHG images. We Fourier transformed the SHG images into the spatial frequency domain following Eq. 1. The epi-SHG signals from muscle fibers are weaker than those from collagen fibers and we want to analyze only the collagen fiber arrangement. Thus, we set a threshold value of the SHG intensity, and the signals from muscle fibers can be filtered before taking Fourier transform if the SHG intensities are lower than the threshold value. The Fourier images of the SHG images of the atrial myocadia are shown in Fig. 5 for NSR and in Fig. 5 for AF tissues. As mentioned, the arrangement of the collagen fibers in NSR tissues is more orderly, hence the distribution of the Fourier image shows a directional pattern in Fig. 5. The Fourier SHG image of AF tissues presents a nondirectional pattern due to randomized arrangement of the collagen fibers, as exampled in Fig. 5.

Fig. 5

Fourier transform images of the SHG images in atrial myocadia for (a) NSR and (b) AF tissues. The arrangement of the collagen fibers in NSR tissues is more orderly, hence the Fourier transform images shows a directional pattern. In contrast, the Fourier transform images in AF tissues present a nondirectional pattern due to randomized arrangement of the collagen fibers.

Furthermore, the arrangement of collagen fibers can be quantified by calculating the angle entropy from the Fourier transform images. To obtain the differences of the collagen fiber arrangements between NSR and AF tissues, we calculated the angle entropy according to the formula in Sec. 2.2 to quantify the arrangement of the collagen fibers in the atrial myocardium. The total number of over 500 SHG images of NSR and AF tissues (279 data for NSR and 251 data for AF) were analyzed. The distribution of angle entropy is shown in a histogram in Fig. 6 , where the mean values of the analyzed angle entropy for the NSR and AF tissues are $2.82\pm 0.40$ and $3.79\pm 0.47$ , respectively. The entropy is a measure of the disorder in the arrangement of the microstructure and the higher the entropy the greater the disorder.⁴⁵ Therefore, the value of angle entropy indicates that the collagen fiber arrangement in AF tissues is much more disordered than in NSR tissues. The result suggests that we succeeded in obtaining the quantification of collagen fiber arrangement by using the Fourier analysis of SHG images and the collagen fibrosis in the atrial myocardium is implicated in the existence of AF.

Fig. 6

Distribution of the angle entropy for NSR and AF tissues. The mean values of angle entropy for the NSR and AF tissues are $2.82\pm 0.40$ and $3.79\pm 0.47$ , respectively. The values of the angle entropy indicate that the arrangement in AF tissues is more disordered than in NSR tissues.