Atrial fibrillation (AF) is the most common arrhythmia and affects more than 2.3 million Americans. Patients with AF suffer from many risks, such as tachycardia-induced atrial dysfunction, cardiomyopathy, thromboembolism, and stroke.1 Proper balance between synthesis and degradation of extracellular matrix molecules is critical for maintaining normal physiologic function. In the human atrial myocardium, the major component of the extracellular matrix is collagen fibers.2 Recently, many studies3, 4, 5 have observed structural remodeling of the extracellular collagen matrix and collagen fibrosis in atrial diseases such as AF. However, the detailed mechanism of AF is still not fully realized and limited reports are available on the relationship between myocardial fibrosis and AF.
Characteristic changes in the organization of fibrillar collagen are known to occur in several diseases6, 7 and could potentially serve as an early diagnostic marker. We propose that collagen fibrosis in the human atrium myocardium is involved in the development of AF. It is desired to find a simple and reproducible method to obtain information concerning collagen fibers for quantitative analysis. Many methods can be used to quantify collagen fibers, such as the weight measurement method8 and the colorimetric method.9 But these methods necessitate destroying the structure of the tissues and the tissues can not be used for further analysis (for example, pathohistological analysis). Optical imaging techniques, such as confocal microscopy10 and second-harmonic generation (SHG) microscopy11 can quantify collagen fibers as well. However, utilizing the confocal microscopy, the tissues must be stained and are still destroyed. Non-fluorescence-based SHG microscopy uses IR excitation wavelengths that minimize the energy deposition and increase the tissue penetration while maintaining intrinsically high spatial resolution without staining.12 Since 1986,13 SHG has emerged as a powerful biological imaging modality for biotissues.14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 However, using SHG microscopy for myocardium observation has never been reported. In this paper, we apply SHG microscopy for collagen fiber imaging in human atrial myocardium samples. Utilizing the SHG images, we can identify the differences in morphology and arrangement of collagen fibers between normal sinus rhythm (NSR) and AF tissues. We further quantify the arrangement of the collagen fibers by using Fourier transform images and calculating the values of angle entropy. Our study indicates that collagen fibrosis in human atrium myocardium is indeed involved in the development of AF. Our study also demonstrates that SHG imaging, a nondestructive and reproducible method to analyze the arrangement of collagen fibers, can provide explicit information about the relationship between myocardial fibrosis and AF and can serve potentially as an early diagnostic marker for AF.
Methods and Materials
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 with a pulse width of , a repetition rate of , and of average output. The Cr:forsterite laser was pumped by of light from a diode-pumped 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 /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.
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 and each pixel can be considered as a spatial function , representing the image intensity at a point , while its spatial Fourier transform is defined byand represent the spatial coordinates of the image; and and indicate the spatial frequency components along and , respectively, in the Fourier domain. Therefore, each spatial Fourier transform function is regarded as a pixel intensity value at a point to form a Fourier image in the Fourier domain. Moreover, to obtain more explicit information about collagen arrangement, we calculated the angle entropy from the Fourier transform images. The probability of the distribution function is given by in the Fourier domain in polar coordinates is defined by is calculated as43
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 . Exclusion criteria include patients with cardiogenic shock, patients receiving major surgery within past , 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 thicknesses. The specimens were incubated directly into optimal cutting temperature (OCT) compound at temperature 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.
Results and Discussion
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 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.
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.
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.
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 and , 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.
We proposed that collagen fibrosis in the human atrium myocardium is involved in the development of AF; thus, it was significant to find a simple and reproducible method to obtain the information about the collagen fibrosis. SHG could provide strong contrasts in the collagen fibers and cardiac muscle fibers of the human atrial myocardium. Thus, the epi-SHG intensities from collagen fibers are stronger than those from muscle fibers and the different arrangements of collagen fibers between NSR and AF tissues were revealed. Furthermore, we quantified the collagen fiber arrangement by using a Fourier transform and calculating angle entropy. The Fourier transform images show the nondirectional and directional patterns in AF and NSR tissues, respectively. By analyzing the Fourier transform images, the angle entropy was calculated. As expected, the higher angle entropy was obtained in AF tissues. We succeeded for the first time in quantifying the arrangement of collagen fibers of the human atrial myocardium in normal and disease states. These results indicate that the random arrangement of collagen fibers in AF tissues and collagen fibrosis in the human atrium myocardium is involved in the development of AF. Therefore, SHG microscopy can serve as a noninvasive tool for collagen fibrosis imaging in the human atrium myocardium and can be an ideal tool for AF diagnosis in the future.
This research is sponsored by the National Health Research Institute of Taiwan (Grant No. NHRI-EX99-9936EI), the National Taiwan University (Grant No. NTU 98R0036-01), the National Science Council (Grant No. NSC 97-2314-B-002-139), the Far-Eastern Memorial Hospital (Grant No. FEMH-96-C-019), and the National Taiwan University Research Center for Medical Excellence. M-T Lo was supported by NSC, Taiwan, Grant No. 98-2627-B-008-005 and joint foundation of CGH and NCU, Grant No. CNJRI-96 CGH-NCU-A3.