Lasers Med Sci DOI 10.1007/s10103-015-1767-9
ORIGINAL ARTICLE
Layer-resolved colorectal tissues using nonlinear microscopy Lianhuang Li 1 & Hongsheng Li 1 & Zhifen Chen 2 & Shuangmu Zhuo 1 & Changyin Feng 3 & Yinghong Yang 3 & Guoxian Guan 2 & Jianxin Chen 1
Received: 4 November 2014 / Accepted: 5 May 2015 # Springer-Verlag London 2015
Abstract In this work, multiphoton microscopy (MPM), based on the nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to evaluate the feasibility of using MPM to distinguish layers of the bowel wall. It was found that MPM has the ability to identify the four-layer microstructures of colorectal tissues including mucosa, submucosa, muscularis propria, and serosa as there are many intrinsic signal sources in each layer. Our results also showed the capability of using the quantitative analyses of MPM images for quantifying some feature parameters including the nuclear area, nuclear-to-cytoplasmic ratio, and optical redox ratio. This work demonstrates that MPM has the potential in noninvasively monitoring the development and progression of colorectal diseases and then guiding effective treatment.
Lianhuang Li, Hongsheng Li and Zhifen Chen contributed equally to this work. * Guoxian Guan [email protected] * Jianxin Chen [email protected] 1
Institute of Laser and Optoelectronics Technology, Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou 350007, China
2
Department of Colorectal Surgery, The Affiliated Union Hospital, Fujian Medical University, Fuzhou 350001, China
3
Department of Pathology, The Affiliated Union Hospital, Fujian Medical University, Fuzhou 350001, China
Keywords Multiphoton microscopy (MPM) . Colorectal tissue . Two-photon excited fluorescence (TPEF) . Second harmonic generation (SHG)
Introduction Colorectal cancer is the third most common cancer in both men and women. An estimated 96,830 cases of colonic cancer as well as 40,000 cases of rectal cancer are expected to occur, and 50,310 deaths from colorectal cancer are expected to occur in 2014, accounting for 9 % of all cancer deaths [1]. In general, mortality declines and treatments that prolong life and improve the quality of life of patients will probably depend on the accurate detection of different diseases. At present, the examination techniques in clinical application mainly include computed tomography (CT), magnetic resonance imaging (MRI), and endorectal ultrasound (ERUS). However, these techniques cannot accurately show various pathologic changes because of the lack of resolution; especially, the major flaw in the use of ERUS is its inability to distinguish tumor from fibrosis, while the accuracy of CT in evaluating colorectal cancer is limited by its ability to distinguish layers of the bowel wall [2, 3]. Therefore, there is widespread interest in developing new methods for detecting colorectal diseases. The advent of novel optical multiphoton microscopy (MPM) techniques opens a new window in exploring colorectal diseases [4, 5]. This nonlinear optical imaging technique is a remarkable evolution of biological science for visualizing cellular and subcellular events within intact tissues with its inherent Boptical sectioning^ capability, deeper penetration, and minimal phototoxicity and photobleaching and has come to occupy a prominent place in modern biomedical research with its ability of high resolution and sensitivity [6–8].
Lasers Med Sci
Materials and methods
specimen. One channel covered the wavelength range from 430 to 716 nm for collection of TPEF signals; another channel covered the wavelength range from 389 to 419 nm for collection of SHG signals at an excitation wavelength of 810 nm. In order to increase the contrast of the TPEF/SHG image, the TPEF images were color coded in red and SHG images were color coded in green. To obtain a large-area image, an optional HRZ 200 fine-focusing stage (HRZ 200 stage, Carl Zeiss) was used to translate the samples after x–y scan of the samples. The images were obtained at 2.56 μs/pixel. All images have a 12bit pixel depth.
Sample preparations
Quantification methods
This study was approved by the Institutional Review Board of the Affiliated Union Hospital of Fujian Medical University. Written informed consent was obtained from every patient prior to study participation. A total of seven normal colorectal tissues were obtained from surgical resection. The specimens were placed in a standard pathologic transport container covered with ice and then sent to pathology laboratory after being removed by surgeons. Two consecutive sections were obtained from each colorectal tissue by cryostat microtome: a 10-μm slice was used for multiphoton microscopic imaging and a 5-μm slice was stained with H&E for histological comparison to the multiphoton microscopic results. Moreover, to avoid dehydration or shrinkage during the imaging process, a little phosphatebuffered saline solution was dripped into the specimen.
In order to quantitatively describe characteristic of cellular architecture and to measure the metabolic state of colorectal tissues, the nuclear area, nuclear-to-cytoplasmic ratio, and redox ratio were calculated, respectively. The nuclear area was obtained by measuring the area of nuclear boundary. The redox ratio was the intensity ratio of cellular NADH to FAD fluorescence, while the nuclear-to-cytoplasmic ratio was obtained by measuring the TPEF pixels of cells and the whole pixels in each image. They were expressed as the form of a mean value followed by its standard deviation (mean±SD). In particular, its standard deviation signified the changed degree of each feature parameter, respectively.
Given the advantages provided by MPM, it has been widely applied to detect colorectal tissues; however, focus of these researches was confined within a specific layer such as mucosa [9–12]. Therefore, the research objective of this work is the whole bowel wall including mucosa, submucosa, muscularis propria, and serosa, and the aim of this study is to investigate whether MPM has the ability to identify the four-layer structures of the bowel wall.
Results Multiphoton microscopic imaging system Nonlinear spectral analysis of normal colorectal tissues MPM images were achieved by a nonlinear optical system which has been described previously [13, 14]. In short, an inverted microscope (LSM 510 META, Zeiss) was equipped with a mode-locked Ti: Sapphire laser (110 fs, 76 MHz) that is tunable from 700 to 980 nm (Mira 900-F, Coherent) for multiphoton excitation. In this work, a high numerical aperture, oil immersion objective (Plan-Apochromat 63×, N.A.1.4, Zeiss) was employed for focusing the excitation beam into tissue samples and also for collecting the backward signals to obtain high-resolution images. The META detector consists of a high-quality reflective grating as a dispersive element and an optimized 32-channel photomultiplier tube (PMT) array detector to collect emission signals. The system has two modes of operation: lambda mode and channel mode. The lambda mode can carry out the nonlinear spectral imaging and acquire emission spectra of regions of interest by plotting the mean intensity of all pixels. The channel mode has eight independent channels, and each channel covers a spectral width of approximately 340 nm, ranging from 377 to 716 nm. In our study, two independent channels were chosen to collect two-photon excited fluorescence (TPEF)/second harmonic generation (SHG) signals from the
Firstly, the focus is on analyzing the origins of multiphoton signals in each layer of colorectal tissues through a nonlinear spectral imaging method and then determining whether the four-layer microstructures of colorectal tissues can be distinguished by the MPM. Therefore, the emission spectroscopic investigations were performed in fresh colorectal tissues. The fresh tissue sections were excited at 810-nm excitation wavelength, and emission signals were collected between 377 and 716 nm using spectral detector under the Lambda mode setting. The normalized multiphoton emission spectra after subtraction of background were displayed in Fig. 1. To be specific, Fig. 1a–d shows representative spectra from mocosa, submucosa, muscular layer, and serosa, respectively. In general, there are seven peaks at approximately 405, 475, 511, 535, 570, 630, and 690 nm. According to previous publications, the fluorescence peaks around 475 and 535 nm were responsible for nicotinamide adenine dinucleotide coenzyme (NADH) and flavin adenine dinucleotide (FAD), respectively [15], while the peaks at 405 nm (half the 810-nm excitation wavelength) and 511 nm were attributed to collagen and cellular structural protein or elastin, respectively [16]. Additionally,
Lasers Med Sci Fig. 1 Nonlinear spectral images of various layers of colorectal tissue obtained with an excitation wavelength of 810 nm, the left column is the spectral imagings of regions of interest and corresponding spectra is in the right. a Emission spectrum of mucosa. b Emission spectrum of submucosa. c Emission spectrum of muscularis propria. d Emission spectrum of fibrous membrane
the fluorescence peaks around 630 and 690 nm corresponded to porphyrin derivatives and the peak wavelength 570 nm possibly originated from eosinophils and lipopigments [17–19]. The origins of intrinsic signals in colorectal tissues were summarized and presented in Table 1. According to the spectroscopic research, we can draw a conclusion that MPM can be used for layer-resolved imaging study of colorectum without the use of exogenous contrast agents as there are many intrinsic signal sources. Figure 2 shows representative MPM
image of the four-layer microstructures of the bowel wall and corresponding H&E light microscopic image. It is clear that MPM has the ability to distinguish layers of the colorectal tissues. The nonlinear spectral analysis can link the biochemical and morphologic properties of tissues to individual patient status and elucidate several parameters describing health level of organisms, whereas morphological image can provide a substrate for the identification and classification of colorectal diseases. Therefore, the following study is to investigate every layer structure features of colorectal tissues by MPM.
Lasers Med Sci Table 1 Intrinsic signals
The origins of intrinsic signals in colorectal tissues TPEF signal
SHG signal
Mucosa
NADH, FAD, elastic fibers, Collagen fibers porphyrin derivatives, eosinophils, and lipopigments Submucosa Elastic fibers, collagen bundles, Collagen fibers, collagen and elastin in the vessel wall bundles, and collagen in the vessel wall Muscularis NADH, FAD, elastic fibers, Muscle fibers, collagen propria collagen bundles, blood fibers, and collagen vessels, myenteric plexus, bundles porphyrin derivatives, eosinophils, and lipopigments Serosa Elastic fibers, collagen bundles, Collagen fibers and and blood vessels collagen bundles
Mucosa Figure 3 displays representative TPEF/SHG images of normal colorectal mucosa and corresponding H&E light microscopic image. It can be clearly seen that this layer is mainly made up of epithelial layer (position 1 in Fig. 3c), colorectal glands (position 3), lamina propria (position 4), and muscularis mucosae (position 5) [20]. The epithelial layer has a singlelayered columnar epithelium mainly including absorptive cells and goblet cells and serves as a protective barrier against pathogens from intestinal lumen. Cellular outline is revealed based on the fluorescent cytoplasmic granules of mitochondria and nonfluorescent nuclei (blue arrows in Fig. 3a). The goblet cells are also found by the obvious mucin droplets (pink arrows in Fig. 3a). Additionally, a thin subepithelial connective tissue (position 2 in Fig. 3c) is identified, which
is primarily composed of collagen and sustains the surface epithelium [21, 22]. As can be seen in Fig. 3, colorectal glands (position 3), which mainly consist of absorptive cells and goblet cells, show a characteristic Brow of test tubes^ appearance, and each gland is a single and nonbranching individual. Some slight differences in morphology can be recognized too. Interestingly, more goblet cells appear in the glands. The lamina propria has a connective tissue structure mainly composed of collagen, and mounting evidence has attested to its functional importance as a diffuse lymphoid organ [22]. It extends from the subepithelial band to muscularis mucosae (position 5 in Fig. 3c) and is easily detected via SHG signal (Fig. 3b). Moreover, many cells (yellow arrows) are also found in lamina propria, which contain plasma cells, T lymphocytes, fibroblasts, macrophages, etc. [20]. The muscularis mucosae (position 5 in Fig. 3c), which is a thin layer and separates the mucosa from submucosa, is constituted of smooth muscle fibers and mainly plays a supportive role. The combination of SHG/TPEF can provide in tandem complementary information on tissue architecture and function shown in Fig. 3c, and all these tissue architecture details readily correlate with the H&E-stained image (Fig. 3d). Submucosa Figure 4 shows representative TPEF/SHG images of normal colorectal submucosa and corresponding H&E light microscopic image. It is well known that the submucosa is a loose connective tissue layer mainly composed of collagen and elastin, which are consistent with our spectroscopic investigations (Fig. 1b). MPM images reveal that this layer contains numerously coarse and spiral collagen bundles (blue arrows in Fig. 4), collagen fibers (white arrows in Fig. 4), ropelike
Fig. 2 Representative MPM image of the four-layer microstructures of the bowel wall and corresponding H&E light microscopic image. (a) MPM image. (b) H&E-stained image
Lasers Med Sci Fig. 3 Representative highresolution large-area MPM images and corresponding H&E light microscopic image from normal colorectal mucosa. a TPEF image (color-coded red), blue arrows, pink arrows, and yellow arrows indicate nonfluorescent nuclei, mucin droplets, and inflammatory cells, respectively. b SHG image (colorcoded green). c TPEF/SHG image overlay, position 1: surface epithelium, position 2: subepithelial band of collagen, position 3: colorectal gland, position 4: lamina propria, position 5: muscularis mucosae. d H&E-stained image (40× magnification). The scale bar represents 200 μm
elastic fibers (pink arrows in Fig. 4), blood vessels (position 1 in Fig. 4), and lymphatic vessels (position 2 in Fig. 4). According to previous study, meshy collagen fibers are visualized distinctively in Fig. 4b via its strong SHG signal because Fig. 4 Representative highresolution large-area MPM images and corresponding H&E light microscopic image from normal colorectal submucosa. a TPEF image (color-coded red), blue arrows and pink arrows represent collagen bundles and elastic fibers, respectively. b SHG image (color-coded green), blue arrows and white arrows indicate collagen bundles and collagen fibers, respectively. c TPEF/SHG image overlay, position 1: blood vessel, position 2: lymphatic vessel. d H&E-stained image (40× magnification). The scale bar represents 200 μm
of its sole non-centrosymmetric structure, while the long, ropelike elastic fibers are displayed very well in Fig. 4a by its TPEF signal. The collagen fibers and elastic fibers interlace with each other but can be identified based on their intrinsic
Lasers Med Sci
signals (Fig. 4c). Interestingly, collagen bundles are in yellow when overlaying SHG image with TPEF image (Fig. 4c), as they can produce comparable SHG and TPEF signals due to certain intramolecular and intermolecular cross-links between collagen bundles [23]. Moreover, MPM can clearly display the blood vessels (position 1 in Fig. 4) through the TPEF signal from elastin in vascular intima [24], whereas the lymphatic vessels (position 2 in Fig. 4) can be identified too by the SHG signal and have a distinctively appearance in comparison with the blood vessels [25]. Apparently, the thinner walls of lymph vessels contain collagen component and this can help differentiate them from blood vessels by using MPM. However, discrepancy between blood vessels and lymphatic vessels is unobvious in the corresponding H&E-stained image (Fig. 4d). Muscularis propria and serosa Figure 5 displays representative TPEF/SHG images of normal colorectal muscularis and corresponding H&E light microscopic image. MPM reveals that the muscular layer mainly consists of internal circular muscle (position 1 in Fig. 5) and external longitudinal muscle (position 2 in Fig. 5). The muscular tissues contain abundant muscle fibers via the SHG signal (Fig. 5b) and elastic fibers by the TPEF signal (Fig. 5a), which agree well with the spectral analysis (Fig. 1c). The myofibers present striated morphology and are parallel with each other, while the elastic fibers are threadlike, spindly, or spiral and express an orientation effect along with the distribution of well-ordered muscle fibers. In addition, the loose connective tissue (position 3 in Fig. 5) and ganglia known as myenteric plexus (position 4 in Fig. 5) inside of the muscular layer can be identified; furthermore, many small blood vessels (white arrows in Fig. 5) and lymphatics (blue arrows in Fig. 5) embedded in the connective tissue are detected too. These same details of architecture correlate readily with the H&Estained image (Fig. 5d). Figure 6 shows representative TPEF/SHG images of normal colorectal serosa and corresponding H&E light microscopic image. According to previous spectrum analysis, serosal layer also contains plenty of collagen and elastin. Indeed, MPM reveals that like the submucosa, serosa mainly composes of elastic fibers (Fig. 6a) and collagen fibers (Fig. 6b). Actually, the serosal layer, also called fibrous membrane, which encases and acts to anchor the intestinal tube, is a layer of connective tissue too. Quantitative analysis Finally, our work is to extract some diagnostic features from the MPM images using the quantitative analysis of image. The alteration of cellular architecture is an important indicator for monitoring colorectal diseases. In order to quantitatively
describe characteristic of normal cell architecture, the nuclear area and nuclear-to-cytoplasmic ratio were calculated, respectively. The quantitative results reveal that the nuclear area of normal epithelial cells was 39.86±8.96 μm2 (n=30), while the nuclear-to-cytoplasmic ratio in normal mucosa was about 0.33±0.09 (n=30). Furthermore, the fluorescence intensity ratio of NADH over FAD was calculated as it can reflect the degree of pathological changes when normal and abnormal tissues are compared in the same condition and has become a good indicator of cellular metabolic state [15]. In this study, the redox ratio was approximately 1.12±0.06 (n=30) that is consistent with the normal metabolism state [26].
Discussion The incidence rate of colorectal cancer is increasing in developing countries with the alteration of lifestyle, particularly the western patterns of diet and physical inactivity. Although there are many examination techniques, such as CT, PET, MRI, ERUS, and they have made much progress, there are also many weaknesses including lack of resolution or need of exogenous contrast agents [2, 27]. Currently, pathological examination is still the gold standard. The process of pathological diagnosis involves removal of tissue, putting it into 10 % buffered formalin, paraffin embedding, sectioning, staining (H&E), and then examination under bright-field white light microscopy. There exist many drawbacks including cost, wait time for results, and the excisional process itself with its inherent risks of adverse events such as bleeding or perforation [10]. Thus, development of new imaging technologies, which are capable of detecting microscopic tumors or precursor lesions, can be of tremendous help. MPM has become one of the most important optical imaging techniques for biomedical research during recent years, which provides a superior effective resolution and an increased penetration depth in comparison with single photon excitation during confocal microscopy. As a result of the effects of nonlinear optics, various molecular components can be discriminated with MPM without use of any contrast agents [4, 9]. The detection of these molecules is based on specific autofluorescence or SHG signals generated by multiphoton excitation. In our study, there are different endogenous fluorophores in various layers of colorectal tissues according to the nonlinear spectral analysis. To be specific, the endogenous fluorophores including elastin, eosinophils, lipofuscin, porphyrin derivative, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate [NADH and NADPH, NAD(P)H], can easily generate strong TPEF signal without the need of contrast agents, while collagen fibers and muscle fibers can produce SHG signal due to their non-centrosymmetric molecular structure [28–31].
Lasers Med Sci Fig. 5 Representative highresolution large-area MPM images and corresponding H&E light microscopic image from normal colorectal muscularis. a TPEF image (color-coded red). b SHG image (color-coded green). c TPEF/SHG image overlay, position 1: circular muscle in inner layer, position 2: longitudinal muscle in outer layer, position 3: connective tissue, position 4: myenteric plexus; d H&E-stained image (40× magnification). The scale bar represents 200 μm
These endogenous signals make it possible to label-freely detect the four-layer microstructures of colorectal tissues based on MPM, which is helpful for clinicians to diagnose and treat colorectal diseases originated from different layers. Our results demonstrate that MPM has the ability not only to identify the epithelial cells, goblet cells, intestinal glands, lamina propria, and muscularis mucosae but also to reveal the collagen fibers, elastin fibers, blood vessel, lymphatic vessel, and myenteric plexus. MPM can also quantify some feature parameters of the cells: the nuclear area, nuclear-tocytoplasmic ratio and optical redox ratio, which are important signs of colorectal diseases. Therefore, it is obvious that MPM can easily distinguish the normal cells from abnormal cells as the tumor cells are characterized by enlarged nuclei and increased nuclear–cytoplasmic ratio. Furthermore, the optical
redox ratio can be used as a diagnostic tool to evaluate cell energy metabolism. It is well known that the metabolic state of the abnormal cells is known to be accelerated compared to normal cells so that comparison of the redox ratios of normal and abnormal tissues from the same patient can reflect the degree of pathological changes. In brief, these results indicate that MPM can determine the components of different layers in colorectal tissues through the nonlinear spectral analysis and can effectively distinguish the layer structure of colorectal tissues by excitation of intrinsic fluorescent molecules. Given the advantages provided by MPM, it is foreseen that MPM can serve as an effective and noninvasive tool for monitoring changes in morphology and biochemistry of colorectal tissues and then guiding the diagnosis and treatment of colorectal diseases.
Lasers Med Sci Fig. 6 Representative highresolution large-area MPM images and corresponding H&E light microscopic image from normal colorectal serosa. a TPEF image (color-coded red); b SHG image (color-coded green); c TPEF/SHG image overlay; d H&E-stained image (40× magnification). The scale bar represents 200 μm
Conclusions In this work, our results demonstrate that multiphoton microscopic imaging technique has the ability to determine the constituents of colorectal tissues by nonlinear spectral analysis and to label-freely identify the four-layer microstructures of fresh colorectal tissue, as well as to provide quantitative information by combining TPEF imaging with SHG imaging. With the continuous advancement of multiphoton-based endoscopic techniques and new laser sources, we have reason to believe that this particularly promising technique will open up many new possibilities for the diagnosis and treatment of colorectal diseases in the near future. Acknowledgments The work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT1115), the National Natural Science Foundation of China (Grant
No. 81271620), the Natural Science Foundation for Distinguished Young Scholars of Fujian Province (Grant No. 2014J06016), and the Youth Scientific Research Foundation of Fujian Provincial Department of Health (2013-2-36), National Clinical Key Specialty Construction Project (General Surgery).
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ORIGINAL ARTICLE
Layer-resolved colorectal tissues using nonlinear microscopy Lianhuang Li 1 & Hongsheng Li 1 & Zhifen Chen 2 & Shuangmu Zhuo 1 & Changyin Feng 3 & Yinghong Yang 3 & Guoxian Guan 2 & Jianxin Chen 1
Received: 4 November 2014 / Accepted: 5 May 2015 # Springer-Verlag London 2015
Abstract In this work, multiphoton microscopy (MPM), based on the nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to evaluate the feasibility of using MPM to distinguish layers of the bowel wall. It was found that MPM has the ability to identify the four-layer microstructures of colorectal tissues including mucosa, submucosa, muscularis propria, and serosa as there are many intrinsic signal sources in each layer. Our results also showed the capability of using the quantitative analyses of MPM images for quantifying some feature parameters including the nuclear area, nuclear-to-cytoplasmic ratio, and optical redox ratio. This work demonstrates that MPM has the potential in noninvasively monitoring the development and progression of colorectal diseases and then guiding effective treatment.
Lianhuang Li, Hongsheng Li and Zhifen Chen contributed equally to this work. * Guoxian Guan [email protected] * Jianxin Chen [email protected] 1
Institute of Laser and Optoelectronics Technology, Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou 350007, China
2
Department of Colorectal Surgery, The Affiliated Union Hospital, Fujian Medical University, Fuzhou 350001, China
3
Department of Pathology, The Affiliated Union Hospital, Fujian Medical University, Fuzhou 350001, China
Keywords Multiphoton microscopy (MPM) . Colorectal tissue . Two-photon excited fluorescence (TPEF) . Second harmonic generation (SHG)
Introduction Colorectal cancer is the third most common cancer in both men and women. An estimated 96,830 cases of colonic cancer as well as 40,000 cases of rectal cancer are expected to occur, and 50,310 deaths from colorectal cancer are expected to occur in 2014, accounting for 9 % of all cancer deaths [1]. In general, mortality declines and treatments that prolong life and improve the quality of life of patients will probably depend on the accurate detection of different diseases. At present, the examination techniques in clinical application mainly include computed tomography (CT), magnetic resonance imaging (MRI), and endorectal ultrasound (ERUS). However, these techniques cannot accurately show various pathologic changes because of the lack of resolution; especially, the major flaw in the use of ERUS is its inability to distinguish tumor from fibrosis, while the accuracy of CT in evaluating colorectal cancer is limited by its ability to distinguish layers of the bowel wall [2, 3]. Therefore, there is widespread interest in developing new methods for detecting colorectal diseases. The advent of novel optical multiphoton microscopy (MPM) techniques opens a new window in exploring colorectal diseases [4, 5]. This nonlinear optical imaging technique is a remarkable evolution of biological science for visualizing cellular and subcellular events within intact tissues with its inherent Boptical sectioning^ capability, deeper penetration, and minimal phototoxicity and photobleaching and has come to occupy a prominent place in modern biomedical research with its ability of high resolution and sensitivity [6–8].
Lasers Med Sci
Materials and methods
specimen. One channel covered the wavelength range from 430 to 716 nm for collection of TPEF signals; another channel covered the wavelength range from 389 to 419 nm for collection of SHG signals at an excitation wavelength of 810 nm. In order to increase the contrast of the TPEF/SHG image, the TPEF images were color coded in red and SHG images were color coded in green. To obtain a large-area image, an optional HRZ 200 fine-focusing stage (HRZ 200 stage, Carl Zeiss) was used to translate the samples after x–y scan of the samples. The images were obtained at 2.56 μs/pixel. All images have a 12bit pixel depth.
Sample preparations
Quantification methods
This study was approved by the Institutional Review Board of the Affiliated Union Hospital of Fujian Medical University. Written informed consent was obtained from every patient prior to study participation. A total of seven normal colorectal tissues were obtained from surgical resection. The specimens were placed in a standard pathologic transport container covered with ice and then sent to pathology laboratory after being removed by surgeons. Two consecutive sections were obtained from each colorectal tissue by cryostat microtome: a 10-μm slice was used for multiphoton microscopic imaging and a 5-μm slice was stained with H&E for histological comparison to the multiphoton microscopic results. Moreover, to avoid dehydration or shrinkage during the imaging process, a little phosphatebuffered saline solution was dripped into the specimen.
In order to quantitatively describe characteristic of cellular architecture and to measure the metabolic state of colorectal tissues, the nuclear area, nuclear-to-cytoplasmic ratio, and redox ratio were calculated, respectively. The nuclear area was obtained by measuring the area of nuclear boundary. The redox ratio was the intensity ratio of cellular NADH to FAD fluorescence, while the nuclear-to-cytoplasmic ratio was obtained by measuring the TPEF pixels of cells and the whole pixels in each image. They were expressed as the form of a mean value followed by its standard deviation (mean±SD). In particular, its standard deviation signified the changed degree of each feature parameter, respectively.
Given the advantages provided by MPM, it has been widely applied to detect colorectal tissues; however, focus of these researches was confined within a specific layer such as mucosa [9–12]. Therefore, the research objective of this work is the whole bowel wall including mucosa, submucosa, muscularis propria, and serosa, and the aim of this study is to investigate whether MPM has the ability to identify the four-layer structures of the bowel wall.
Results Multiphoton microscopic imaging system Nonlinear spectral analysis of normal colorectal tissues MPM images were achieved by a nonlinear optical system which has been described previously [13, 14]. In short, an inverted microscope (LSM 510 META, Zeiss) was equipped with a mode-locked Ti: Sapphire laser (110 fs, 76 MHz) that is tunable from 700 to 980 nm (Mira 900-F, Coherent) for multiphoton excitation. In this work, a high numerical aperture, oil immersion objective (Plan-Apochromat 63×, N.A.1.4, Zeiss) was employed for focusing the excitation beam into tissue samples and also for collecting the backward signals to obtain high-resolution images. The META detector consists of a high-quality reflective grating as a dispersive element and an optimized 32-channel photomultiplier tube (PMT) array detector to collect emission signals. The system has two modes of operation: lambda mode and channel mode. The lambda mode can carry out the nonlinear spectral imaging and acquire emission spectra of regions of interest by plotting the mean intensity of all pixels. The channel mode has eight independent channels, and each channel covers a spectral width of approximately 340 nm, ranging from 377 to 716 nm. In our study, two independent channels were chosen to collect two-photon excited fluorescence (TPEF)/second harmonic generation (SHG) signals from the
Firstly, the focus is on analyzing the origins of multiphoton signals in each layer of colorectal tissues through a nonlinear spectral imaging method and then determining whether the four-layer microstructures of colorectal tissues can be distinguished by the MPM. Therefore, the emission spectroscopic investigations were performed in fresh colorectal tissues. The fresh tissue sections were excited at 810-nm excitation wavelength, and emission signals were collected between 377 and 716 nm using spectral detector under the Lambda mode setting. The normalized multiphoton emission spectra after subtraction of background were displayed in Fig. 1. To be specific, Fig. 1a–d shows representative spectra from mocosa, submucosa, muscular layer, and serosa, respectively. In general, there are seven peaks at approximately 405, 475, 511, 535, 570, 630, and 690 nm. According to previous publications, the fluorescence peaks around 475 and 535 nm were responsible for nicotinamide adenine dinucleotide coenzyme (NADH) and flavin adenine dinucleotide (FAD), respectively [15], while the peaks at 405 nm (half the 810-nm excitation wavelength) and 511 nm were attributed to collagen and cellular structural protein or elastin, respectively [16]. Additionally,
Lasers Med Sci Fig. 1 Nonlinear spectral images of various layers of colorectal tissue obtained with an excitation wavelength of 810 nm, the left column is the spectral imagings of regions of interest and corresponding spectra is in the right. a Emission spectrum of mucosa. b Emission spectrum of submucosa. c Emission spectrum of muscularis propria. d Emission spectrum of fibrous membrane
the fluorescence peaks around 630 and 690 nm corresponded to porphyrin derivatives and the peak wavelength 570 nm possibly originated from eosinophils and lipopigments [17–19]. The origins of intrinsic signals in colorectal tissues were summarized and presented in Table 1. According to the spectroscopic research, we can draw a conclusion that MPM can be used for layer-resolved imaging study of colorectum without the use of exogenous contrast agents as there are many intrinsic signal sources. Figure 2 shows representative MPM
image of the four-layer microstructures of the bowel wall and corresponding H&E light microscopic image. It is clear that MPM has the ability to distinguish layers of the colorectal tissues. The nonlinear spectral analysis can link the biochemical and morphologic properties of tissues to individual patient status and elucidate several parameters describing health level of organisms, whereas morphological image can provide a substrate for the identification and classification of colorectal diseases. Therefore, the following study is to investigate every layer structure features of colorectal tissues by MPM.
Lasers Med Sci Table 1 Intrinsic signals
The origins of intrinsic signals in colorectal tissues TPEF signal
SHG signal
Mucosa
NADH, FAD, elastic fibers, Collagen fibers porphyrin derivatives, eosinophils, and lipopigments Submucosa Elastic fibers, collagen bundles, Collagen fibers, collagen and elastin in the vessel wall bundles, and collagen in the vessel wall Muscularis NADH, FAD, elastic fibers, Muscle fibers, collagen propria collagen bundles, blood fibers, and collagen vessels, myenteric plexus, bundles porphyrin derivatives, eosinophils, and lipopigments Serosa Elastic fibers, collagen bundles, Collagen fibers and and blood vessels collagen bundles
Mucosa Figure 3 displays representative TPEF/SHG images of normal colorectal mucosa and corresponding H&E light microscopic image. It can be clearly seen that this layer is mainly made up of epithelial layer (position 1 in Fig. 3c), colorectal glands (position 3), lamina propria (position 4), and muscularis mucosae (position 5) [20]. The epithelial layer has a singlelayered columnar epithelium mainly including absorptive cells and goblet cells and serves as a protective barrier against pathogens from intestinal lumen. Cellular outline is revealed based on the fluorescent cytoplasmic granules of mitochondria and nonfluorescent nuclei (blue arrows in Fig. 3a). The goblet cells are also found by the obvious mucin droplets (pink arrows in Fig. 3a). Additionally, a thin subepithelial connective tissue (position 2 in Fig. 3c) is identified, which
is primarily composed of collagen and sustains the surface epithelium [21, 22]. As can be seen in Fig. 3, colorectal glands (position 3), which mainly consist of absorptive cells and goblet cells, show a characteristic Brow of test tubes^ appearance, and each gland is a single and nonbranching individual. Some slight differences in morphology can be recognized too. Interestingly, more goblet cells appear in the glands. The lamina propria has a connective tissue structure mainly composed of collagen, and mounting evidence has attested to its functional importance as a diffuse lymphoid organ [22]. It extends from the subepithelial band to muscularis mucosae (position 5 in Fig. 3c) and is easily detected via SHG signal (Fig. 3b). Moreover, many cells (yellow arrows) are also found in lamina propria, which contain plasma cells, T lymphocytes, fibroblasts, macrophages, etc. [20]. The muscularis mucosae (position 5 in Fig. 3c), which is a thin layer and separates the mucosa from submucosa, is constituted of smooth muscle fibers and mainly plays a supportive role. The combination of SHG/TPEF can provide in tandem complementary information on tissue architecture and function shown in Fig. 3c, and all these tissue architecture details readily correlate with the H&E-stained image (Fig. 3d). Submucosa Figure 4 shows representative TPEF/SHG images of normal colorectal submucosa and corresponding H&E light microscopic image. It is well known that the submucosa is a loose connective tissue layer mainly composed of collagen and elastin, which are consistent with our spectroscopic investigations (Fig. 1b). MPM images reveal that this layer contains numerously coarse and spiral collagen bundles (blue arrows in Fig. 4), collagen fibers (white arrows in Fig. 4), ropelike
Fig. 2 Representative MPM image of the four-layer microstructures of the bowel wall and corresponding H&E light microscopic image. (a) MPM image. (b) H&E-stained image
Lasers Med Sci Fig. 3 Representative highresolution large-area MPM images and corresponding H&E light microscopic image from normal colorectal mucosa. a TPEF image (color-coded red), blue arrows, pink arrows, and yellow arrows indicate nonfluorescent nuclei, mucin droplets, and inflammatory cells, respectively. b SHG image (colorcoded green). c TPEF/SHG image overlay, position 1: surface epithelium, position 2: subepithelial band of collagen, position 3: colorectal gland, position 4: lamina propria, position 5: muscularis mucosae. d H&E-stained image (40× magnification). The scale bar represents 200 μm
elastic fibers (pink arrows in Fig. 4), blood vessels (position 1 in Fig. 4), and lymphatic vessels (position 2 in Fig. 4). According to previous study, meshy collagen fibers are visualized distinctively in Fig. 4b via its strong SHG signal because Fig. 4 Representative highresolution large-area MPM images and corresponding H&E light microscopic image from normal colorectal submucosa. a TPEF image (color-coded red), blue arrows and pink arrows represent collagen bundles and elastic fibers, respectively. b SHG image (color-coded green), blue arrows and white arrows indicate collagen bundles and collagen fibers, respectively. c TPEF/SHG image overlay, position 1: blood vessel, position 2: lymphatic vessel. d H&E-stained image (40× magnification). The scale bar represents 200 μm
of its sole non-centrosymmetric structure, while the long, ropelike elastic fibers are displayed very well in Fig. 4a by its TPEF signal. The collagen fibers and elastic fibers interlace with each other but can be identified based on their intrinsic
Lasers Med Sci
signals (Fig. 4c). Interestingly, collagen bundles are in yellow when overlaying SHG image with TPEF image (Fig. 4c), as they can produce comparable SHG and TPEF signals due to certain intramolecular and intermolecular cross-links between collagen bundles [23]. Moreover, MPM can clearly display the blood vessels (position 1 in Fig. 4) through the TPEF signal from elastin in vascular intima [24], whereas the lymphatic vessels (position 2 in Fig. 4) can be identified too by the SHG signal and have a distinctively appearance in comparison with the blood vessels [25]. Apparently, the thinner walls of lymph vessels contain collagen component and this can help differentiate them from blood vessels by using MPM. However, discrepancy between blood vessels and lymphatic vessels is unobvious in the corresponding H&E-stained image (Fig. 4d). Muscularis propria and serosa Figure 5 displays representative TPEF/SHG images of normal colorectal muscularis and corresponding H&E light microscopic image. MPM reveals that the muscular layer mainly consists of internal circular muscle (position 1 in Fig. 5) and external longitudinal muscle (position 2 in Fig. 5). The muscular tissues contain abundant muscle fibers via the SHG signal (Fig. 5b) and elastic fibers by the TPEF signal (Fig. 5a), which agree well with the spectral analysis (Fig. 1c). The myofibers present striated morphology and are parallel with each other, while the elastic fibers are threadlike, spindly, or spiral and express an orientation effect along with the distribution of well-ordered muscle fibers. In addition, the loose connective tissue (position 3 in Fig. 5) and ganglia known as myenteric plexus (position 4 in Fig. 5) inside of the muscular layer can be identified; furthermore, many small blood vessels (white arrows in Fig. 5) and lymphatics (blue arrows in Fig. 5) embedded in the connective tissue are detected too. These same details of architecture correlate readily with the H&Estained image (Fig. 5d). Figure 6 shows representative TPEF/SHG images of normal colorectal serosa and corresponding H&E light microscopic image. According to previous spectrum analysis, serosal layer also contains plenty of collagen and elastin. Indeed, MPM reveals that like the submucosa, serosa mainly composes of elastic fibers (Fig. 6a) and collagen fibers (Fig. 6b). Actually, the serosal layer, also called fibrous membrane, which encases and acts to anchor the intestinal tube, is a layer of connective tissue too. Quantitative analysis Finally, our work is to extract some diagnostic features from the MPM images using the quantitative analysis of image. The alteration of cellular architecture is an important indicator for monitoring colorectal diseases. In order to quantitatively
describe characteristic of normal cell architecture, the nuclear area and nuclear-to-cytoplasmic ratio were calculated, respectively. The quantitative results reveal that the nuclear area of normal epithelial cells was 39.86±8.96 μm2 (n=30), while the nuclear-to-cytoplasmic ratio in normal mucosa was about 0.33±0.09 (n=30). Furthermore, the fluorescence intensity ratio of NADH over FAD was calculated as it can reflect the degree of pathological changes when normal and abnormal tissues are compared in the same condition and has become a good indicator of cellular metabolic state [15]. In this study, the redox ratio was approximately 1.12±0.06 (n=30) that is consistent with the normal metabolism state [26].
Discussion The incidence rate of colorectal cancer is increasing in developing countries with the alteration of lifestyle, particularly the western patterns of diet and physical inactivity. Although there are many examination techniques, such as CT, PET, MRI, ERUS, and they have made much progress, there are also many weaknesses including lack of resolution or need of exogenous contrast agents [2, 27]. Currently, pathological examination is still the gold standard. The process of pathological diagnosis involves removal of tissue, putting it into 10 % buffered formalin, paraffin embedding, sectioning, staining (H&E), and then examination under bright-field white light microscopy. There exist many drawbacks including cost, wait time for results, and the excisional process itself with its inherent risks of adverse events such as bleeding or perforation [10]. Thus, development of new imaging technologies, which are capable of detecting microscopic tumors or precursor lesions, can be of tremendous help. MPM has become one of the most important optical imaging techniques for biomedical research during recent years, which provides a superior effective resolution and an increased penetration depth in comparison with single photon excitation during confocal microscopy. As a result of the effects of nonlinear optics, various molecular components can be discriminated with MPM without use of any contrast agents [4, 9]. The detection of these molecules is based on specific autofluorescence or SHG signals generated by multiphoton excitation. In our study, there are different endogenous fluorophores in various layers of colorectal tissues according to the nonlinear spectral analysis. To be specific, the endogenous fluorophores including elastin, eosinophils, lipofuscin, porphyrin derivative, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate [NADH and NADPH, NAD(P)H], can easily generate strong TPEF signal without the need of contrast agents, while collagen fibers and muscle fibers can produce SHG signal due to their non-centrosymmetric molecular structure [28–31].
Lasers Med Sci Fig. 5 Representative highresolution large-area MPM images and corresponding H&E light microscopic image from normal colorectal muscularis. a TPEF image (color-coded red). b SHG image (color-coded green). c TPEF/SHG image overlay, position 1: circular muscle in inner layer, position 2: longitudinal muscle in outer layer, position 3: connective tissue, position 4: myenteric plexus; d H&E-stained image (40× magnification). The scale bar represents 200 μm
These endogenous signals make it possible to label-freely detect the four-layer microstructures of colorectal tissues based on MPM, which is helpful for clinicians to diagnose and treat colorectal diseases originated from different layers. Our results demonstrate that MPM has the ability not only to identify the epithelial cells, goblet cells, intestinal glands, lamina propria, and muscularis mucosae but also to reveal the collagen fibers, elastin fibers, blood vessel, lymphatic vessel, and myenteric plexus. MPM can also quantify some feature parameters of the cells: the nuclear area, nuclear-tocytoplasmic ratio and optical redox ratio, which are important signs of colorectal diseases. Therefore, it is obvious that MPM can easily distinguish the normal cells from abnormal cells as the tumor cells are characterized by enlarged nuclei and increased nuclear–cytoplasmic ratio. Furthermore, the optical
redox ratio can be used as a diagnostic tool to evaluate cell energy metabolism. It is well known that the metabolic state of the abnormal cells is known to be accelerated compared to normal cells so that comparison of the redox ratios of normal and abnormal tissues from the same patient can reflect the degree of pathological changes. In brief, these results indicate that MPM can determine the components of different layers in colorectal tissues through the nonlinear spectral analysis and can effectively distinguish the layer structure of colorectal tissues by excitation of intrinsic fluorescent molecules. Given the advantages provided by MPM, it is foreseen that MPM can serve as an effective and noninvasive tool for monitoring changes in morphology and biochemistry of colorectal tissues and then guiding the diagnosis and treatment of colorectal diseases.
Lasers Med Sci Fig. 6 Representative highresolution large-area MPM images and corresponding H&E light microscopic image from normal colorectal serosa. a TPEF image (color-coded red); b SHG image (color-coded green); c TPEF/SHG image overlay; d H&E-stained image (40× magnification). The scale bar represents 200 μm
Conclusions In this work, our results demonstrate that multiphoton microscopic imaging technique has the ability to determine the constituents of colorectal tissues by nonlinear spectral analysis and to label-freely identify the four-layer microstructures of fresh colorectal tissue, as well as to provide quantitative information by combining TPEF imaging with SHG imaging. With the continuous advancement of multiphoton-based endoscopic techniques and new laser sources, we have reason to believe that this particularly promising technique will open up many new possibilities for the diagnosis and treatment of colorectal diseases in the near future. Acknowledgments The work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT1115), the National Natural Science Foundation of China (Grant
No. 81271620), the Natural Science Foundation for Distinguished Young Scholars of Fujian Province (Grant No. 2014J06016), and the Youth Scientific Research Foundation of Fujian Provincial Department of Health (2013-2-36), National Clinical Key Specialty Construction Project (General Surgery).
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