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Barrett’s esophagus (BE) is a metaplastic disorder where dysplastic and early cancerous changes are invisible to the naked eye and where the practice of blind biopsy is hampered by large sampling errors. Multi-photon microscopy (MPM) has emerged as an alternative solution for fast and label-free diagnostic capability for identifying the histological features with sub-micron accuracy. We developed a compact, inexpensive MPM system by using a handheld mode-locked fiber laser operating at 1560nm to study mucosal biopsies of BE. The combination of back-scattered THG, back-reflected forward THG and SHG signals generate images of cell nuclei and collagen, leading to label-free diagnosis in Barrett’s.
© 2015 Optical Society of America
1. Introduction
The incidence of adenocarcinoma of the esophagus is rising faster than that of any other cancer in the U.S [1]. An estimated 16,980 new cases and 15,590 deaths are predicted in 2015 [2]. More than eighty percent of patients with invasive esophageal adenocarcinoma die within five years of diagnosis [3]. Barrett's esophagus is a complication of gastro-esophageal reflux disease (GERD) where the normal squamous epithelium of the esophagus is replaced by columnar epithelium with goblet cells (intestinal metaplasia); it is the major risk factor for esophageal adenocarcinoma [4]. Barrett’s is a common disorder, affecting an estimated 5.6% of adults in the United States [5]. Regular endoscopic surveillance for dysplasia in Barrett's esophagus (BE) is recommended as early identification and therapy of dysplasia and adenocarcinoma has been associated with improved survival [6, 7]. If small foci of intra-mucosal adenocarcinoma can be accurately located, current endoscopic resection techniques can lead to an excellent long-term prognosis, evidenced by the remission rate of 93.8% after a mean follow-up of 57 months [8].
Detection of dysplasia within Barrett’s esophagus is difficult, as dysplasia is not visible to the naked eye and is randomly distributed throughout the Barrett's epithelium. High-grade dysplasia and superficial adenocarcinoma can be multifocal, tiny and easily missed [9]. To further complicate matters, there is lack of intra- and inter-observer agreement among pathologists in distinguishing dysplasia from reactive epithelial changes to reflux esophagitis, and in grading the severity of dysplastic change [10–12]. Furthermore, islands of Barrett’s buried under the normal squamous epithelium have been reported, which can contain or progress to dysplasia or adenocarcinoma [13–15]. Buried BE glands are also found to a varying degree under the regenerative neo-squamous epithelium after treatment of Barrett’s tissue with radiofrequency ablation and argon plasma coagulation [15, 16]. Thus, imaging below the epithelium would be valuable for the identification of buried glands and for their management.
Although new techniques are being developed to facilitate image directed biopsies, the current reliance on routine histology delays treatment [17–19]. Confocal microscopy can provide microscopic images at endoscopy; however, this requires a contrast agent and current clinical devices do not image tissue well below the surface and are unable to identify buried glands [20]. Optical coherence tomography (OCT) can identify buried glands, but does not have the resolution to facilitate an accurate histologic diagnosis [21]. Auto-fluorescence microscopy under UV excitation can present high-resolution images in order to establish optical patterns for diagnosing dysplasia in BE [22]. Although AF microscopy is a label-free and high-resolution technique for visualization of cellular structures with no need of sample preparation, due to the short excitation the penetration depth is limited to the epithelium preventing to detect buried glands.
Multi-photon microscopy (MPM) has emerged as an alternative solution that combines high resolution with the capability of increased imaging depth and short acquisition time [23, 24]. Various multi-photon imaging (MPI) modalities such as second harmonic generation (SHG), two- and three-photon excitation fluorescence (2PEF and 3PEF), and third harmonic generation (THG) have been utilized. For instance, the 2PEF signal has been used to study the endogenous fluorescence of normal, pre-cancerous, and cancerous squamous epithelial tissues as well as the associated morphological features [25]. Recently, 2PEF was applied as a probe to characterize the normal esophageal squamous and gastric columnar mucosa biopsy to assess MPM [26]. Moreover, a miniaturized multiphoton probe was developed to examine fresh, unfixed and unstained gastrointestinal mucosa biopsies by using the 2PEF signal [27]. Further, it has been suggested that quantification of the SHG signal from fibrosis can be used as an indicator of epithelial tumor progression [28, 29]. Additionally, THG microscopy is an important label-free technique that can be applied to study a broad range of biological specimens [30–43], as the signal is sensitive to interfaces and is able to image inhomogeneous structures where the refractive index changes. By using excitation wavelengths in the 1300-1700 nm range, image depth can be increased due to reduced scattering. It also facilitates the collection and detection of the THG signal.
In this report, we assess the feasibility of label-free MPM (at 1560nm) in endoscopically obtained Barrett’s tissue and provide herein what we believe to be the first THG images of Barrett’s mucosa as well as the first description of thickened basement membrane collagen with neoplastic progression in BE at telecommunication wavelength excitation. The aim of our study is to determine if SHG and THG signals can be used to identify histologic features that can identify Barrett’s metaplasia and differentiate between different grades of dysplasia.
2. Experimental setup
2.1. Multi-photon microscopy system
Figure 1 depicts the schematic diagram of the in-house designed and assembled MPM system [43]. The excitation source is a compact femtosecond fiber laser mode-locked by a carbon nanotube saturable absorber operating at 1560 nm with an 8MHz pulse repetition rate and 150fs pulse duration. The design of the excitation source is similar to that reported in [44]; the difference here is that the laser output is linearly polarized. The laser output is delivered to the microscope via a PM fiber patch cord and is collimated by using a fiber collimator (Thorlabs, F240APC-1550); the collimated beam is then raster scanned on the sample attached to an xyz stage by employing a 2D galvo-scanner system. A telescope consisting of scan and tube lenses expands the beam to approximately illuminate the entire back aperture of objective lens (New Focus, 5724-H-C) so that the smallest possible laser spot size is achieved (THG resolution = 1.3µm, see [43] for resolution measurement). The back-scattered signal from the sample is separated from the excitation source by a long-pass dichroic mirror with 870nm cut-off wavelength. We use a non-descanned detection scheme enabled by a dichroic mirror (λcut-off = 552nm, Semrock) and two similar high gain and sensitive PMTs (Hamamatsu H10720-21) for simultaneously recording in two channels. The longer wavelength channel collects the 2PEF, 3PEF or SHG signal by using appropriate filters before the PMT. On the other hand, the shorter wavelength channel collects the THG signal, where a narrow band pass filter centered at 525/30nm (Semrock) was embedded in front of the PMT. Two low-noise current amplifiers(Stanford Research Systems, SR570) amplify the signal generated by the PMTs before digitization by a data acquisition card (NI PCI-6110). In-house laser scanning software based on LabVIEW was developed to acquire images, control the image acquisition process and adjust the xyz translation stage.
Fig. 1 Schematic diagram of the in-house MPM system (left) and the multiphoton spectrum of Barrett Esophagus tissue (right). Red: Optical spectrum of the multiphoton generated signal from a normal tissue showing a strong THG signal at ~520nm. Inset: zoom-in SHG spectrum.
Each image frame was acquired and saved with 512 × 512 pixels and a 250 × 250µm2 field of view (FOV). The frame acquisition time was 8s (pixel dwell time of ~30µs) with 15mW average power on the sample. Since the FOV was small in comparison to the region of interest, several frames were acquired and corrected for non-uniform illumination. The correction was performed by normalizing each frame by a reference frame outside the region where the sample resides. Finally, the sequentially ordered frames were stitched by using the Stitching [45] plugin in Fiji [46].
2.2. Methods and sample preparation
Cross-sectional imaging was performed on fixed tissue samples obtained by endoscopy from patients with Barrett’s esophagus. Tissue samples were sectioned at 8µm, stained with hematoxylin and eosin (H&E) and evaluated independently by two gastrointestinal pathologists. Unstained samples were obtained from a section residing 8µm below the section used for histology, placed on a glass slide with a mounting medium, and covered with a glass cover slip for MP imaging. Representative samples were classified as Barrett’s esophagus (BE) with no dysplasia (51 year old Caucasian male), BE with low-grade dysplasia (75 year old Caucasian man) and BE with high-grade dysplasia (72 year old Caucasian male). The institutional review board of the University of Arizona approved the study.
3. Results
The THG images presented herein (Figs. 2–4) demonstrate that these label-free images are of sufficient resolution and contrast to depict the histologic features of BE with and without dysplasia that are seen with standard H&E stains. Of interest, the SHG images displayed increasing thickness of the basement membrane collagen with progression of the dysplasia, which is normally not seen in H&E stains, but generally requires specific stains for displaying fibrous tissue [47–49]. It should be noted that although obtained from the same biopsy specimen and from the same patient, the multi-photon images are not as an exact a match as the H&E images. Due to the 3-dimensional nature of the tissue fragments suspended in paraffin, sequential 8µm sectioning of the tissue results in non-identical tissue shapes and sizes, and thus a variation of appearances seen from the same biopsy on separate slides. However, the H&E and multi-photon images depict the same histological features from the same tissue biopsy.
Fig. 2 Comparison between multi-photon microscopy and conventional light microscopy of BE tissue that is negative for dysplasia. (a) H&E conventional light microscopy image showing intestinal metaplasia with goblet cells. There is surface maturation, low nuclear to cytoplasmic ratio and no dysplasia. (b-d) magnified regions shown in (a). (e) High resolution THG signal from MPM and (f-h) the magnified regions in (e). (i) SHG signal recorded simultaneously with the THG signal showing collagen distribution in the stroma and blood vessels. (j-l) magnified marked regions in the SHG image to show collagen in the basement membrane. The THG signal has a clear correlation to the H&E light-microscope image. The architectural structure of nuclei indicates that the tissue has no dysplastic feature. The yellow arrows show the cells with no evidence of dysplasia. (FOV in (e) and (i): 1000 × 750 µm2, acquisition time: ~2min).
3.1. Negative for dysplasia
The THG images (Figs. 2(e)-4(e)) show tissue structure with cell nuclei displayed with high contrast and resolution that is comparable to standard, H&E stained microscopic images. Figures 2(a)-2(d) and Figs. 2(e)-2(h) show the H&E light-microscopic images and multi-photon THG images from a sample of Barrett’s without dysplasia. The generation of TH signal from the surface of a microscope slide creates a bright background in THG images. Since the THG signal is phase-matched in the forward direction, the generated forward TH signal is larger than the backward THG signal. To detect this signal, we used a mirror underneath the thin section of the tissue to redirect the forward THG signal to the PMT.
As is typical for non-dysplastic Barrett’s, the cells are positioned uniformly in a single row along the basement membrane (Figs. 2(f)-2(h)). The nuclei are of normal size with a normal nucleus to cytoplasm ratio. The cells with no evidence of dysplasia are shown with yellow arrows in Fig. 2. The SHG images (Figs. 2(i)-2(l)) show very fine strands of sub-epithelial collagen, which cannot be discerned in H&E images.
3.2. Low-grade dysplasia
Figure 3 shows Barrett’s with low-grade dysplasia; the cells are not all positioned in a single uniform line and the pattern of the glands remains normal. Nuclei are stratified and mildly hyperchromatic with an increased nucleus to cytoplasm ratio (Figs. 3(f)-3(h)). These changes are shown with high contrast and clarity in the THG images that depict all the features of the H&E findings. The yellow arrows in Fig. 3 represent the cells with low-grade dysplasia. The SHG images shows thicker strands of sub-epithelial (basement-membrane) collagen than in Barrett’s without dysplasia (Figs. 2(i)-2(l)). Note that collagen cannot be seen in any of the color H&E images.
Fig. 3 Multi-photon and conventional light microscopy images for low-grade dysplastic BE tissue. (a) Conventional light microscopy image of the tissue stained with H&E; there is intestinal metaplasia with goblet cells, surface luminal cells displaying nuclear stratification, hyperchromasia, and an increased nuclear to cytoplasmic ratio (N: C). (b-d) magnified regions marked in the H&E image. (e-h) High resolution THG images that show features of low-grade dysplasia in the same biopsy. (i-l) corresponding simultaneous SHG signals of the THG images that display the presence of collagen. The spatial distribution of cell nuclei is consistent with low-grade dysplasia. The cells with low-grade dysplasia are marked with arrows. (FOV in (e) and (i): 750 × 1000 um2, acquisition time: ~2min). The vertical white lines in (j)-(l) are the PMT artifacts.
3.3. High-grade dysplasia
In Fig. 4, the H&E and THG images exemplify high-grade dysplasia characterized by marked disruption of the tissue architecture with cellular disorganization. The nuclei are hyperchromatic and pleomorphic, with a greater increase in the nucleus to cytoplasm ratio as well as showing loss of polarity (Figs. 4(e)-4(h)). Label-free THG imaging with comparable contrast and resolution renders the features of the H&E images. The yellow arrows in Fig. 4 point to the cells identified as high-grade dysplasia. In addition, the SHG images of the basement membrane show thicker strands of sub-epithelial collagen than in Barrett’s with low-grade dysplasia and non-dysplastic Barrett’s. Moreover, collagen is not seen in the H&E views.
Fig. 4 MPM and conventional light microscopy images of high-grade dysplastic tissue. (a) Conventional light microscopy image of the tissue after labeling with H&E. There is a progressive dysplastic change in a background of intestinal metaplasia. Cells have marked hyperchromatic nuclei nuclear stratification, loss of polarity and a markedly increased N:C ratio, considerably more than in low grade dysplasia. (b-d) magnified squared regions marked in (a). (e) High resolution THG and (f-h) corresponding magnified regions. (i) High resolution SHG image and (j-l) the magnified regions marked in to show basement membrane collagen (i). The dense nuclei with variable shapes and sizes as well as the loss of orderly arrangement of the cells are consistent with high-grade dysplasia. Arrows represent the cells with high-grade dysplasia. (FOV in (e) and (i): 500 × 500 um2, acquisition time: <1min).
3.4. Basement membrane collagen
The SHG images acquired simultaneously with the THG images in this study show thickening of sub-epithelial (basement membrane) collagen with advancing stages of dysplasia. Figure 5 shows the same SHG images of different stages of the dysplasia shown in Figs. 2–4 when contrast adjusted by using Fiji. In each image (Figs. 5(a)-5(c)), three areas of sub-epithelial collagen were chosen (A, B, C) and the thickness measured increased progressively with advancing grades of dysplasia, with marked thickening in high-grade dysplasia.
Fig. 5 SHG signal from Barrett’s with non-dysplastic dysplasia (a), low grade dysplasia (b) and high grade dysplasia (c). Corresponding graphs to the right show the thickness of the basement membrane collagen measured at each of the three chosen points per image, which confirm increasing thickness with progression of dysplasia.
4. Discussion
This initial work indicates the potential of MPM in generating label-free images of Barrett’s mucosa with contrast and clarity that is equal, if not superior, to traditional H&E microscopy enabling a histologic diagnosis to be made. Furthermore, the imaging of the sub-epithelial basement membrane evidences increasing thickness of collagen with progression of dysplasia, most prominently in the high-grade form. The increased thickness of basement membrane collagen may represent the body’s response to carcinogenesis by attempting to prevent invasion of cells into deeper layers as the disease progresses to cancer. Noting that this observation has been made in a small sample and the findings need to be verified in a larger study, we believe that the implied capability of the SHG MPI to selectively image basement membrane collagen may enable it to become a quantifiable tool for the identification of high-grade dysplasia, which would be of great clinical value.
Histological sample preparation and examination by a pathologist to identify the morphological features of a suspicious biopsy in conventional H&E light microscopy are not only time-consuming and burdensome; a diagnosis cannot be made in-vivo or at the point of care. MPM has the advantage of being able to provide high quality images ex-vivo or in-vivo and at the point of care. Furthermore, MPM should be able to image sub-epithelial buried glands and detect early invasion of cancer in reconstructed 3D images as well as 2D views.
5. Conclusion
We believe these to be the first reported THG images of Barrett’s esophagus for demonstration of MPI to identify diagnostic features of low- and high-grade dysplasia in Barrett’s. We also report on thickened basement membrane collagen in dysplastic Barrett’s epithelium in SHG images, which may prove to be a complementary diagnostic marker of disease stage.
Our preliminary results suggest that label-free THG and SHG images may provide not only a substitution for H&E histology in Barrett’s, but the future construction of a fiber-optic probe may enable in-vivo detection of unseen dysplasia or cancer during endoscopy and guide therapy, such as endoscopic mucosal resection and radio-frequency ablation.
Acknowledgments
Support from the CIAN NSF ERC under grant #EEC-0812072, the State of Arizona’s Photonics TRIF, and the Barrett’s Cancer Imaging grant are gratefully acknowledged.
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