Label-Free In Vivo Histopathology of Experimental Colitis via 3-Channel Multiphoton Endomicroscopy

Abstract

An analysis of the microscopic tissue morphology is essential for a diagnosis of inflammatory bowel disease (IBD).1Bryant R.V. Burger D.C. Delo J. et al.Beyond endoscopic mucosal healing in UC: histological remission better predicts corticosteroid use and hospitalisation over 6 years of follow-up.Gut. 2016; 65: 408-414Crossref PubMed Scopus (236) Google Scholar Multiphoton microscopy (MPM) is an advanced optical technology for label-free imaging of native tissues2Zipfel W.R. Williams R.M. Webb W.W. Nonlinear magic: multiphoton microscopy in the biosciences.Nature Biotechnology. 2003; 21: 1368-1376Crossref Scopus (3149) Google Scholar and has already been applied to experimental colitis models3Makino T. Jain M. Montrose D.C. et al.Multiphoton tomographic imaging: a potential optical biopsy tool for detecting gastrointestinal inflammation and neoplasia.Cancer Prev Res. 2012; 5: 1280-1290Crossref PubMed Scopus (28) Google Scholar as well as to biopsies of patients with IBD.4Schurmann S. Foersch S. Atreya R. et al.Label-free imaging of inflammatory bowel disease using multiphoton microscopy.Gastroenterology. 2013; 145: 514-516Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Recently, multiphoton imaging has been transferred to endoscopic applications for the in vivo investigation of the mucosal microstructure in rodents5Dilipkumar A. Al-Shemmary A. Kreiss L. et al.Label-free multiphoton endomicroscopy for minimally invasive in vivo imaging.Adv Sci. 2019; 6Crossref Scopus (35) Google Scholar and several prototypes of flexible endomicroscopes have already been reported.6Liang W.X. Hall G. Messerschmidt B. et al.Nonlinear optical endomicroscopy for label-free functional histology in vivo.Light-Science & Applications. 2017; 6Crossref PubMed Scopus (78) Google Scholar Compared with confocal laser endomicroscopy,7Goetz M. Ziebart A. Foersch S. et al.In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor.Gastroenterology. 2010; 138: 435-446Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar multiphoton endomicroscopy (MPEM) enables label-free imaging at cellular resolution with reasonable penetration depth. In contrast with previous systems,5Dilipkumar A. Al-Shemmary A. Kreiss L. et al.Label-free multiphoton endomicroscopy for minimally invasive in vivo imaging.Adv Sci. 2019; 6Crossref Scopus (35) Google Scholar we have engineered a new technical device for endoscopic multiphoton imaging with 3 imaging channels that allows more accurate recognition of different cell types. For a comparison to imaging with contrast agents, 100 μL of fluorescein solution (1 mg/mL) were injecting intravenously into mice before imaging with the new MPEM system. For label-free in vivo investigation of colitis, 2 different experimental models were tested. The first model of acute DSS colitis was used to systematically compare MPEM to ex vivo MPM and hematoxylin and eosin (H&E) histology. Therefore, 20 mice received 2% dextran sulfate sodium (DSS) in the drinking water. On days 0, 3, 6, and 9, one-quarter of the mice were investigated by MPEM, while receiving isoflurane anesthesia (1.5%–2.0%). Afterward, the animals were humanely killed and 3 tissue samples from the distal end of the colon from each animal were collected. These were kept in ice-cold phosphate-buffered saline, and MPM was performed on the same day from 1 to 3 locations per sample. Finally, the samples were analyzed by H&E histology. Furthermore, MPEM was applied to a transfer colitis model. Therefore, 5 Rag–/– mice received an intraperitoneal injection of 106 CD4+CD25– T cells. Here, the disease was studied repeatedly in the same animals over 3 weeks. MPM and H&E histology were performed at the end point. Three-dimensional image stacks of label-free MPEM were recorded with an axial spacing of 3 μm from 3 to 6 measurement spots per animal, with approximately 1 frame per second and 20 to 40 seconds per stack. Images are displayed to the user in real time. For ex vivo MPM, a research microscope (Lavision Biotec, Bielefeld, Germany; TriM-Scope II) was used in combination with a high-NA water immersion objective (Leica HC FLUOTAR L 25×/0.95 W VISIR; Wetzlar, Germany) and a femtosecond-pulsed Ti-Sapphire laser. MPM stacks were recorded at an axial spacing of 2 μm. The optical filters for MPEM and MPM were chosen to target collagen-I, NADH, and FAD. The image contrast was adjusted manually upon visual inspection. No other image processing has been used. Images from MPEM, MPM, and H&E were randomized and the file names were hidden before scoring. In the case of DSS colitis, scoring was performed by an experienced pathologist. After this validation, a nonexpert was able to score the randomized and blinded results of transfer colitis. The newly developed multiphoton colitis score was applied to MPEM and MPM images. For H&E images, an established murine colitis score was used.8Koelink P.J. Wildenberg M.E. Stitt L.W. et al.Development of reliable, valid and responsive scoring systems for endoscopy and histology in animal models for inflammatory bowel disease.J Crohns Colitis. 2018; 12: 794-803Crossref PubMed Scopus (48) Google Scholar Additionally, the number of crypts was counted in each image. Video sequences and 3-dimensional renderings were done using Fiji (ImageJ, National Institutes of Health, Bethesda, MD). Statistical analysis and plotting were performed in Matlab (Matlab 2016a, The MathWorks, Natick, MA). The video was edited using Powerpoint, Movie Maker (Office 2016, Microsoft Corporation, Redmond, WA), and Audacity (v2.3.1, GNU General Public License). The video begins by introducing the clinical assessment of IBD, fundamental contrast mechanism of label-free multiphoton imaging,2Zipfel W.R. Williams R.M. Webb W.W. Nonlinear magic: multiphoton microscopy in the biosciences.Nature Biotechnology. 2003; 21: 1368-1376Crossref Scopus (3149) Google Scholar as well as previous studies on patients with IBD4Schurmann S. Foersch S. Atreya R. et al.Label-free imaging of inflammatory bowel disease using multiphoton microscopy.Gastroenterology. 2013; 145: 514-516Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar and technical approaches for in vivo imaging.5Dilipkumar A. Al-Shemmary A. Kreiss L. et al.Label-free multiphoton endomicroscopy for minimally invasive in vivo imaging.Adv Sci. 2019; 6Crossref Scopus (35) Google Scholar Afterward, the new technical device is presented. In the experimental section, the image contrast from label-free endomicroscopy is compared with fluorescein-based imaging. Thereafter, the novel multiphoton colitis score is presented. The morphologic results from in vivo and ex vivo multiphoton imaging of the DSS model are discussed in greater detail. In conclusion, the results from H&E histopathology and label-free multiphoton imaging are in good agreement, as seen in Figure 1. This is also accurately quantified by the multiphoton colitis score and the detected crypt density, both of which are strong indicators for the disease activity. Imaging results from the transfer colitis model show disorders of the epithelial layer and an increase in the number of nonepithelial cells. In contrast with the DSS model, the crypt architecture in deeper tissue layers remains intact. Again, H&E histology and ex vivo MPM confirm these observations. The video concludes by summarizing the main findings and pointing towards possible future applications of multiphoton imaging for in vivo histopathology in patients. In this article, we present the first systematic, experimental validation of label-free multiphoton imaging in a truly endoscopic in vivo application. For this purpose, a new 3-channel multiphoton endomicroscope in combination with a novel multiphoton colitis score for quantification have been applied to different experimental colitis models. This allowed a precise evaluation and grading of the disease activity, as well as of the differences between both models. As the technology advances, label-free MPEM might be a key technology for in vivo histopathology in patients. We thank Angelika Böhm for excellent technical assistance during experiments. This work is part of the Doctoral thesis of LK. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI4YTAwYzlmNjdiNDE4YTE5ZDUxMGViZTJmNmZmNjgwNSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4ODE1MTI0fQ.YEZZPwzTnWZR5cjwGcgsWw6houqe9d9GLm0mtmdk3MWY3aRTWTqr2QN9GyoUzafAtE3nQ6SUBXJMgPloz8YBV1MxF5meXrQHEGtv4OyUKAoqIblvXk1Rv9yNI5lV7OfSJ7hN9DZBrQzi0kII2yyCZrVpyJ87l-k5EDwsL78mdSoI9Sa6ZBJFHOVgQFiIy43Q_Y_x1UF_ySkcdArkmRu2Hzx4fbQmlyBM7cf0ChRGh9r6GUK-zFc4za36pd0BMizevPSnhXaJdsvA_r1cjqeKbPC4TSxbqYLzuktSAIzAEa2ykzAJThVsuMW5BxsTiM9DpUcOs97js7U6NMMe_UJmNg Download .mp4 (126.37 MB) Help with .mp4 files Video CME Exam 1: Label-Free In Vivo Histopathology of Experimental Colitis via 3-Channel Multiphoton EndomicroscopyGastroenterologyVol. 159Issue 3Preview Full-Text PDF Open Access