Label-Free Imaging of Inflammatory Bowel Disease Using Multiphoton Microscopy
2013; Elsevier BV; Volume: 145; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2013.06.054
ISSN1528-0012
AutoresSebastian Schürmann, Sebastian Foersch, Raja Atreya, Helmut Neumann, Oliver Friedrich, Markus F. Neurath, Maximilian J. Waldner,
Tópico(s)Photodynamic Therapy Research Studies
ResumoMultiphoton microscopy (MPM) became one of the most important optical in vivo imaging techniques for basic research during recent years. In comparison with single photon excitation during confocal microscopy, MPM provides a superior effective resolution in thick tissue samples and an increased penetration depth.1Zipfel W.R. et al.Nat Biotechnol. 2003; 21: 1369-1377Crossref PubMed Scopus (3107) Google Scholar Because of the effects of nonlinear optics, various molecular components can be discriminated with MPM in vivo and in vitro without the need to apply fluorophores. The detection of these molecules is based on specific autofluorescence or second harmonic generation (SHG) signals generated by multiphoton excitation.These characteristics clearly suggest MPM for in vivo diagnostics of human disease. For instance, MPM of autofluorescence and SHG has been used in initial clinical studies for the diagnostics of human skin diseases such as melanoma, angioma, psoriasis and others in vivo.2Perry S.W. et al.Ann Biomed Engineer. 2012; 40: 277-291Crossref PubMed Scopus (148) Google Scholar Similarly, MPM could be used for the endoscopic analysis of human gastrointestinal diseases.In this article, with an accompanying video, we show that MPM allows the visualization of pathologic changes in tissue samples from patients with inflammatory bowel disease (IBD) without requiring exogenous fluorophores.Description of TechnologyFresh tissue specimens were collected from control patients or patients with IBD during operative procedures or during endoscopy according to ethical guidelines. All tissue samples were kept in ice-cold phosphate-buffered saline, and MPM was performed within 3 hours after sampling of mucosal tissue. The MPM system used for single-color imaging (Leica TCS MP5; Leica Microsystems, Wetzlar, Germany) was equipped with a 25× 0.95NA water immersion objective and a femtosecond-pulsed Ti-Sapphire laser. For label-free single-color imaging, tissue samples were excited at 800 nm wavelength and emitted signals were detected from 390nm to 580 nm. For multicolor imaging with spectral separation, performed on a second MPM system (LaVision BioTec TriM-Scope II; LaVision BioTec GmbH, Bielefeld, Germany) with 40× 1.1NA objective, SHG signals were detected from 395nm to 415nm wavelength (shown in blue), NADH from 435nm to 465nm (green), and FAD from 540nm to 580 nm (red) (Figure 1A–C).All images were acquired as Z-stacks with a section thickness of 3 or 1 μm and penetration depth up to 150 μm. Magnifications, video sequences, and 3-dimensional (3D) renderings were calculated using the Fiji package of ImageJ software (National Institutes of Health, Bethesda, MD). The video was edited with Premiere Elements Software (Adobe, Redmond, WA).Video DescriptionThe video starts with a short description of fundamental optical principles of MPM. Two-photon excited fluorescence is based on the molecular absorption of 2 infrared photons at the same time. SHG is a nonlinear scattering effect, which occurs only in highly ordered and non-centrosymmetrical structures, for example fibers of collagen-I. Because multiphoton effects are only visible in a small volume in the objective focus (∼1 μm³), the technique is perfectly suited for the acquisition of 3D image stacks.Subsequently, image stacks, still images, and 3D-rendered volumes of human control and IBD tissue samples are presented. The section begins with multiphoton imaging of colon tissue from a control patient. MPM allows a detailed visualization of the mucosal architecture, including the lamina propria, with a penetration depth of up to 120 μm and subcellular resolution. The colonic mucosa is covered by an intact epithelial layer, crypts are distributed uniformly, and individual goblet cells can be identified owing to a darker cytoplasm in comparison with normal enterocytes. Below the epithelial layer, collagen matrix fibers surrounding colonic crypts and individual infiltrating cells emit a bright signal.The next part shows inflamed colon tissue from active ulcerative colitis and active Crohn's disease, respectively. In contrast with control colon tissue, MPM reveals epithelial erosions and a tremendous infiltration of the lamina propria with bright fluorescent cells. Whereas the crypt architecture is completely abrogated in the ulcerative colitis sample, as also shown by 3D rendered volumes, it is preserved in the Crohn's disease sample.The next part of the video provides a short description of spectral differences of SHG and autofluorescence signals, which can be used during MPM imaging to provide even more detail about tissue morphology. As previously shown, multiphoton-excited autofluorescence of the gastrointestinal mucosa can mainly be attributed to NADH and FAD molecules.3Rogart J.N. et al.Clin Gastroenterol Hepatol. 2008; 6: 95-101Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Because most epithelial cell types provide strong NADH-dependent autofluorescence and various immune cell subtypes contain large amounts of flavins (eg, FAD), the spectral discrimination of these molecules can allow the discrimination of individual cell types during image acquisition.4Zipfel W.R. et al.Proc Natl Acad Sci U S A. 2003; 100: 7075-7080Crossref PubMed Scopus (1516) Google ScholarTo take advantage of these effects, the last part of the video shows multicolor images with spectral separation of SHG (blue), NADH (green), and FAD (red) signals during MPM of control and IBD tissue. In the control tissue sample, NADH signals can mainly be collected from the epithelial layer, whereas SHG signals show collagen fibers at the basal lamina. Interstitial cells of the lamina propria contain a bright FAD signal and therefore seem to be resident or infiltrating immune cells. Again, multicolor MPM of active Crohn's disease tissue shows an increased number of interstitial cells in the lamina propria.Take Home MessageIn this video article, we show that MPM allows a detailed 3D analysis of intestinal tissue without the need of exogenous fluorophore administration. Based on the detection of autofluorescence and SHG signals, comprehensive morphologic aspects of intestinal inflammation can be visualized at the subcellular level for the evaluation of gastrointestinal pathology. Therefore, MPM seems to be an ideal technique for a future endoscopic acquisition of in vivo histology.During recent years, single-photon excitation based confocal laser endomicroscopy of gastrointestinal disease has entered clinical endoscopy.5Neumann H. et al.Gastroenterology. 2010; 139 (392.e1–2): 388-392Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar Although an increasing amount of applications are continuously being discovered for confocal laser endomicroscopy, the technique is generally restricted by the detection of exogenous fluorophores (so far, fluorescein sodium, acriflavine hydrochloride, and cresyl violet have been approved for the use in humans).Therefore, an endoscopic multiphoton endomicroscopy device shall be a helpful addition for the diagnosis of human gastrointestinal disease. Since first fiber-based multiphoton endoscopy systems have been developed for preclinical research,6Myaing M.T. et al.Optics Lett. 2006; 31: 1076-1078Crossref PubMed Scopus (275) Google Scholar, 7Wu Y. et al.Optics Express. 2009; 17: 7907-7915Crossref PubMed Scopus (138) Google Scholar, 8Rivera D.R. Brown C.M. et al.Proc Natl Acad Sci U S A. 2011; 108: 17598-17603Crossref PubMed Scopus (220) Google Scholar the translation of this technique for clinical usage seems to be feasible and worthwhile. Multiphoton microscopy (MPM) became one of the most important optical in vivo imaging techniques for basic research during recent years. In comparison with single photon excitation during confocal microscopy, MPM provides a superior effective resolution in thick tissue samples and an increased penetration depth.1Zipfel W.R. et al.Nat Biotechnol. 2003; 21: 1369-1377Crossref PubMed Scopus (3107) Google Scholar Because of the effects of nonlinear optics, various molecular components can be discriminated with MPM in vivo and in vitro without the need to apply fluorophores. The detection of these molecules is based on specific autofluorescence or second harmonic generation (SHG) signals generated by multiphoton excitation. These characteristics clearly suggest MPM for in vivo diagnostics of human disease. For instance, MPM of autofluorescence and SHG has been used in initial clinical studies for the diagnostics of human skin diseases such as melanoma, angioma, psoriasis and others in vivo.2Perry S.W. et al.Ann Biomed Engineer. 2012; 40: 277-291Crossref PubMed Scopus (148) Google Scholar Similarly, MPM could be used for the endoscopic analysis of human gastrointestinal diseases. In this article, with an accompanying video, we show that MPM allows the visualization of pathologic changes in tissue samples from patients with inflammatory bowel disease (IBD) without requiring exogenous fluorophores. Description of TechnologyFresh tissue specimens were collected from control patients or patients with IBD during operative procedures or during endoscopy according to ethical guidelines. All tissue samples were kept in ice-cold phosphate-buffered saline, and MPM was performed within 3 hours after sampling of mucosal tissue. The MPM system used for single-color imaging (Leica TCS MP5; Leica Microsystems, Wetzlar, Germany) was equipped with a 25× 0.95NA water immersion objective and a femtosecond-pulsed Ti-Sapphire laser. For label-free single-color imaging, tissue samples were excited at 800 nm wavelength and emitted signals were detected from 390nm to 580 nm. For multicolor imaging with spectral separation, performed on a second MPM system (LaVision BioTec TriM-Scope II; LaVision BioTec GmbH, Bielefeld, Germany) with 40× 1.1NA objective, SHG signals were detected from 395nm to 415nm wavelength (shown in blue), NADH from 435nm to 465nm (green), and FAD from 540nm to 580 nm (red) (Figure 1A–C).All images were acquired as Z-stacks with a section thickness of 3 or 1 μm and penetration depth up to 150 μm. Magnifications, video sequences, and 3-dimensional (3D) renderings were calculated using the Fiji package of ImageJ software (National Institutes of Health, Bethesda, MD). The video was edited with Premiere Elements Software (Adobe, Redmond, WA). Fresh tissue specimens were collected from control patients or patients with IBD during operative procedures or during endoscopy according to ethical guidelines. All tissue samples were kept in ice-cold phosphate-buffered saline, and MPM was performed within 3 hours after sampling of mucosal tissue. The MPM system used for single-color imaging (Leica TCS MP5; Leica Microsystems, Wetzlar, Germany) was equipped with a 25× 0.95NA water immersion objective and a femtosecond-pulsed Ti-Sapphire laser. For label-free single-color imaging, tissue samples were excited at 800 nm wavelength and emitted signals were detected from 390nm to 580 nm. For multicolor imaging with spectral separation, performed on a second MPM system (LaVision BioTec TriM-Scope II; LaVision BioTec GmbH, Bielefeld, Germany) with 40× 1.1NA objective, SHG signals were detected from 395nm to 415nm wavelength (shown in blue), NADH from 435nm to 465nm (green), and FAD from 540nm to 580 nm (red) (Figure 1A–C). All images were acquired as Z-stacks with a section thickness of 3 or 1 μm and penetration depth up to 150 μm. Magnifications, video sequences, and 3-dimensional (3D) renderings were calculated using the Fiji package of ImageJ software (National Institutes of Health, Bethesda, MD). The video was edited with Premiere Elements Software (Adobe, Redmond, WA). Video DescriptionThe video starts with a short description of fundamental optical principles of MPM. Two-photon excited fluorescence is based on the molecular absorption of 2 infrared photons at the same time. SHG is a nonlinear scattering effect, which occurs only in highly ordered and non-centrosymmetrical structures, for example fibers of collagen-I. Because multiphoton effects are only visible in a small volume in the objective focus (∼1 μm³), the technique is perfectly suited for the acquisition of 3D image stacks.Subsequently, image stacks, still images, and 3D-rendered volumes of human control and IBD tissue samples are presented. The section begins with multiphoton imaging of colon tissue from a control patient. MPM allows a detailed visualization of the mucosal architecture, including the lamina propria, with a penetration depth of up to 120 μm and subcellular resolution. The colonic mucosa is covered by an intact epithelial layer, crypts are distributed uniformly, and individual goblet cells can be identified owing to a darker cytoplasm in comparison with normal enterocytes. Below the epithelial layer, collagen matrix fibers surrounding colonic crypts and individual infiltrating cells emit a bright signal.The next part shows inflamed colon tissue from active ulcerative colitis and active Crohn's disease, respectively. In contrast with control colon tissue, MPM reveals epithelial erosions and a tremendous infiltration of the lamina propria with bright fluorescent cells. Whereas the crypt architecture is completely abrogated in the ulcerative colitis sample, as also shown by 3D rendered volumes, it is preserved in the Crohn's disease sample.The next part of the video provides a short description of spectral differences of SHG and autofluorescence signals, which can be used during MPM imaging to provide even more detail about tissue morphology. As previously shown, multiphoton-excited autofluorescence of the gastrointestinal mucosa can mainly be attributed to NADH and FAD molecules.3Rogart J.N. et al.Clin Gastroenterol Hepatol. 2008; 6: 95-101Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Because most epithelial cell types provide strong NADH-dependent autofluorescence and various immune cell subtypes contain large amounts of flavins (eg, FAD), the spectral discrimination of these molecules can allow the discrimination of individual cell types during image acquisition.4Zipfel W.R. et al.Proc Natl Acad Sci U S A. 2003; 100: 7075-7080Crossref PubMed Scopus (1516) Google ScholarTo take advantage of these effects, the last part of the video shows multicolor images with spectral separation of SHG (blue), NADH (green), and FAD (red) signals during MPM of control and IBD tissue. In the control tissue sample, NADH signals can mainly be collected from the epithelial layer, whereas SHG signals show collagen fibers at the basal lamina. Interstitial cells of the lamina propria contain a bright FAD signal and therefore seem to be resident or infiltrating immune cells. Again, multicolor MPM of active Crohn's disease tissue shows an increased number of interstitial cells in the lamina propria. The video starts with a short description of fundamental optical principles of MPM. Two-photon excited fluorescence is based on the molecular absorption of 2 infrared photons at the same time. SHG is a nonlinear scattering effect, which occurs only in highly ordered and non-centrosymmetrical structures, for example fibers of collagen-I. Because multiphoton effects are only visible in a small volume in the objective focus (∼1 μm³), the technique is perfectly suited for the acquisition of 3D image stacks. Subsequently, image stacks, still images, and 3D-rendered volumes of human control and IBD tissue samples are presented. The section begins with multiphoton imaging of colon tissue from a control patient. MPM allows a detailed visualization of the mucosal architecture, including the lamina propria, with a penetration depth of up to 120 μm and subcellular resolution. The colonic mucosa is covered by an intact epithelial layer, crypts are distributed uniformly, and individual goblet cells can be identified owing to a darker cytoplasm in comparison with normal enterocytes. Below the epithelial layer, collagen matrix fibers surrounding colonic crypts and individual infiltrating cells emit a bright signal. The next part shows inflamed colon tissue from active ulcerative colitis and active Crohn's disease, respectively. In contrast with control colon tissue, MPM reveals epithelial erosions and a tremendous infiltration of the lamina propria with bright fluorescent cells. Whereas the crypt architecture is completely abrogated in the ulcerative colitis sample, as also shown by 3D rendered volumes, it is preserved in the Crohn's disease sample. The next part of the video provides a short description of spectral differences of SHG and autofluorescence signals, which can be used during MPM imaging to provide even more detail about tissue morphology. As previously shown, multiphoton-excited autofluorescence of the gastrointestinal mucosa can mainly be attributed to NADH and FAD molecules.3Rogart J.N. et al.Clin Gastroenterol Hepatol. 2008; 6: 95-101Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Because most epithelial cell types provide strong NADH-dependent autofluorescence and various immune cell subtypes contain large amounts of flavins (eg, FAD), the spectral discrimination of these molecules can allow the discrimination of individual cell types during image acquisition.4Zipfel W.R. et al.Proc Natl Acad Sci U S A. 2003; 100: 7075-7080Crossref PubMed Scopus (1516) Google Scholar To take advantage of these effects, the last part of the video shows multicolor images with spectral separation of SHG (blue), NADH (green), and FAD (red) signals during MPM of control and IBD tissue. In the control tissue sample, NADH signals can mainly be collected from the epithelial layer, whereas SHG signals show collagen fibers at the basal lamina. Interstitial cells of the lamina propria contain a bright FAD signal and therefore seem to be resident or infiltrating immune cells. Again, multicolor MPM of active Crohn's disease tissue shows an increased number of interstitial cells in the lamina propria. Take Home MessageIn this video article, we show that MPM allows a detailed 3D analysis of intestinal tissue without the need of exogenous fluorophore administration. Based on the detection of autofluorescence and SHG signals, comprehensive morphologic aspects of intestinal inflammation can be visualized at the subcellular level for the evaluation of gastrointestinal pathology. Therefore, MPM seems to be an ideal technique for a future endoscopic acquisition of in vivo histology.During recent years, single-photon excitation based confocal laser endomicroscopy of gastrointestinal disease has entered clinical endoscopy.5Neumann H. et al.Gastroenterology. 2010; 139 (392.e1–2): 388-392Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar Although an increasing amount of applications are continuously being discovered for confocal laser endomicroscopy, the technique is generally restricted by the detection of exogenous fluorophores (so far, fluorescein sodium, acriflavine hydrochloride, and cresyl violet have been approved for the use in humans).Therefore, an endoscopic multiphoton endomicroscopy device shall be a helpful addition for the diagnosis of human gastrointestinal disease. Since first fiber-based multiphoton endoscopy systems have been developed for preclinical research,6Myaing M.T. et al.Optics Lett. 2006; 31: 1076-1078Crossref PubMed Scopus (275) Google Scholar, 7Wu Y. et al.Optics Express. 2009; 17: 7907-7915Crossref PubMed Scopus (138) Google Scholar, 8Rivera D.R. Brown C.M. et al.Proc Natl Acad Sci U S A. 2011; 108: 17598-17603Crossref PubMed Scopus (220) Google Scholar the translation of this technique for clinical usage seems to be feasible and worthwhile. In this video article, we show that MPM allows a detailed 3D analysis of intestinal tissue without the need of exogenous fluorophore administration. Based on the detection of autofluorescence and SHG signals, comprehensive morphologic aspects of intestinal inflammation can be visualized at the subcellular level for the evaluation of gastrointestinal pathology. Therefore, MPM seems to be an ideal technique for a future endoscopic acquisition of in vivo histology. During recent years, single-photon excitation based confocal laser endomicroscopy of gastrointestinal disease has entered clinical endoscopy.5Neumann H. et al.Gastroenterology. 2010; 139 (392.e1–2): 388-392Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar Although an increasing amount of applications are continuously being discovered for confocal laser endomicroscopy, the technique is generally restricted by the detection of exogenous fluorophores (so far, fluorescein sodium, acriflavine hydrochloride, and cresyl violet have been approved for the use in humans). Therefore, an endoscopic multiphoton endomicroscopy device shall be a helpful addition for the diagnosis of human gastrointestinal disease. Since first fiber-based multiphoton endoscopy systems have been developed for preclinical research,6Myaing M.T. et al.Optics Lett. 2006; 31: 1076-1078Crossref PubMed Scopus (275) Google Scholar, 7Wu Y. et al.Optics Express. 2009; 17: 7907-7915Crossref PubMed Scopus (138) Google Scholar, 8Rivera D.R. Brown C.M. et al.Proc Natl Acad Sci U S A. 2011; 108: 17598-17603Crossref PubMed Scopus (220) Google Scholar the translation of this technique for clinical usage seems to be feasible and worthwhile. The authors thank Prof. N. Sauer for access to his multiphoton system during the initial image acquisition phase of this project. 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