Multiphoton morpho-functional imaging of healthy colon mucosa, adenomatous polyp and adenocarcinoma

Riccardo Cicchi; Alessandro Sturiale; Gabriella Nesi; Dimitrios Kapsokalyvas; Giovanni Alemanno; Francesco Tonelli; Francesco S. Pavone

doi:10.1364/BOE.4.001204

Multiphoton morpho-functional imaging of healthy colon mucosa, adenomatous polyp and adenocarcinoma

Riccardo Cicchi, Alessandro Sturiale, Gabriella Nesi, Dimitrios Kapsokalyvas, Giovanni Alemanno, Francesco Tonelli, and Francesco S. Pavone

Biomedical Optics Express
Vol. 4,
Issue 7,
pp. 1204-1213
(2013)
•https://doi.org/10.1364/BOE.4.001204

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Riccardo Cicchi, Alessandro Sturiale, Gabriella Nesi, Dimitrios Kapsokalyvas, Giovanni Alemanno, Francesco Tonelli, and Francesco S. Pavone, "Multiphoton morpho-functional imaging of healthy colon mucosa, adenomatous polyp and adenocarcinoma," Biomed. Opt. Express 4, 1204-1213 (2013)

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Related Topics
Table of Contents Category
- Functional Imaging
Optics & Photonics Topics
?

The topics in this list come from the OSA Optics and Photonics Topics applied to this article.
Previously assigned OCIS codes
- Medical and biological imaging (170.3880)
- Nonlinear microscopy (180.4315)

About this Article
History
- Original Manuscript: April 16, 2013
- Revised Manuscript: May 9, 2013
- Manuscript Accepted: May 9, 2013
- Published: June 24, 2013

Accessible

Open Access

Abstract

Two-photon spectral resolved imaging was used to image fresh human biopsies of colon tissue and to characterize healthy colon mucosa, adenomatous polyp and adenocarcinoma by means of a morpho-functional analysis. Morphological examination, performed using endogenous tissue fluorescence, discriminated adenomatous and adenocarcinoma tissues from normal mucosa in terms of cellular asymmetry and nucleus-to-cytoplasm ratio. Good agreement was found between multiphoton images and histological examination performed on the same samples. Further characterization, performed by means of spectral-resolved analysis of NADH and FAD fluorescence, demonstrated an altered metabolic activity in both adenomatous and adenocarcinoma tissues compared to healthy mucosa. This morpho-functional approach may represent a powerful method to be used in combination with endoscopy for in vivo optical diagnosis of colon cancer and may be extended to other tissues.

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[Crossref]
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2012 (7)

C. Hirche, Z. Mohr, S. Kneif, S. Doniga, D. Murawa, M. Strik, and M. Hünerbein, “Ultrastaging of colon cancer by sentinel node biopsy using fluorescence navigation with indocyanine green,” Int. J. Colorectal Dis. 27(3), 319–324 (2012).
[Crossref] [PubMed]

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[Crossref] [PubMed]

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[Crossref] [PubMed]

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[Crossref] [PubMed]

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[Crossref] [PubMed]

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[Crossref] [PubMed]

W. Hu, G. Zhao, C. Wang, J. Zhang, and L. Fu, “Nonlinear optical microscopy for histology of fresh normal and cancerous pancreatic tissues,” PLoS ONE 7(5), e37962 (2012).
[Crossref] [PubMed]

2011 (10)

R. Cicchi and F. Saverio Pavone, “Non-linear fluorescence lifetime imaging of biological tissues,” Anal. Bioanal. Chem. 400(9), 2687–2697 (2011).
[Crossref] [PubMed]

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(43), 17598–17603 (2011).
[Crossref] [PubMed]

X. Shao, W. Zheng, and Z. Huang, “In vivo diagnosis of colonic precancer and cancer using near-infrared autofluorescence spectroscopy and biochemical modeling,” J. Biomed. Opt. 16(6), 067005 (2011).
[Crossref] [PubMed]

X. Shao, W. Zheng, and Z. Huang, “Near-infrared autofluorescence spectroscopy for in vivo identification of hyperplastic and adenomatous polyps in the colon,” Biosens. Bioelectron. 30(1), 118–122 (2011).
[Crossref] [PubMed]

S. M. Yoon, S. J. Myung, I. W. Kim, E. J. Do, B. D. Ye, J. H. Ryu, K. Park, K. Kim, I. C. Kwon, M. J. Kim, D. H. Moon, D. H. Yang, K. J. Kim, J. S. Byeon, S. K. Yang, and J. H. Kim, “Application of near-infrared fluorescence imaging using a polymeric nanoparticle-based probe for the diagnosis and therapeutic monitoring of colon cancer,” Dig. Dis. Sci. 56(10), 3005–3013 (2011).
[Crossref] [PubMed]

M. J. Waldner, S. Wirtz, C. Neufert, C. Becker, and M. F. Neurath, “Confocal laser endomicroscopy and narrow-band imaging-aided endoscopy for in vivo imaging of colitis and colon cancer in mice,” Nat. Protoc. 6(9), 1471–1481 (2011).
[Crossref] [PubMed]

S. J. Miller, B. P. Joshi, Y. Feng, A. Gaustad, E. R. Fearon, and T. D. Wang, “In vivo fluorescence-based endoscopic detection of colon dysplasia in the mouse using a novel peptide probe,” PLoS ONE 6(3), e17384 (2011).
[Crossref] [PubMed]

H. K. Roy, M. J. Goldberg, S. Bajaj, and V. Backman, “Colonoscopy and optical biopsy: bridging technological advances to clinical practice,” Gastroenterology 140(7), 1863–1867 (2011).
[Crossref] [PubMed]

L. G. Lim, M. Bajbouj, S. von Delius, and A. Meining, “Fluorescein-enhanced autofluorescence imaging for accurate differentiation of neoplastic from non-neoplastic colorectal polyps: a feasibility study,” Endoscopy 43(5), 419–424 (2011).
[Crossref] [PubMed]

S. Zhuo, J. Yan, G. Chen, J. Chen, Y. Liu, J. Lu, X. Zhu, X. Jiang, and S. Xie, “Label-free monitoring of colonic cancer progression using multiphoton microscopy,” Biomed. Opt. Express 2(3), 615–619 (2011).
[Crossref] [PubMed]

2010 (4)

R. Cicchi, A. Crisci, A. Cosci, G. Nesi, D. Kapsokalyvas, S. Giancane, M. Carini, and F. S. Pavone, “Time- and spectral-resolved two-photon imaging of healthy bladder mucosa and carcinoma in situ,” Opt. Express 18(4), 3840–3849 (2010).
[Crossref] [PubMed]

B. Mayinger, F. Neumann, C. Kastner, T. Haider, and D. Schwab, “Hexaminolevulinate-induced fluorescence colonoscopy versus white light endoscopy for diagnosis of neoplastic lesions in the colon,” Endoscopy 42(1), 28–33 (2010).
[Crossref] [PubMed]

K. Inoue, N. Wakabayashi, Y. Morimoto, K. Miyawaki, A. Kashiwa, N. Yoshida, K. Nakano, H. Takada, Y. Harada, N. Yagi, Y. Naito, T. Takamatsu, and T. Yoshikawa, “Evaluation of autofluorescence colonoscopy for diagnosis of superficial colorectal neoplastic lesions,” Int. J. Colorectal Dis. 25(7), 811–816 (2010).
[Crossref] [PubMed]

R. Cicchi, D. Kapsokalyvas, D. Stampouli, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “In-vivo tissue imaging using a compact mobile non-linear microscope,” Proc. SPIE 7569, 756930, 756930–756939 (2010).
[Crossref]

2008 (5)

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[Crossref] [PubMed]

J. N. Rogart, J. Nagata, C. S. Loeser, R. D. Roorda, H. Aslanian, M. E. Robert, W. R. Zipfel, and M. H. Nathanson, “Multiphoton imaging can be used for microscopic examination of intact human gastrointestinal mucosa ex vivo,” Clin. Gastroenterol. Hepatol. 6(1), 95–101 (2008).
[Crossref] [PubMed]

B. Mayinger, F. Neumann, C. Kastner, K. Degitz, E. G. Hahn, and D. Schwab, “Early detection of premalignant conditions in the colon by fluorescence endoscopy using local sensitization with hexaminolevulinate,” Endoscopy 40(2), 106–109 (2008).
[Crossref] [PubMed]

A. L. McCallum, J. T. Jenkins, D. Gillen, and R. G. Molloy, “Evaluation of autofluorescence colonoscopy for the detection and diagnosis of colonic polyps,” Gastrointest. Endosc. 68(2), 283–290 (2008).
[Crossref] [PubMed]

W. R. Kessler, “Autofluorescence colonoscopy: a green light on the long road to “real-time” histology,” Gastrointest. Endosc. 68(2), 291–293 (2008).
[Crossref] [PubMed]

2007 (2)

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[Crossref] [PubMed]

R. Cicchi, D. Massi, S. Sestini, P. Carli, V. De Giorgi, T. Lotti, and F. S. Pavone, “Multidimensional non-linear laser imaging of Basal Cell Carcinoma,” Opt. Express 15(16), 10135–10148 (2007).
[Crossref] [PubMed]

2005 (3)

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[Crossref] [PubMed]

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, “Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra,” J. Biomed. Opt. 10(2), 024016 (2005).
[Crossref] [PubMed]

E. Dekker and P. Fockens, “New imaging techniques at colonoscopy: tissue spectroscopy and narrow band imaging,” Gastrointest. Endosc. Clin. N. Am. 15(4), 703–714 (2005).
[Crossref] [PubMed]

2003 (2)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[Crossref] [PubMed]

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8(3), 432–439 (2003).
[Crossref] [PubMed]

2002 (1)

J. C. Malone, A. F. Hood, T. Conley, J. Nürnberger, L. A. Baldridge, J. L. Clendenon, K. W. Dunn, and C. L. Phillips, “Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy,” J. Cutan. Pathol. 29(8), 453–458 (2002).
[Crossref] [PubMed]

2000 (2)

J. Gahlen, J. Stern, J. Pressmar, J. Böhm, R. Holle, and C. Herfarth, “Local 5-aminolevulinic acid application for laser light-induced fluorescence diagnosis of early staged colon cancer in rats,” Lasers Surg. Med. 26(3), 302–307 (2000).
[Crossref] [PubMed]

D. A. Lieberman, D. G. Weiss, J. H. Bond, D. J. Ahnen, H. Garewal, W. V. Harford, D. Provenzale, S. Sontag, T. Schnell, T. E. Durbin, D. B. Nelson, S. L. Ewing, G. Triadafilopoulos, F. C. Ramirez, J. G. Lee, J. F. Collins, M. B. Fennerty, T. K. Johnston, C. L. Corless, K. R. McQuaid, R. E. Sampliner, T. G. Morales, R. Fass, R. Smith, Y. Maheshwari, and G. Chejfec, “Use of colonoscopy to screen asymptomatic adults for colorectal cancer. Veterans Affairs Cooperative Study Group 380,” N. Engl. J. Med. 343(3), 162–168 (2000).
[Crossref] [PubMed]

1998 (2)

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Biomed. Opt. Express (1)

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BMC Gastroenterol. (1)

Cancer Res. (1)

Clin. Gastroenterol. Hepatol. (1)

Dig. Dis. Sci. (1)

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Gastroenterology (1)

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Fig. 1 Histological images taken using a 20X objective on H&E stained tissue sections of healthy colon mucosa (A), adenomatous polyp (C), and adenocarcinoma (E). Corresponding TPF images (taken with a 20X objective - 0.9 NA) acquired from ex vivo fresh biopsies of healthy colon mucosa (B), adenomatous polyp (D), and adenocarcinoma (F). Scale bars: 40 μm. Histological images taken using a 40X objective on H&E stained tissue sections of healthy colon mucosa (G), adenomatous polyp (J), and adenocarcinoma (L). Corresponding TPF images (taken with a 40X objective – 1.3 NA) acquired from ex vivo fresh biopsies of healthy colon mucosa (H), adenomatous polyp (K), and adenocarcinoma (M). Scale bars: 30 μm.

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Fig. 2 TPF images at high resolution (taken with a 40X objective - 1.3 NA) of healthy colon mucosa (A), adenomatous polyp (B), and adenocarcinoma (C) acquired at 30 μm depth from tissue surface on ex-vivo fresh biopsies. Images have been acquired using 740 nm as excitation wavelength. Scale bars: 10 μm. (D) Distribution of the cellular/nuclear size ratio for healthy mucosa, adenomatous polyp, and adenocarcinoma. (E) Distribution of the cellular asymmetry for healthy mucosa, adenomatous polyp, and adenocarcinoma. In the graphs dots represent single data points, while the box is enclosing the data within SD. All the values for both Cellular/Nuclear Ratio and Cellular Asymmetry, taken in couples, were found to be statistically different at the 0.05 level after a two-sample statistical t-test.

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Fig. 3 Red-Ox Ratio color-coded maps obtained after processing of NADH-images (740 nm excitation; 440 – 480 nm detection) and FAD-images (890 nm excitation; 480 – 540 nm detection) for healthy tissue (A-C), adenomatous polyp (D-F), and adenocarcinoma (G-I). The depth of recording from tissue surface is indicated in the down-left corner. Scale bars: 10 μm. (J) Red-Ox Ratio distribution for healthy tissue (blue), adenomatous polyp (yellow), and adenocarcinoma (green) with the color-coded scale used in Red-Ox Ratio mapping superimposed. Distributions have been obtained by averaging over 6 images per samples and 5 samples per tissue type. All the values for RedOx Ratio, taken in couples, were found to be statistically different at the 0.05 level after a two-sample statistical t-test.

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Tables (1)

Table 1 Average Values of Cellular/Nuclear Ratio (Left Column) and Cellular Asymmetry (Right Column) Calculated on a Population of 125 Cells of Healthy Mucosa, Adenomatous Polyp, and Adenocarcinoma

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Equations (1)

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Red-Ox Ratio = \frac{F A D - N A D H}{F A D + N A D H} .

Cellular/Nuclear Ratio		Cellular Asymmetry
Healthy Mucosa:	6.7 ± 1.8	Healthy Mucosa:	1.3 ± 0.2
Adenomatous Polyp:	3.3 ± 0.8	Adenomatous Polyp:	2.6 ± 0.6
Adenocarcinoma:	2.1 ± 0.3	Adenocarcinoma:	1.7 ± 0.3