Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues.
This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting.
Skala
Melissa C
MC
Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, USA.
Squirrell
Jayne M
JM
Vrotsos
Kristin M
KM
Eickhoff
Jens C
JC
Gendron-Fitzpatrick
Annette
A
Eliceiri
Kevin W
KW
Ramanujam
Nirmala
N
D015127:9,10-Dimethyl-1,2-benzanthracene; D000818:Animals; D002273:Carcinogens; D002278:Carcinoma in Situ / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology*; D002610:Cheek; D006224:Cricetinae; D004847:Epithelial Cells / Q000473:pathology; D005453:Fluorescence; D007633:Keratins / Q000378:metabolism; D008297:Male; D008647:Mesocricetus; D018613:Microscopy, Confocal / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009062:Mouth Neoplasms / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology*; D011230:Precancerous Conditions / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology*
D002273:Carcinogens; D015127:9,10-Dimethyl-1,2-benzanthracene; D007633:Keratins
Opt Lett. 2002 Jun 15;27(12):1031-3
18026355
Nat Biotechnol. 1999 Aug;17(8):763-7
10429240
Appl Opt. 2000 Mar 1;39(7):1194-201
18338003
Science. 1990 Apr 6;248(4951):73-6
2321027
Opt Lett. 2003 Jun 1;28(11):902-4
12816240
Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11014-9
12177437
Int J Exp Pathol. 1996 Feb;77(1):7-14
8664146
Lasers Surg Med. 2004;35(2):96-103
15334611
Photochem Photobiol. 2001 Jun;73(6):636-41
11421069
Exp Physiol. 2002 Nov;87(6):737-45
12447453
Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7075-80
12756303
Opt Lett. 2002 Sep 1;27(17):1513-5
18026490
Photochem Photobiol. 2000 Jun;71(6):730-6
10857369
Opt Lett. 1997 Sep 1;22(17):1344-6
18188234
Rev Sci Instrum. 2003 Jan;74(1):193-201
18607511
Neoplasia. 2000 Jan-Apr;2(1-2):89-117
10933071
J Oral Pathol. 1981 Jun;10(3):186-91
6798180
Opt Lett. 2003 Sep 1;28(17):1552-4
12956376
Biotechnol Appl Biochem. 2003 Feb;37(Pt 1):45-50
12578551
Photochem Photobiol. 2003 May;77(5):550-5
12812299
Biophys J. 1998 Oct;75(4):2015-24
9746543
J Biomed Opt. 2003 Jul;8(3):432-9
12880349
Lasers Surg Med. 2004;34(1):25-38
14755422
Appl Opt. 2002 Apr 1;41(10):1852-7
11936781
Opt Express. 2001 Mar 12;8(6):335-43
19417824
Opt Express. 2000 Dec 4;7(12):436-46
19407895
J Oral Pathol. 1984 Jun;13(3):244-54
6429298
Nat Med. 2003 Jun;9(6):796-800
12754503
Diagn Histopathol. 1981 Oct-Dec;4(4):307-33
6802623
Ann N Y Acad Sci. 1998 Feb 9;838:58-67
9511795
J Oral Pathol Med. 1998 Nov;27(10):470-4
9831958