1. Hyperspectral two-photon near- infrared cancer imaging at depth Nikolay S. Makarov, Jean Starkey, Mikhail Drobizhev, Aleksander Rebane, Montana State University, Bozeman, MT Commercially available dye, Styryl-9M, can be used as a fluorescent probe for detection of cancer cell colonies embedded in a biological tissue phantom The ratio of fluorescence signals obtained at 1100 and 1200 excitation wavelength nm depends on the composition of the phantoms The ratio can be used as a quantitative measure to distinguish between samples without cells, with normal cells and with cancer cells This imaging allows cancer tumor localization within 1 mm 3 The proposed method is a promising tool for non-invasive deep photodetection of cancer

2. Two-photon absorption measurement setup Correction curve: Relative 2PA spectrum:Absolute 2PA cross section:

3. 2PA spectrum of Styryl-9M N.S. Makarov, M. Drobizhev, A. Rebane, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range”, Opt. Expr., 16, 4029-4047 (2008). A. Rebane, N.S. Makarov, M. Drobizhev, B. Spangler, E.S. Tarter, B.D. Reeves, C.W. Spangler, F. Meng, Z. Suo, “Quantitative prediction of two-photon absorption cross section based on linear spectroscopic properties”, J. Phys. Chem. C, 112, 7997-8004 (2008).

4. Sensing local environment: 1PA vs. 2PA N.S. Makarov, E. Beuerman, M. Drobizhev, J. Starkey, A. Rebane, “Environment-sensitive two-photon dye”, Proc. SPIE, 7049, 70490Y (2008). Advantageous determination of the local environment polarity via 2PA excitation as compared to the 1PA excitation: Only two selected excitation wavelengths while observation at one fixed fluorescence wavelength are enough to distinguish between solvents of different polarities. The polarity dependence is nonlinear, in contrast with the linear dependence upon 1PA excitation, which increases sensitivity of 2PA excitation. The difference between the observed 2PA-excited intensity ratio for the most and least polar solvents is about 9 folds, while the same difference for the absorption peak position, determined based on 1PA-excitation is less than 15%. Furthermore, longer wavelengths used for 2PA excitation offer less scattering, which is important in imaging of the tissue phantoms.

5. Sensing the environment of cancer cells

6. Spectrum decomposition for cancer detection

7. Cell detection in phantoms: experimental 6 3 2 7 4 5 1 (1) entrance diaphragm for laser beam (2) focusing lens (3) sample inside holder (4) 75 mm f/1.4 lens (5) variable wavelength filter (6) – CCD camera (7) He-Ne laser 10 mm Sample preparation: 0.3 ml of setting solution, 1.5 ml collagen solution, 10 5 cells, 1.5 ml tissue culture medium carefully layered over the gel. Stylyl-9 dye was added (7 μl of a 10 mg/ml DMSO solution per phantom) 16 hours before imaging. The following tumor cell lines were used: NIHOVCAR3, human ovarian cancer; MDA-MB-231, human mammary carcinoma; 4T1, mouse mammary carcinoma; +SA mammary carcinoma. N.P. Robertson, J.R. Starkey, S. Hamner, G.G. Meadows, "Tumor cell invasion of three-dimensional matrices of defined composition: evidence for a specific role for heparin sulfate in rodent cell lines", Cancer Res., 49, 1816-1823 (1989). U.K. Ehmann, W.D. Peterson Jr., D.S. Misfeldt, "To grow mouse mammary epithelial cells in culture", J Cell Biol., 98, 1026-1032 (1984). K.G. Danielson, L.W. Anderson, H.L. Hosick, "Selection and characterization in culture of mammary tumor cells with distinctive growth properties in vivo", Cancer Res., 40, 1812- 1819 (1980).

8. Cancer detection in phantoms

9. Cancer cells localization in phantoms

10. Commercially available dye, Styryl-9M, can be used as a fluorescent probe for detection of local polarity via one- or two-photon excitation 2PA excitation increases sensitivity of local polarity detection Longer wavelengths used for 2PA excitation offer less scattering, which is important in imaging of the tissue phantoms. 2PA can be used for detection of cancer cell colonies embedded in a biological tissue phantom The ratio of fluorescence signals excited at 1100 and 1200 nm depends on the composition of phantoms The ratio can be used as a quantitative measure to distinguish between samples without cells, with normal cells and with cancer cells The cancer imaging allows tumor localization within 1 mm 3 The proposed method is a promising tool for non-invasive deep photodetection of cancer Conclusions Acknowledgement This work is supported by MBRCT