Laser microscope-spectrum analyzer for studying intracellular accumulation of near infrared emitting photosensitizers in vitro

Measuring system based on the binocular microscope for analysis of intracellular accumulation of infrared IR photosenstizers allowing to obtain graphic data about state of analyzed objects, location of fluorescence foci and to obtain details of spectral profile of fluorescence emission centers in IR spectral region was developed. According to image of fluorescence signal distribution the location of photosensitizer accumulation in the cell may be detected accurately and the spectrum of fluorescence signal of near IR-range in the targeted point may be obtained. The developed system is quite comprehensive because there is an opportunity to choose technical parameters, operating modes, measuring methods and analysis. The advantage of the developed microscope-spectrum analyzer is an opportunity to focus emission and create high power intensity on the irradiated area by means of laser source with small-angle beam spreading, all this allow to perform ultra-precise operations with cells. Particularly, tunable size of the diaphragm opening in the far field allows to register fluorescence signal on certain cell organoids. By means of developed system the studies of accumulation of the new bacteriochlorine photosensitizers on HeLa cell line were performed. The system allowed to register accumulation of cancer cells with definite sites of selectively accumulated photosensitizer. The sites of fluorescence were the centers of accumulation of bacteriochlorine photosensitizer, this suggests that studied photosensitizer has a tendency for local accumulation in cellular organoids. The authors suggested that the developed system allowed to perform the effective and rapid screening of new photosensitizers, particularly IR bacteriochlorine photosensitizers.

1. Loschenov V.B., Konov V.I., Prokhorov A.M. Photodynamic therapy and fluorescence diagnostics, Laser Physics, 2000, Vol. 10, No. 6, pp. 1188–1207.

2. Filonenko E.V. Flyuorestsentnaya diagnostika i fotodinamicheskaya terapiya – obosnovanie primeneniya i vozmozhnosti v onkologii (Fluorescence diagnosis and photodynamic therapy – justification of use and opportunities in oncology), Fotodinamicheskaya terapiya i fotodiagnostika, 2014, No. 1, pp. 3–7.

3. Lin'kov K.G., Berezin A.N., Loshchenov V.B. Apparatura dlya FD i FDT (Devices for FD and PDT), Ross. bioterapevt. zhurnal, 2004, T. 3, No. 2, p. 54.

4. Meerovich I. G., Grin M. A., Meerovich G. A. i dr. Novye fotosensibilizatory blizhnego infrakrasnogo diapazone na osnove proizvodnykh bakteriokhlorina: predvaritel'nye rezul'taty izucheniya in vivo (New bacteriochlorine-based photosensitizers of near infrared range: preliminary results of study in vivo), Ross. bioterapevt. zhurnal, 2006, T. 5, No. 2, pp. 73–76.

5. Vasilchenko S.Yu., Volkova A.I., Ryabova A.V. et al. Application of aluminum phthalocyanine nanoparticles for fluorescent diagnostics in dentistry and skin autotransplantology, J. Biophoton., 2010, Т. 3, No. 5–6, pp. 336–346.

6. Breymayer J., Rück A., Ryabova A.V., Loschenov V.B., Steiner R.W. Fluorescence Investigation of the Effect of Monocytes/Macrophages and Skin Cells on Aluminium Phthalocyanine Nanoparticles, Journal Photodiagnosis and Photodynamic Therapy, 2014, Vol. 11 (3), pp. 380–390.

7. Ryabova A.V., Pominova D.V., Klimov A.I. i dr. Effektivnost' generatsii singletnogo kisloroda ftalotsianinami Al i Zn i ikh fotodinamicheskaya aktivnost' pri kon"yugirovanii s nanochastitsami Au, Ag, Fe2O3, Si (Efficiency of singlet oxygen generation by Al and Zn phthalocyanines and its photodynamic activity for conjugation with nano-particles of Au, Ag, Fe2O3, Si), Rossiiskii khimicheskii zhurnal, 2013, T. LVII, No. 5, pp. 25–34.

8. Oertel, M., Schastak S. I., Tannapfel A. et al. Novel bacteriochlorin for high tissue-penetration: photodynamic properties in human biliary tract cancer cells in vitro and in a mouse tumour model, J. Photochem. Photobiol., 2003, Vol. 71, pp. 1–10.

9. Mazor O., Brandis A., Plaks, V., Neumark, E. et al. WST11, a novel water-soluble bacteriochlorophyll derivative; cellular uptake, pharmacokinetics, biodistribution, and vascular targeted photodynamic activity against melanoma tumors, Photochem. Photobiol., 2005, Vol. 81, pp. 342–351.

10. Rovers J. P., de Jode M.L., Rezzoug H., Grahn M.F. In vivo photodynamic characteristics of the near-infrared photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin, Photochem. Photobiol., 2000, Vol. 72, pp. 358–364.

11. Mironov A.F., Grin M.A., Karmakova T.A. i dr. Novye fotosensibilizatory dlya FDT raka na osnove prirodnogo bakteriokhlorofilla a (New natural bacteriochlorophyl a based photosensitizers for PDT cancer), Ross. bioterapevt. zhurnal, 2003, T. 2, No. 1, pp. 33–34.

12. Yakubovskaya R.I., Plotnikova E.A., Morozova N.B., Grin M.A., Mironov A.F. Aminoamidy v ryadu bakteriokhlorofilla a i ikh fotoindutsirovannaya aktivnost' v sistemakh in vitro i in vivo (Aminoamids in the range of bacteriochlorophyl a and its photoinduced activity in systems in vitro and in vivo), Fotodinamicheskaya terapiya i fotodiagnostika, 2013, No. 3, pp. 29–30.

13. Grin M.A., Pantyushenko I.V., Plotnikova E.A. i dr. Novye fotosensibilizatory na osnove bakteriopurpurinimida i ikh fotoindutsirovannaya protivoopukholevaya aktivnost' (New bacteriopurpurinimide-based photosensitizers and its photoinduced anti-cancer activity), Fotodinamicheskaya terapiya i fotodiagnostika, 2013, No. 3, pp. 33–34.