1. Victoria Zherdeva A.N.Bach Institute of Biochemistry of the Russian Academy of Science 28th Feb – 1st March 2013 New Delhi International Seminar

2. Molecular biology Cell biologyHistology Physiology Medicine Information level ProteinsAnimalHuman ProteomicsGenomicsCytomics Phenomic Clinic DNACellsTissue CT no US no NMR 10 -3 - 10 -5 М Fl 10 -9 - 10 -12 М Tomography Sesitivity to malecular events $$ $$$$ $-$$

3. A summary of modalities used for molecular imaging. Nature Reviews: Drug discovery. 7: 591-606; J. K. Willmann et al., Molecular imaging in drug development

5. fusion proteins mRNA DNA targe t reporter stop in vivo microscopy cellular level Whole-body imaging

6. Functional studies of proteins in living cellsFunctional studies of proteins in living cells Construct target-GFP fusion proteinConstruct target-GFP fusion protein Examine at high resolution the behaviour of the protein in living cellsExamine at high resolution the behaviour of the protein in living cells

7. 2008 Nobel Prize in Chemistry "for the discovery and development of the green fluorescent protein, GFP“. Osamu Shimomura Marine Biological Laboratory Martin Chalfie Columbia University Roger Y. Tsien University of California, San Diego

8. The technology of transgenic models obtaining Trancduced human Tumor cell line with gene of FP Xenotransplantation of the fluorescent cell line to the Nude mouse Fluorescent imaging techniques Transfection (liposomal or lentiviral) of cancer cells with fluorescent repoter gene

9. Fluorescence imaging techniques Laser spectrometer with optic fiber zond Fluorescence diffuse tomography iBox UVP

10. In vivo visualization of subcutaneous transduced models of lung adenocarcinoma А549 -TagRFP on iBox (USA, UVP) on the 7- th, 15 - th, 20 - th day after tumor cells inoculation. Ex. filter 502-547 nm, em. filter 570-640 nm. Exposure time - 1 s 7- th day 15 - th day20 - th day

12. Monitoring of subcutaneous transduced models of lung adenocarcinoma А549 -TagRFP (А) and А549 -TRK 23( B ) with laser spectrometer SpectrClaster (Russia) on the 1- st, 7- th, 15- th, 20- th, 27- th, 32- d day after tumor cells inoculation. AB

13. mel Kor-Turbo-RFP cells Photosensitizer (PS) in vitro Preliminary screening of phototoxic action of PS on monolayers of fluorescent tumor cells in vivo BalbC/Nu mice, female, 18-20 g, 8 week Cell inoculation Tumor growth monitoring Diffuse fluorescent tomograph iBox excitation 502-547 nm, emission 570-640 nm

14. Tumor fluorescence Fluorescence of “Tiosense” excitation 600-645 nm, emission > 700 nm excitation 502-547 nm, emission 570-640 nm

15. Laser irradiation 730 nm, 260 mWt/sm 2, 20 min 4 mg/kg of bodyweight

17. TagRFP FRET eficciency FRET 51,1% * - Rusanov AL. et al. Lifetime imaging of FRET between red fluorescent proteins. J Biophotonics. 2010; 3:774-83

18. А549-ТagRFPА549-ТRK23 KFP TagRFP GTGGSGG DEVD GTGGSGDPPVAT P CMV IE Lentivirus transfection

19. FLIM FLIM- FRET Life-time of donor, ns 12 MicroTime 200, PicoQuant

20. Intact cells A549-TRK23 After apoptosis induction 800 мкм H 2 O 2, after 24 ч 1.8-2.1 нс 2.4 -2. 6 нс

21. Small animal FLIM-FRET whole body imaging Red Fluorescent proteins (RFP)

22. Semiconducter core: Cd/Se, Cd/Te, and Ga/N shell: Zn/S, Cd/Se biomolecule: polymer, protein, lipid Quantum dots (QD) are nanometers size ( 1 – 10nm) semiconductor nanostructured materials with the tuneable size-dependent emission, high photoluminescence (PL) quantum yields, long PL lifetimes (10 – 50ns) and narrow symmetric emission bands. Task 4: biodistribution of QD

23. Qd application Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S: Quantum Dots for Live Cells, in Vivo Imaging, and Diagnositics. Science 2005 307(5709): 538-544.

24. QDs MPA λ em 611nм or 630 nм d ~ 8 – 11нм QY 10-20% QDs PolyT λ em 626 нм d ~ 15 – 16 нм QY 10-30% QDs PolyT- APS λ em 678 нм d ~ 36 нм QY 5-20%

25. In vivo monitoring of Qd fluorescence in digestion tract of mice Excititation 502-547 nм, registration 570-640 нм, exposition 25 s. per os

26. QDs Stomach after 2 h Intestine after 2 h Intestine after 4 h Intestine after 6 h MPA+--- PolyT++±- PolyT-APS++++ The relative estimation: QDs were not detected (-), low amount of QDs (±), well detectable amount of QDs (+). Fiber optical fluorescence spectroscopy Fluorescence spectra of feces probes 24 h after per os administration QDs MPA black curve - feces control, red curve - feces after administration of QDs. QDs PolyT-APS QDs PolyT

27. 1. A. L. Rusanov, T. V. Ivashina, L. M. Vinokurov, I. I. Fiks, A. G. Orlova, I. V. Turchin, I. G. Meerovich, V. V. Zherdeva, and A. P. Savitsky. Lifetime imaging of FRET between red fluorescent proteins. J. Biophotonics, 2010, v. 3(12), p. 774-783. 2. A.L. Rusanov, A.P. Savitsky. Fluorescence resonance energy transfer between fluorescent proteins as powerful toolkits for in vivo studies. Las. Phys. Lett., 2011, v. 8(2), p. 91-102. 3. Rusanov A.L., Mironov V.A., Goryashenko A.S., Grigorenko B.L., Nemukhin A.V., Savitsky A.P. «Conformational partitioning in pH- induced fluorescence of the kindling fluorescent protein (KFP)» // J Phys Chem B. (2011);115(29):9195-201. 4. Alexander L. Rusanov, Tatiana V. Ivashina, Leonid M. Vinokurov, Alexander S. Goryashenko, Victoria V. Zherdeva, Alexander P. Savitsky «FRET-sensor for imaging with lifetime resolution» // Laser Applications in Life Sciences, edited by Matti Kinnunen; Risto Myllylä. Proceedings of the SPIE, Volume 7376, pp. 737611-1-6 (2010). 5. Alexander P. Savitsky, Alexander L. Rusanov, Victoria V. Zherdeva, Tatiana V. Gorodnicheva, Maria G. Khrenova and Alexander V. Nemukhin. FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins. Theranostics. (2012) v. 2, №2, pp.215- 226. doi:10.7150/thno.3885 Publications:

28. 6. Loginova Y.F., Kazachkina N.I., Zherdeva V.V., Rusanov A.L., Shirmanova M.V., Zagaynova E.V., Sergeeva E.A., Dezhurov S.V., Wakstein M.S., Savitsky A.P. Biodistribution of intact fluorescent CdSe/CdS/ZnS quantum dots coated by mercaptopropionic acid after intravenous injection into mice. – J. Biophotonics, 2012, vol. 11-12, pp. 848- 859. 7. Loginova Y.F., Dezhurov S.V., Zherdeva V.V., Kazachkina N.I., Wakstein M.S., Savitsky A.P. Biodistribution and stability of CdSe core quantum dots in mouse digestive tract following per os administration: Advantages of double polymer/silica coated nanocrystals. – Biochem. Biophys. Res. Comm., 2012, vol. 419 (1), pp. 54–59 8. Salykina Y.F., Zherdeva V.V., Dezhurov S.V., Wakstein M.S., Shirmanova M.V., Zagaynova E.V., Martyanov A.A., Savitsky A.P. Biodistribution and clearance of quantum dots in small animals. – Proc. SPIE, 2011, vol. 7999, pp. 799908 – 799908-10.

29. Thank you