The development of new organic probes for

1. The development of new organic probes for two-photon induced fluorescence microscopy application Mykhailo V. Bondar and Olga V. PrzhonskaInstitute of Physics, Prospect Nauki, 46, Kiev-28, 03028, Ukraine

2. Two-photon absorbing organic molecules with efficient stimulated emission depletion (STED) for bioimaging Outline • Stimulated and spontaneous transitions in organic molecules • Linear spectral characterization • Two-photon absorption spectra • Transient absorption spectroscopy and superfluorescence • Stimulated emission depletion properties • Conclusions

3. Stimulated and spontaneous transitions in organic molecule Simplified molecular electronic model Sn ESA NR S1 hv, 2hv Stimulated Emission Abs Fluor NR S0 01()  3.8*10-21() [M-1cm-1] ~ 10-16 cm2; 10() ~ 10-16 cm2; 2PA() ~ 10-48 cm4s K.D. Belfield, et al.,J. Phys. Chem. B, 2009,113, 1701.

4. Fluorescence quenching method S4 I(r,t) = I0exp[-(r2/r02 + t2/ 2)] S3 S2 ESA pump quench S1 pump 1hv quench 2hv quench Delay  10 ps S0 Ifl0~Ip; (1-Ifl/Ifl0) ~Iq • dN1(r,t)/dt = N001I(r,t) - N1[1/ + 10I(r,t)] • N0(r,t) + N1(r,t) = NC • N1(r,t)dt = N1(r,p) • dN1(r,t)/dt = N1(r,t)2PEI2q(r,t) Ifl0 ~ N1(r,p)rdrd Ifl ~ N1q(r,p)rdrd N1q(r,p) = N1(r,p)[1- 2PE(/2)1/2qI2q(r)] (1 - Ifl/Ifl0) = 2PE{q(/8)1/2[rq2/(rp2+rq2)]}Iq2 J.R. Lakowicz,et al.,J. Photochem. Photobiol., 1994,60, 546.

5. Fluorescence quenching method I(r,t) = I0exp[-(r2/r02 + t2/ 2)] probe pump Delay  10 ps K.D. Belfield, et al.,J. Phys. Chem. B, 2009,113, 1701.

6. Experimental setup for pump-probe and single beam experiments Sample Ocean Optics DL 800 nm, 76 MHz, 200 fs PD DC SF MIRA 900-F SM 400 nm Z F 800 nm, 1kHz PD S Verdi-V10 240–20000 nm PD OPA OperaSolo Legend Elite P ~ 100 fs F WP P SF K.D. Belfield, et al.,ChemPhysChem, 2011,12, 2755.

7. Linear photophysical and photochemical properties FPh 410-7 IRF ~ 80 ps lexc= 440 nm t = tR  QY; A = 1 / tR K.D. Belfield, et al.,ChemPhysChem, 2011,12, 2755.

8. Two-photon absorption spectra FD SQ

9. Transient absorption spectroscopy SQ #44 TOL #44 DCM #44 TOL K.D. Belfield, et al.,ChemPhysChem, 2013,14, 1-14.

10. Transient absorption spectroscopy FD in СН2Cl2 FD K.D. Belfield et al., J. Phys. Chem. C,2013, 117, 11941.

11. Superfluorescence properties Pump 650 nm, 100 fs, 1 кHz С  7.5·10-5 М (a, b) С  1.8·10-3М (c, d) K.D. Belfield, et al.,ChemPhysChem, 2013,14, 1-14.

12. 1hv STED 2hv STED 2PA Two-photon absorption and STED spectra (A. Penzkofer,et al.,Chem. Phys., 1990,142, 123.) K.D. Belfield, et al.,ChemPhysChem, 2011,12, 2755.

13. Two-photon absorption and STED spectra #40 in Toluene Abs Em Em 1hv quench # 40 TOL CHF Abs Em 2hv quench 2PA 2PE K.D. Belfield et al., ChemPhysChem,2012, 13, 3481.

14. Bioimaging application of new fluorene derivative Images of Hela cells incubated with SNP-DBF-NHFA (20 μM, 2 h). (b) 3D reconstruction from overlaid two-photon fluorescence images (Ex: 740 nm; Power: 30 mW; Em. short-pass filter 690 nm) 10 μm grid, (c) 2P-FLIM image (Ex: 740 nm; Power: 30 mW). Images of HCT 116 cells incubated with 1. (A) DIC; (B) One photon fluorescence image; (C) 3D reconstruction from overlaid two-photon fluorescence images (Ex: 940 nm; Power: 120 mW; Em. short-pass filter 800 nm). K.D. Belfield, et al.,ChemPhysChem, 2011,12, 2755. X. Wang, et al.,Biomed. Opt. Express, 2010,1, 453.

15. Conclusions • Fluorescence quenching methodology is a promising technique for STED investigations • Two-photon stimulated emission spectra were obtained in a broad spectral range and high STED cross sections were observed for fluorene molecule • New fluorene derivatives with large two-photon absorption, • efficient STED and high photostability has a good potential • for application in fluorescence bioimaging