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

2. Іnstitute of Physics NASU 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 Two-photon absorbing organic molecules with efficient stimulated emission depletion (STED) for bioimaging 2

3. Іnstitute of Physics NASU Stimulated and spontaneous transitions in organic molecule AbsFluor S0S0 S1S1 SnSn ESA hv, 2hv Stimulated Emission NR  01 ( )  3.8*10 -21  ( ) [M -1 cm -1 ] ~ 10 -16 cm 2 ;  10 ( ) ~ 10 -16 cm 2 ;  2PA ( ) ~ 10 -48 cm 4  s Simplified molecular electronic model 3 K.D. Belfield, et al., J. Phys. Chem. B, 2009, 113, 1701.

4. Іnstitute of Physics NASU S1S1 S0S0 S2S2 S3S3 S4S4 1hv quench pump I fl 0 ~ Ip; (1-I fl /I fl 0 ) ~ I q pumpquench I(r,t) = I 0  exp[-(r 2 /r 0 2 + t 2 /  2 )] Delay  10 ps dN 1 (r,t)/dt = N 0  01  I(r,t) - N 1  [1/  +  10  I(r,t)] N 0 (r,t) + N 1 (r,t) = N C  N 1 (r,t)dt = N 1 (r,  p ) dN 1 (r,t)/dt = N 1 (r,t)  2PE  I 2 q (r,t) (1 - I fl /I fl 0 ) =  2PE  {  q  (  /8) 1/2  [r q 2 /(r p 2 +r q 2 )]}  I q 2 I fl 0 ~  N 1 (r,  p )  r  dr  d  I fl ~  N 1 q (r,  p )  r  dr  d  N 1 q (r,  p ) = N 1 (r,  p )  [1-  2PE  (  /2) 1/2  q  I 2 q (r)] J.R. Lakowicz, et al., J. Photochem. Photobiol., 1994, 60, 546. 2hv quench ESA Fluorescence quenching method 4

5. Іnstitute of Physics NASU pumpprobe I(r,t) = I 0  exp[-(r 2 /r 0 2 + t 2 /  2 )] Delay  10 ps Fluorescence quenching method 5 K.D. Belfield, et al., J. Phys. Chem. B, 2009, 113, 1701.

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

7. Іnstitute of Physics NASU Linear photophysical and photochemical properties  Ph  4  10 -7  =  R  QY; A = 1 /  R IRF ~ 80 ps exc = 440 nm 7 K.D. Belfield, et al., ChemPhysChem, 2011, 12, 2755.

8. Іnstitute of Physics NASU Two-photon absorption spectra SQ FD 8

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

10. Іnstitute of Physics NASU Transient absorption spectroscopy 10 FD in СН 2 Cl 2 K.D. Belfield et al., J. Phys. Chem. C, 2013, 117, 11941. FD

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

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

13. Іnstitute of Physics NASU  #40 in Toluene 1hv quench Abs Em Em CHFTOL K.D. Belfield et al., ChemPhysChem, 2012, 13, 3481. 2hv quench Abs Em 2PA2PE Two-photon absorption and STED spectra 13

14. Іnstitute of Physics NASU Bioimaging application of new fluorene derivative 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). 14 K.D. Belfield, et al., ChemPhysChem, 2011, 12, 2755.X. Wang, et al., Biomed. Opt. Express, 2010, 1, 453. 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).

15. Іnstitute of Physics NASU  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 Conclusions 15