the nature of one- and two - photon stimulated transition processes in organic molecules is a subject of both scientific and technological interest due to a number of nonlinear optical properties , such as two - photon induced fluorescence ( 2pf ) , nonlinear transmittance , two - photon absorption ( 2pa ) processes , and light amplification of stimulated emission ( lasing ) .
several different types of stimulated transitions play important roles in the mechanisms of electric fieldmolecular structure interactions and should be considered in a broad variety of nonlinear optical measurements . for example , possible effects of saturation and stimulated emission(12 ) should be taken into account in the determination of 2pa cross sections by the 2pf method when sufficiently large pulse energies are used and the excitation wavelength overlaps with the fluorescence spectrum of the fluorophore . in the case of 2pa cross section measurements by the open - aperture z - scan method,(13 ) possible excited - state absorption ( esa ) and stimulated one - photon emission also need to be analyzed , as they can make significant contributions to the observed data.(14 ) excited - state dynamics and stimulated transitions that occur in organic molecules can be studied by fluorescence - quenching methodology , as described previously by lakowicz and co - workers .
this methodology allows modification of the molecular orientational distribution in the excited states and creates anisotropic molecular ensembles with specific fluorescence properties.(17 ) as an example , the fluorescence anisotropy properties of 4-dimethylamino-4-cyanostilbene and tetraphenylbutadiene in the presence of stimulated light quenching have been described . in some cases , a noticeable deviation from a quadratic dependence of fluorescence emission on excitation power was observed .
quite significantly , the use of stimulated emission transitions has provided a means to overcome the diffraction limit in fluorescence microscopy , resulting in the development of high resolution ( 60-nm ) stimulated emission depletion ( sted ) microscopy . herein , the nature of one- and two - photon stimulated emission transitions for a new sulfonyl - containing fluorene , 2,7-bis(4-(phenylsulfonyl)styryl)-9,9-didecyl-9h - fluorene ( 1 , figure 1a ) , is investigated in toluene and tetrahydrofuran ( thf ) at room temperature by a fluorescence quenching methodology based on different fluorescence excitation conditions .
one - photon fluorescence excitation followed by one- or two - photon quenching was performed using a picosecond pumpprobe technique and two laser beams at different wavelengths .
two - photon induced fluorescence along with concurrent one - photon quenching was detected using a single picosecond laser beam .
fluorene 1 is a particularly attractive molecule for such a study because it is a highly fluorescent , photochemically stable compound that exhibits efficient one- and two - photon stimulated transitions .
( a ) normalized absorption ( 1 , 2 ) and emission ( 1 , 2 ) spectra of 1 in toluene ( 1 , 1 ) and thf ( 2 , 2 ) .
( bd ) electronic molecular model of 1 and corresponding stimulated transitions : ( b ) one - photon excitation ( pump ) laser beam at 355 nm , followed by delayed one - photon quenching laser beam at 532 nm ; ( c ) one - photon excitation ( 355 nm ) and delayed two - photon quenching ( 1064 nm ) ; ( d ) two - photon excitation and simultaneous one - photon quenching by a single laser beam at 532 nm .
precursors 2 , 3 , 5 , and 6 were prepared according to the literature.(26 ) compound 4 was obtained from the oxidation of 3 , using a modified literature procedure.(27 ) hornerwadsworthemmons coupling was performed as described below , affording 1 in 38% yield .
the synthesis of ( 4-(bromomethyl)phenyl)(phenyl ) sulfone ( 4 ) was performed according to a modified procedure from hsiao et al.(27 ) hydrogen peroxide ( 30% , 0.100 g , 0.80 mmol ) was added to a solution of ( 4-(bromomethyl)phenyl)(phenyl ) sulfide ( 3 , 0.141 g , 0.51 mmol ) in glacial acetic acid ( 0.200 g ) .
the mixture was mildly heated until the exothermic reaction was initiated , and an additional 0.100 g of 30% hydrogen peroxide was added .
the mixture was then taken to 100 c and monitored by thin layer chromatography ( tlc ) every 15 min . upon completion of the reaction ( 2 h ) , the mixture was cooled to room temperature , and the acetic acid was evaporated in vacuo .
the crude product was dissolved in et2o , washed twice with saturated nahco3 ( aqueous ) , washed twice with saturated nacl ( aqueous ) , dried over anhydrous mgso4 , and filtered .
after concentration of the filtrate in vacuo , the resulting product was recrystallized ( 1:1 acetone / water ) to obtain 0.155 g ( 98% ) of a colorless solid ; mp 135136 c ( lit .
132 c).(28)h nmr ( 300 mhz , cdcl3 ) : 7.94 ( m , 4h , ar 
h ) , 4.45 ( s , 2h , ch2).c nmr ( 75 mhz , cdcl3 ) 143.3 ( c ) , 141.6 ( c ) , 141.4 ( c ) , 133.6 ( ch ) , 130.1 ( ch ) , 129.6 ( ch ) , 128.4 ( ch ) , 127.9 ( ch ) , 31.8 ( ch2 ) . in a two - neck , 250-ml round - bottom flask , 0.245 g ( 0.79 mmol ) of ( 4-(bromomethyl)phenyl)(phenyl ) sulfone ( 4 )
the mixture was taken to reflux and monitored by tlc until complete conversion of the starting material was observed ( 5 h ) .
unreacted p(oet)3 was evaporated by vacuum distillation , affording a viscous , pale yellow oil that was used for the hornerwadsworthemmons reaction without further purification .
the phosphonate was dissolved in 10 ml of dry dimethylformamide ( dmf ) , and 0.228 g of nah was added portionwise to the solution .
the mixture was stirred at room temperature for 1 h. a solution of 2,7-diformylflourene , 6 , ( 0.198 g , 0.39 mmol ) in 5 ml of dry dmf was added dropwise to the solution , which was then monitored by tlc until no 6 was observed ( 24 h ) .
once the reaction had reached completion , the solution was added dropwise to 150 ml of ice - chilled water .
the resulting yellow oil was extracted with ch2cl2 ( twice ) , washed ( three times ) with water , and dried over mgso4 .
the crude product was purified by column chromatography on silica gel ( under n2 ) using a 1:1 mixture of ethyl acetate / hexane as the eluent .
a yellow solid was obtained ( 0.142 g , 38% yield for two steps ) ; mp 150.9151.5 c .
h nmr ( 300 mhz , cdcl3 ) : 7.96 ( m , 8h ) , 7.63 ( m , 16h ) , 7.28 ( d , 2h ) , 7.12 ( d , 16.5 hz , 2h ) , 1.99 ( m , 4h ) , 1.10 ( m , 28h ) , 0.77 ( t , 0.6.3 hz , 6h ) , 0.63 ( m , 0.4h ) .
c nmr ( 75 mhz , cdcl3 ) : 151.5 ( c ) , 142.3 ( c ) , 141.5 ( c ) , 139.4 ( c ) , 135.3 ( c ) , 132.9 ( c ) , 132.8 ( ch ) , 131.2 ( ch ) , 129.1 ( ch ) , 128.0 ( ch ) , 127.3 ( ch ) , 126.7 ( c ) , 126.7 ( ch ) , 121.0 ( c ) , 120.2 ( c ) . 108.1 ( c ) , 55.0 ( c ) , 31.8 ( c ) , 29.9 ( c ) , 29.5 ( c ) , 29.2 ( c ) , 23.7 ( c ) , 22.6 ( c ) , 14.0 ( c ) . hrms for ( c61h70o4s2 ) : theoretical [ m + h ] , 931.4788 ; [ m + nh4 ] , 948.5054 ; [ m + na ] , 953.4608 . found : [ m + h ] , 931.4769 ; [ m + nh4 ] , 948.5057 ; [ m + na ] , 953.4604 .
the absorption spectra were obtained with an agilent 8453 uvvisible spectrophotometer in 10-mm - path - length quartz cuvettes for concentrations , c , of (12 )  10 m. the steady - state fluorescence spectra were recorded with a pti quantamaster spectrofluorimeter in 10-mm spectrofluorometric quartz cuvettes for dilute solutions ( c  10 m ) . all spectra were corrected for the spectral responsivity of the pti detection system .
the fluorescence quantum yields of 1 were determined relative to 9,10-diphenylanthracene in cyclohexane (  = 0.95).(29 ) fluorescence lifetimes were measured with a picoharp300 time - correlated single - photon - counting system and 76 mhz femtosecond excitation ( mira 900 , coherent ) .
optical stimulated transitions in 1 were investigated using a picosecond nd : yag laser ( pl 2143 b ekspla ) operating with exit wavelengths of 1064 nm ( base ) , 532 nm ( second harmonic ) , and 355 nm ( third harmonic ) ; a pulse duration of   21 ps ( hw1/em ) ; nearly gaussian time and space pulse intensity distributions ; and a 10 hz repetition rate .
two separate laser beams were used ( pumpprobe method(25 ) ) for one - photon excitation and one- or two - photon induced quenching .
excitation laser pulses at p = 355 nm with ep  12 j were focused into a 1-mm quartz cuvette ( concentration c  5  10 m ) with a waist of radius r0  0.15 mm .
this fluorescence was partially quenched by another laser pulse at q = 532 nm ( one - photon quenching , ep  50 j ) , propagating in the same direction , fully overlapped in space with the excitation volume and delayed for d  80 ps relative to the excitation pulse .
the amount of spontaneously emitted fluorescence photons during delay time d was negligible given the condition d  f .
after the quenching pulse , the energy of the rest of the collected fluorescence photons was measured with a silicon photodetector . for two - photon
induced quenching , the laser beam at q = 1064 nm ( ep  200 j ) was used at the same excitation conditions as for one - photon quenching : p = 355 nm , ep  12 j , r0  0.15 mm , d  80 ps , and full spatial overlap . experimental setup : ( 1 ) picosecond laser , ( 2 ) beam splitters , ( 3 ) polarizers , ( 4 ) wave plate /2 , ( 5 ) 100% reflection mirror , ( 6 ) neutral density filters , ( 7 ) calibrated silicon photodetectors , ( 8) time delay line , ( 9 ) focusing lens ( 25 cm ) , ( 10 ) 1- or 10-mm quartz cuvettes with investigated solutions , ( 11 ) optical collection system , ( 12 ) set of neutral and interferometric filters , ( 13 ) fiber - optic spectrometer . optical elements 3 and 4 were used for ease of changing the pulse energies for the wavelengths used .
a single laser beam at p = 532 nm was used in the case of two - photon excitation and simultaneous one - photon induced quenching of the fluorescence emission of 1 . according to figure 1a , the wavelength p = 532 nm is within the fluorescence emission spectral range of 1 and the losses of pulse energy ep , determined by the ground - state one - photon absorption , are negligible . in this case , the laser beam with ep  200 j was focused in a 10-mm quartz cuvette ( concentration c  3  10 m ) to a waist of radius r0  0.3 mm .
it should be mentioned that , in all measurements , the polarization of the laser beams was vertical , and the quality of the filtered scattered excitation and quenching light was controlled by a fiber - optic spectrometer usb2000 ( ocean optics inc . ) .
the linear absorption and steady - state fluorescence spectra of 1 in toluene and thf at room temperature are presented in figure 1a .
the absorption spectra were nearly independent of solvent properties , whereas the fluorescence spectra exhibited a slight solvatochromic dependence .
fluorene 1 has nearly identical extinction coefficients ( molar absorptivity ) in toluene and thf [ absmax  ( 9295 )  10 m cm ] and a single exponential fluorescence decay with lifetimes of f  0.85 ns ( toluene ) and f  0.74 ns ( thf ) .
the fluorescence quantum yields of 1 were   1 and independent of the excitation wavelength in all solvents , as shown in figure 3a .
a comprehensive analysis of the linear spectral properties of 1 , including excitation anisotropy spectra , is reported separately.(30 ) ( a ) spectral dependence of the fluorescence quantum yield  ( 1 ) and normalized absorption spectrum of 1 in toluene .
( c , d ) dependences 1  if / if0 = f(ep ) for one - photon fluorescence quenching at q = 532 nm in ( c ) toluene and ( d ) thf .
( e , f ) two - photon fluorescence quenching at q = 1064 nm in ( e ) toluene and ( f ) thf . one - photon induced fluorescence of 1 , and its delayed one- and two - photon quenching , can be analyzed from the data presented in figure 3bf .
the dependences of the integrated fluorescence emission ifl on the excitation pulse energy ep at p = 355 nm ( figure 3b ) revealed a linear one - photon population of the fluorescent level s1 over the energy range used .
a linear dependence ifl = f(ep ) confirmed the validity of assumptions ii , iii , and v. the one - photon quenching wavelength q = 532 nm was chosen to be within the fluorescence emission spectral range of 1 , and the quenching pulse was delayed to d  80 ps > 2 to avoid overlap with the excitation pulse .
the dependences of 1  if / if0 on the quenching pulse energy ep for one - photon excitation at 355 nm and one - photon quenching at 532 nm are presented in figure 3c , d . from these data ,
the values of 1  if / if0 were found to exhibit a linear dependence on ep in both solvents , in agreement with eq 6 .
the values of the one - photon stimulated transition cross sections , 10(q ) , were estimated from the slopes of these dependences .
for example , 10(532 nm )  2.5  10 cm in toluene , which is close to the corresponding value of 10(532 nm )  3  10 cm calculated from the known steady - state absorption abs( ) and fluorescence ifl( ) spectra .
the values of 1  if / if0 exhibited a quadratic dependence on the quenching pulse energy ep ( see figure 3e , f ) in the case of two - photon fluorescence quenching at q = 1064 nm , which is in good agreement with eq 9 .
it should be mentioned that q = 1064 nm is outside the fluorescence emission spectral range of 1 and , thus , one - photon stimulated transitions at this wavelength are negligible . possible esa processes at q = 1064 nm from the s1 electronic state do not affect the fluorescence intensity , as discussed previously . as a result , the quadratic dependences 1  if / if0
the values of the corresponding cross sections were estimated from the slopes of the dependences in figure 3e , f : 2pe(1064 nm )  240 and 280 gm for 1 in toluene and thf , respectively . the nature of the stimulated transitions under two - photon fluorescence excitation and simultaneous one - photon fluorescence quenching at 532 nm was analyzed from the dependences presented in figure 4 .
the quenching wavelength is located sufficiently far from the linear absorption bands and can be absorbed only through the 2pa mechanism .
the dependences of the integrated fluorescence emission ifl of 1 on excitation pulse energy ep exhibited nearly quadratic character at low excitation irradiance ( ep  15 j ) , as shown in the insets of figure 4 .
these results are in good agreement with eq 11 , describing a pure 2pa mechanism when the stimulated transitions s1  s0 are neglected . as can be seen in figure 4 ,
the dependence ifl = f(ep ) becomes close to linear in the intermediate range of excitation irradiance ( ep > 15 j ) , providing evidence for the contribution of one - photon quenching at 532 nm .
further increase in the pulse energy to ep > 200 j , along with an expected saturation effect , was not realized because of laser damage of the cuvette . for comparison , the same dependence ifl = f(ep )
the fluorescence spectrum of this compound ( curve 2 in figure 5a ) did not overlap with p = 532 nm .
as can be seen in figure 4b , the dependence ifl = f(ep ) was close to quadratic over the entire energy range . these results for p - terphenyl in toluene at p
= 532 nm were anticipated , reflecting the two - photon excitation origin of the induced fluorescence .
dependences of the integrated fluorescence intensity of 1 in ( a ) toluene and ( b ) thf on excitation pulse energy ep at p = 532 nm .
( a ) normalized absorption ( 1 ) and fluorescence ( 2 ) spectra of p - terphenyl in toluene .
( b ) dependence ifl = f(ep ) at p = 532 nm for p - terphenyl in toluene .
a comprehensive investigation of stimulated transitions in sulfonyl - containing fluorene 1 in toluene and thf was accomplished with a picosecond fluorescence quenching method at room temperature .
the fluorescence quenching processes exhibited good agreement with theoretical predictions and can be used to study the nature of stimulated transitions and to determine the corresponding one- and two - photon quenching cross sections .
 s0 with 2pe(q )  240280 gm were shown for 1 at q = 1064 nm , representing the first report of two - photon sted in a molecular system . a complex , nonquadratic dependence of two - photon induced fluorescence intensity on excitation pulse energy was obtained at p = 532 nm , providing compelling evidence for the contribution of one - photon induced quenching upon two - photon excitation .
the determination of the spectral dependences and absolute values of one- and two - photon stimulated emission cross sections is not only fundamentally important but might help develop the tenets for a number of nonlinear optical applications that can exploit sted . in particular , the two - photon stimulated emission processes can be harnessed to further increase the spatial resolution of fluorescence microscopy imaging .
based on the results of this study , sulfonyl - containing fluorene derivative 1 , exhibiting a high fluorescence quantum yield and photochemical stability , is a promising fluorescence probe for one- and two - photon sted microscopy , a subject of further investigation in our laboratory .
 one- and two - photon stimulated emission transitions were investigated by the fluorescence quenching of a sulfonyl - containing fluorene compound , 2,7-bis(4-(phenylsulfonyl)styryl)-9,9-didecyl-9h - fluorene ( 1 ) , in solution at room temperature using a picosecond pumpprobe technique . 
 the nature of stimulated transitions under various fluorescence excitation and quenching conditions was analyzed theoretically , and good agreement with experimental data was demonstrated . 
 two - photon stimulated transitions s1 
  s0 were shown for 1 at q = 1064 nm , representing the first report of two - photon stimulated emission depletion ( sted ) in a molecular system . 
 the two - photon stimulated emission cross section , 2pe(q ) , of fluorene 1 was estimated to be 240280 gm , suggesting that this compound may be a good candidate for use in two - photon sted microscopy . 
Introduction
Experimental Section
Results and Discussion
Conclusion