A. Fischer, C. Cremer, and E. H. K. Stelzer, "Fluorescence of coumarins and xanthenes after two-photon absorption with a pulsed titanium–sapphire laser," Appl. Opt. 34, 1989-2003 (1995)
Fluorescence emission after two-photon absorption of coumarins and xanthenes in an alcoholic solution was measured in the tuning range of a femtosecond-pulsed titanium–sapphire laser (750–840 nm). Xanthenes, which have a low one-photon absorption in the near UV, show a higher fluorescence signal after two-photon absorption than the UV-excitable coumarins. When fluxes of 1028 photons/(cm2 s) are used, the two-photon absorption cross sections for xanthenes are 1 order of magnitude higher than the two-photon absorption cross sections of the coumarins. Absolute cross sections have been estimated for three coumarins and three xanthenes. For the xanthenes a significant wavelength-dependent departure from the expected fluorescence intensity square law was observed. The coumarins follow the square-law dependence. The consequences of the findings are discussed for analytic and diagnostic methods. An especially important result is that the resolution in two-photon microscopy of xanthenes is worse than expected.
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Relative Fluorescence Signals after Two-Photon Absorption of the 10−3-M Solutions in Methanola
Substance
λabs* (nm)
λem* (nm)
ɛ (L mole−1 cm−1)
Fluorescence Signal (%)
λem (nm)
Coumarin 120
352
428
1.70 × 104
1.6
429
Coumarin 1
374
450
2.54 × 104
11.7
455
Coumarin 138
365
447
2.23 × 104
8.7
457
Coumarin 151
377
479
1.70 × 104
15.9
483
Coumarin 152
394
496
1.94 × 104
3.6
510
Coumarin 153
423
532
1.47 × 104
10.2
536
Fluorescein
498
518
6.39 × 104
3.2
532
Rhodamine 6G
528
555
11.6 × 104
100
579
Rhodamine B
545
565
10.6 × 104
28.3
605
Chresyl-Violett
593
615
8.3 × 104
0
Oxazine 4
610
625
10.3 × 104
0
Oxazine 170
620
637
8.3 × 104
0
Nilblue A
627
660
7.68 × 104
0
Fluorescence signal of Rhodamine 6G is set equal to 100%. The excitation wavelength of the titanium–sapphire laser was 784 nm. λabs* and λem* are taken from the literature.14,15 The measured value λem is shifted to longer wavelengths because of self-absorption. For the bottom four dyes no fluorescence emission could be found.
Table 2
Quantum Efficiencies (QE’s) Relative to Rhodamine 6G for Excitation Wavelengths between 375 and 425 nma
Substance
QE
Absolute Error
Relative Error (%)
Rhodamine 6G
0.95
—
—
Rhodamine B
0.499
0.038
7.6
Fluorescein
0.496
0.074
14.9
Coumarin 1
0.529
0.034
6.4
Coumarin 120
0.514
0.096
18.6
Coumarin 151
0.582
0.032
5.5
Values were calculated from the fluorescence signals after one-photon absorption at six excitation wavelengths between 375 and 425 nm shown in Fig. 7. The one-photon absorption cross sections for these excitation wavelengths are presented in Fig. 8.
Table 3
Ratio of the Quantum Efficiencies for Excitation Wavelengths 375–425 nm and 514.5 nm Compared with Values Found by Other Authors
Estimation of the Two-Photon Absorption Cross Section for the Solutions in Ethanol Excited with the Titanium–Sapphire Lasera
Substance
λexc (nm)
δ × 10−50 (cm4 s)
Coumarin 120
754.5
19.3
774.5
6.8
797.0
2.0
814.5
0.01
840.5
—
Coumarin 1
754.5
103.5
774.5
75.6
797.0
28.4
814.5
9.0
840.5
1.4
Coumarin 151
754.0
47.1
774.5
40.0
797.0
24.1
813.5
16.5
840.0
11.6
Fluorescein
754.5
188.6
774.5
210.5
796.5
170.2
813.5
75.4
840.5
27.3
Rhodamine 6G
754.0
197.0
774.5
221.5
795.5
243.5
814.0
169.1
840.0
155.1
Rhodamine B
754.5
421.1
774.5
532.9
797.0
719.9
814.0
474.7
840.5
796.7
Values were calculated with Eq. (16). The fluorescence signal after two-photon absorption of the xanthenes was believed to be quenched (Fig. 4). The quenching factors used are shown in Table 6. Comparison with the values found by other authors shows that the values measured by us are systematically one order of magnitude higher.
Table 5
Values for the Two-Photon Absorption Cross Section Taken from the Literaturea
Measurements were all made with ruby or neodymium lasers with nanosecond or picosecond pulses. The fluxes used were of the same order of magnitude or higher because giant pulse lasers were used. When only ns (nanoseconds) or ps (picoseconds) are indicated for a particular case, it means that specific pulse-width values were not available.
Table 6
Estimated Excited-State Absorption Coefficients σ2nof the Xanthenes for Excitation Wavelengths 770 and 825 nma
Substance
σ2n × 10−16 (cm2)
Δ Quenching Factor
λexc = 770 nm
λexc = 825 nm
Fluorescein
3.0
—
1.4
Rhodamine 6G
1.7
8.0
1.8
Rhodamine B
5.2
6.3
1.8
Values are the results of the fits of the sum N1(t) + N2(t) according to Eq. (13) to the experimental data (Fig. 4). We assumed the constant values τ21 = 1 ps for the relaxation time and t = 100 fs for the pulse duration. The resulting quenching factors are calculated for a flux of (2.7 ± 1) × 1028 photons/(cm2 s), which was used in the experimental determination of the two-photon absorption cross sections (Fig. 10). The slopes of the experimental data were nearly equal for the same excitation wavelength. Since this result is not fulfilled for the results of the simple fit, we averaged the results of the fits. We therefore assumed a quenching factor of 1.8 for all xanthenes and all excitation wavelengths.
Tables (6)
Table 1
Relative Fluorescence Signals after Two-Photon Absorption of the 10−3-M Solutions in Methanola
Substance
λabs* (nm)
λem* (nm)
ɛ (L mole−1 cm−1)
Fluorescence Signal (%)
λem (nm)
Coumarin 120
352
428
1.70 × 104
1.6
429
Coumarin 1
374
450
2.54 × 104
11.7
455
Coumarin 138
365
447
2.23 × 104
8.7
457
Coumarin 151
377
479
1.70 × 104
15.9
483
Coumarin 152
394
496
1.94 × 104
3.6
510
Coumarin 153
423
532
1.47 × 104
10.2
536
Fluorescein
498
518
6.39 × 104
3.2
532
Rhodamine 6G
528
555
11.6 × 104
100
579
Rhodamine B
545
565
10.6 × 104
28.3
605
Chresyl-Violett
593
615
8.3 × 104
0
Oxazine 4
610
625
10.3 × 104
0
Oxazine 170
620
637
8.3 × 104
0
Nilblue A
627
660
7.68 × 104
0
Fluorescence signal of Rhodamine 6G is set equal to 100%. The excitation wavelength of the titanium–sapphire laser was 784 nm. λabs* and λem* are taken from the literature.14,15 The measured value λem is shifted to longer wavelengths because of self-absorption. For the bottom four dyes no fluorescence emission could be found.
Table 2
Quantum Efficiencies (QE’s) Relative to Rhodamine 6G for Excitation Wavelengths between 375 and 425 nma
Substance
QE
Absolute Error
Relative Error (%)
Rhodamine 6G
0.95
—
—
Rhodamine B
0.499
0.038
7.6
Fluorescein
0.496
0.074
14.9
Coumarin 1
0.529
0.034
6.4
Coumarin 120
0.514
0.096
18.6
Coumarin 151
0.582
0.032
5.5
Values were calculated from the fluorescence signals after one-photon absorption at six excitation wavelengths between 375 and 425 nm shown in Fig. 7. The one-photon absorption cross sections for these excitation wavelengths are presented in Fig. 8.
Table 3
Ratio of the Quantum Efficiencies for Excitation Wavelengths 375–425 nm and 514.5 nm Compared with Values Found by Other Authors
Estimation of the Two-Photon Absorption Cross Section for the Solutions in Ethanol Excited with the Titanium–Sapphire Lasera
Substance
λexc (nm)
δ × 10−50 (cm4 s)
Coumarin 120
754.5
19.3
774.5
6.8
797.0
2.0
814.5
0.01
840.5
—
Coumarin 1
754.5
103.5
774.5
75.6
797.0
28.4
814.5
9.0
840.5
1.4
Coumarin 151
754.0
47.1
774.5
40.0
797.0
24.1
813.5
16.5
840.0
11.6
Fluorescein
754.5
188.6
774.5
210.5
796.5
170.2
813.5
75.4
840.5
27.3
Rhodamine 6G
754.0
197.0
774.5
221.5
795.5
243.5
814.0
169.1
840.0
155.1
Rhodamine B
754.5
421.1
774.5
532.9
797.0
719.9
814.0
474.7
840.5
796.7
Values were calculated with Eq. (16). The fluorescence signal after two-photon absorption of the xanthenes was believed to be quenched (Fig. 4). The quenching factors used are shown in Table 6. Comparison with the values found by other authors shows that the values measured by us are systematically one order of magnitude higher.
Table 5
Values for the Two-Photon Absorption Cross Section Taken from the Literaturea
Measurements were all made with ruby or neodymium lasers with nanosecond or picosecond pulses. The fluxes used were of the same order of magnitude or higher because giant pulse lasers were used. When only ns (nanoseconds) or ps (picoseconds) are indicated for a particular case, it means that specific pulse-width values were not available.
Table 6
Estimated Excited-State Absorption Coefficients σ2nof the Xanthenes for Excitation Wavelengths 770 and 825 nma
Substance
σ2n × 10−16 (cm2)
Δ Quenching Factor
λexc = 770 nm
λexc = 825 nm
Fluorescein
3.0
—
1.4
Rhodamine 6G
1.7
8.0
1.8
Rhodamine B
5.2
6.3
1.8
Values are the results of the fits of the sum N1(t) + N2(t) according to Eq. (13) to the experimental data (Fig. 4). We assumed the constant values τ21 = 1 ps for the relaxation time and t = 100 fs for the pulse duration. The resulting quenching factors are calculated for a flux of (2.7 ± 1) × 1028 photons/(cm2 s), which was used in the experimental determination of the two-photon absorption cross sections (Fig. 10). The slopes of the experimental data were nearly equal for the same excitation wavelength. Since this result is not fulfilled for the results of the simple fit, we averaged the results of the fits. We therefore assumed a quenching factor of 1.8 for all xanthenes and all excitation wavelengths.