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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 13 — Jun. 18, 2012
  • pp: 13669–13676
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Investigation of two-photon excited fluorescence increment via crosslinked bovine serum albumin

Chun-Yu Lin, Chi-Hsiang Lien, Keng-Chi Cho, Chia-Yuan Chang, Nan-Shan Chang, Paul J. Campagnola, Chen Yuan Dong, and Shean-Jen Chen  »View Author Affiliations


Optics Express, Vol. 20, Issue 13, pp. 13669-13676 (2012)
http://dx.doi.org/10.1364/OE.20.013669


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Abstract

The two-photon excited fluorescence (TPEF) increments of two dyes via bovine serum albumin (BSA) microstructures fabricated by the two-photon crosslinking technique were investigated. One is Rose Bengal (RB) with a high non-radiative decay rate, while the other is Eosin Y with a low non-radiative decay rate. Experimental results demonstrate that the quantum yield and lifetime of RB are both augmented via crosslinked BSA microstructures. Compared with theoretical analysis, this result indicates that the non-radiative decay rate of RB is decreased; hence, the quenched effect induced by BSA solution is suppressed. However, the fluorescence lifetime of Eosin Y is acutely abated despite the augmented quantum yield for the two-photon crosslinking processing from BSA solution. This result deduces that the radiative decay rate increased. Furthermore, the increased TPEF intensity and lifetime of RB correlated with the concentration of fabricated crosslinked BSA microstructures through pulse selection of the employed femtosecond laser is demonstrated and capable of developing a zone-plate-like BSA microstructure.

© 2012 OSA

1. Introduction

Since multiphoton excited (MPE) photochemistry is confined to the focal volume, spatially-precise, sub-micron microstructures can be created three dimensionally (3D) [1

1. S. Kawata, H. B. Sun, T. Tanaka, and K. Takada, “Finer features for functional microdevices,” Nature 412(6848), 697–698 (2001). [CrossRef] [PubMed]

5

5. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]

]. In MPE photochemistry, Rose Bengal (RB) is utilized as the photoinitiator in two-photon polymerization (TPP) processing and the photoactivator in two-photon crosslinking (TPC) processing [6

6. D. C. Neckers, “Rose Bengal,” J. Photochem. Photobiol. A 47(1), 1–29 (1989). [CrossRef]

] to develop 3D microstructures of ethoxylated trimethylolpropane triacrylate (ethoxylated TMPTA) and bovine serum albumin (BSA), respectively. Also, RB is used as a chromophoric dye for two-photon excited fluorescence (TPEF) [7

7. K.-C. Cho, C.-H. Lien, C.-Y. Lin, C.-Y. Chang, L. L. H. Huang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Enhanced two-photon excited fluorescence in three-dimensionally crosslinked bovine serum albumin microstructures,” Opt. Express 19(12), 11732–11739 (2011). [CrossRef] [PubMed]

]. The TPEF of RB was significantly increased in crosslinked BSA microstructures but reduced in polymerized TMPTA microstructures [7

7. K.-C. Cho, C.-H. Lien, C.-Y. Lin, C.-Y. Chang, L. L. H. Huang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Enhanced two-photon excited fluorescence in three-dimensionally crosslinked bovine serum albumin microstructures,” Opt. Express 19(12), 11732–11739 (2011). [CrossRef] [PubMed]

]. This seems to be an augmentation mechanism for TPEF of RB via crosslinked BSA microstructures.

TPEF is a nonlinear phenomenon and its intensity is proportional to the two-photon absorption (TPA) cross-section, square of excitation power, and fluorescence quantum yield [8

8. C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13(3), 481–491 (1996). [CrossRef]

]. Generally speaking, the intensity of TPEF is very weak, which results from the extremely low TPA cross-section. As such, enhancement of the TPEF signal has generated interest and led to several techniques that increase its intensity. Two such techniques include the enhancement of localized excitation power and the modification of the intrinsic characteristics of the fluorescent dye by increasing the TPA cross-section and fluorescence quantum yield. For instance, the periodic structures, based on waveguide resonance, were used to enhance localized excitation power 120 times [9

9. J. Y. Ye, M. Ishikawa, Y. Yamane, N. Tsurumachi, and H. Nakatsuka, “Enhancement of two-photon excited fluorescence using one-dimensional photonic crystals,” Appl. Phys. Lett. 75(23), 3605–3607 (1999). [CrossRef]

], while designed organic molecules, based on symmetric charge transfer, can enlarge the TPA cross-section 400 times [10

10. M. Albota, D. Beljonne, J. L. Brédas, J. E. Ehrlich, J. Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X. L. Wu, and C. Xu, “Design of organic molecules with large two-photon absorption cross sections,” Science 281(5383), 1653–1656 (1998). [CrossRef] [PubMed]

].

In this study, the TPEF increments of two dyes via BSA microstructures fabricated by the two-photon crosslinking technique were investigated. One is RB with a high non-radiative decay rate, while the other is Eosin Y with a low non-radiative decay rate. TPEF increments are due to enlargements of their quantum yields and can be confirmed from the variations of their fluorescence lifetimes. Experimental results demonstrate that the quantum yield and lifetime of RB are both augmented via crosslinked BSA microstructures resulting from reducing the non-radiative decay rate and suppressing the quenched effect induced by the BSA solution. For Eosin Y, the fluorescence lifetime is acutely abated despite the augmented quantum yield, which indicates that the radiative decay rate is increased. Both simulation and experimental results confirm that a fluorescent dye with a high non-radiative decay rate, such as RB, has a large TPEF augmentation. Furthermore, the concentration of crosslinked BSA can be modified by controlling the pulse number of the employed femtosecond laser. A zone-plate-like microstructure as a function of the concentration of crosslinked BSA correlating with the TPEF intensity and lifetime of RB was developed.

2. Theoretical analysis

2.1. Quantum yield proportional to variation of TPEF intensity

Investigation of the fluorescent dye TPEF increment relative to its quantum yield enlargement requires that the quantum yield and TPEF intensity are connected. The TPEF intensity is proportional to the square of excitation power. The time-averaged fluorescence F can be expressed as [8

8. C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13(3), 481–491 (1996). [CrossRef]

]
FP2τpfσ(λ)CQ,
(1)
where P is the excitation power of the employed femtosecond laser, τp the pulse width, and f the pulse repetition rate. Further, σ(λ) is the TPA cross-section of fluorescent dye, C the concentration, and Q the fluorescence quantum yield. Equation (1) can be modified as the slope S of the differentiation of the TPEF intensity divided by the differentiation of the square of the excitation power. The slope S is then proportional to the quantum yield as

S=ΔFΔP21τpfσ(λ)CQ.
(2)

To evaluate the fluorescence quantum yield according to the measured TPEF intensity and the standard of the quantum yield under same femtosecond laser excitation condition, an estimated quantum yield Q can be calculated by
Q=σrσCrCSSrQr,
(3)
where r denotes the fluorescence standard.

2.2. Quantum yield and lifetime based on radiative and non-radiative decay rates

Fluorescence quantum yield and lifetime are both major parameters when investigating the emission characteristics of fluorescent dyes in diverse environments. Fluorescence quantum yields are usually the ratio of the emitted photons to the absorbed photons. The quantum yield Q is also described by electron decay rates and can be expressed as [16

16. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd Edition (Springer, 2010).

]
Q=ΓΓ+k,
(4)
where Γ is the radiative decay rate and k is the non-radiative decay rate. When the non-radiative decay rate is smaller than the radiative decay rate, the quantum yield approaches unity. On the other hand, the quantum yield moves toward zero if the non-radiative decay rate is greater than the radiative decay rate. Fluorescence lifetime is usually defined as the average time required for an electron in the excited state to decay to the ground state. The fluorescence lifetime τ can be also relative to the decay rates and described as [16

16. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd Edition (Springer, 2010).

]

τ=1Γ+k.
(5)

The quantum yield enlarges with an increase in the radiative decay rate or a decrease in the non-radiative decay rate. However, the lifetime lengthens when decreasing either the radiative or non-radiative decay rate. Hence, increasing the radiative decay rate leads to an increase of the fluorescence quantum yield and a decrease in its lifetime. Consequently, increasing the non-radiative decay rate results in decreases of both the fluorescence quantum yield and lifetime.

RB and Eosin Y were chosen to explore whether the decay rates influence the fluorescence quantum yield and lifetime. RB has a very low quantum yield of 0.01 and a short lifetime of 80 ps in deionized water [17

17. M. A. Montenegro, M. A. Nazareno, E. N. Durantini, and C. D. Borsarelli, “Singlet molecular oxygen quenching ability of carotenoids in a reverse-micelle membrane mimetic system,” Photochem. Photobiol. 75(4), 353–361 (2002). [CrossRef] [PubMed]

]; by comparison, Eosin Y has a higher quantum yield of 0.2 and a longer lifetime of 0.95 ns in deionized water [18

18. A. Penzkofer, A. Beidoun, and M. Daiber, “Intersystem-crossing and excited-state absorption in eosin Y solutions determined by picosecond double pulse transient absorption measurements,” J. Lumin. 51(6), 297–314 (1992). [CrossRef]

]. The original radiative and non-radiative decay rates of RB are 1.25 × 108 s−1 and 1.24 × 1010 s−1, respectively. As can be seen, the non-radiative decay rate is two orders higher than the radiative decay rate. On the other hand, the original radiative decay rate of Eosin Y is 2.11 × 108 s−1, which is close to the non-radiative decay rate of 8.42 × 108 s−1. As a consequence of a fluorescent dye with a relatively large non-radiative decay rate such as RB, variations in the quantum yield and lifetime are dominated by the changes of the non-radiative decay rate. Alternatively, the quantum yield and lifetime of Eosin Y are both affected by the radiative and non-radiative decay rates; however, the quantum yield of which does not apparently change under the radiative and non-radiative decay rates. Hence, the increment of TPEF intensity can be observed for fluorescent dyes with low quantum yields. For example, the TPEF intensity of RB can be maximally increased by up to 100 times compared to that in solution.

3. Microfabrication setup and experimental results

3.1. Sample preparation and optical setup

3.2. Experiment results for fluorescent dyes with low and high quantum yields

In comparison, the fluorescence quantum yield and lifetime of Eosin Y are both diminished in the BSA solution in deionized water (see Figs. 2(c) and 2(d)); hence, Eosin Y is quenched by the BSA molecule. This indicates that the non-radiative decay rate of Eosin Y is increased at this moment, and so the quenching effect is augmented for both RB and Eosin Y in the BSA solution. Nevertheless, the quantum yield increases and the lifetime decreases when BSA is crosslinked, as shown in Figs. 2(c) and 2(d), which implies that the radiative decay rate of Eosin Y is enlarged by BSA TPC processing. This is not, however, the same result as RB. The quantum yield and lifetime are both increased when the fabrication power is increased. The phenomenon is due to the reduction of the non-radiative decay rate in crosslinked BSA. The relative values of the quantum yield based on the quantity in deionized water are 0.18, 0.34, 0.42, and 0.42 for the BSA solution, and for the crosslinked BSA microstructures with the fabrication powers of 2.13, 2.40, and 2.67 mW, respectively. Compared with Eosin Y in the BSA solution, the highest quantum yield increment in the crosslinked BSA microstructures is only 2.3 times. Overall, the experimental results demonstrate that the quantum yields and lifetimes of RB and Eosin Y in the crosslinked BSA microstructures are increased due to the reduction of the quenching effect resulting from decreasing the non-radiative decay rate, particularly under high fabrication power.

3.3. Lifetime imaging for RB in crosslinked BSA microstructures

Figure 4
Fig. 4 Images of zone-plate-like BSA microstructure fabricated by the four pulse numbers of 60,000, 51,200, 44,000, and 40,000 from inside to outside: (a) TPEF intensity, (b) fluorescence lifetime, and (c) combination of (a) & (b).
shows a zone-plate-like BSA crosslinked microstructure that was fabricated by modulating pulse numbers. Pulse numbers were 60,000 (75%), 51,200, 44,000 and 40,000 (50%) pulses for the zero-order, first-order, second-order, and third-order, sequentially. The radiuses of the concentric circles from the inside to the outside are 6.89, 9.74, 11.93, 13.78, 15.4, 16.87, and 18.23 μm, respectively. The average TPEF intensities are around 2,670, 3,970, 5,380, and 7,400 photon counts in Fig. 4(a), while Fig. 4(b) indicates average fluorescence lifetimes of about 100, 120, 140, and 160 ps for the third-order, second-order, first-order, and zero-order, respectively. The average TPEF intensity of RB measures only about 40 photon counts in BSA solution, it is significantly increased via the crosslinked BSA microstructure, the increment of which can reach well over 60 times. Obviously, the TPEF intensity and fluorescence lifetime are both increased with increasing pulse numbers, which also results in increasing the concentration of the crosslinked BSA. Therefore, the quenching effect gradually decreases according to the reduction in the non-radiative decay rate. Figure 4(c) shows an image of the zone-plate-like BSA microstructure based on a combination of the TPEF intensity and fluorescence lifetime. Colors denote the different fluorescence lifetimes, while brightness indicates the TPEF intensity.

4. Conclusions

The TPEF intensity (i.e. the quantum yield) of RB is augmented via crosslinked BSA microstructures due to a reduction in the quenching effect. Therefore, increments in the fluorescence lifetime are observed while non-radiative decay rates decrease. However, the fluorescence lifetime of Eosin Y is acutely abated despite the augmented quantum yield for TPC processing from the BSA solution, which implies that the radiative decay rate is increased. Theoretical analysis and experimental results both reveal that the TPEF intensity of low quantum yield fluorescent dye can have large increments induced via crosslinked BSA microstructures. Compared with the TPEF intensity of RB in BSA solution, a maximal increment of 180 times can be achieved via the crosslinked BSA microstructures. The increased TPEF intensity and lifetime of RB correlated with the concentration of crosslinked BSA microstructures through modulation of the femtosecond laser pulse number is demonstrated and capable of developing a zone-plate-like BSA microstructure.

Acknowledgments

This work was supported by the National Science Council (NSC) in Taiwan with the grant numbers of NSC 99-2627-B-006-017, NSC 99-3111-B-006-004, and NSC 100-2623-E-006-016-D.

References and links

1.

S. Kawata, H. B. Sun, T. Tanaka, and K. Takada, “Finer features for functional microdevices,” Nature 412(6848), 697–698 (2001). [CrossRef] [PubMed]

2.

P. Galajda and P. Ormos, “Complex micromachines produced and driven by light,” Appl. Phys. Lett. 78(2), 249–251 (2001). [CrossRef]

3.

T. Tanaka, H. B. Sun, and S. Kawata, “Rapid sub-diffraction-limit laser micro/nanoprocessing in a threshold material system,” Appl. Phys. Lett. 80(2), 312–314 (2002). [CrossRef]

4.

M. Miwa, S. Juodkazis, T. Kawakami, S. Matsuo, and H. Misawa, “Femtosecond two-photon stereo-lithography,” Appl. Phys., A Mater. Sci. Process. 73(5), 561–566 (2001). [CrossRef]

5.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]

6.

D. C. Neckers, “Rose Bengal,” J. Photochem. Photobiol. A 47(1), 1–29 (1989). [CrossRef]

7.

K.-C. Cho, C.-H. Lien, C.-Y. Lin, C.-Y. Chang, L. L. H. Huang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Enhanced two-photon excited fluorescence in three-dimensionally crosslinked bovine serum albumin microstructures,” Opt. Express 19(12), 11732–11739 (2011). [CrossRef] [PubMed]

8.

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13(3), 481–491 (1996). [CrossRef]

9.

J. Y. Ye, M. Ishikawa, Y. Yamane, N. Tsurumachi, and H. Nakatsuka, “Enhancement of two-photon excited fluorescence using one-dimensional photonic crystals,” Appl. Phys. Lett. 75(23), 3605–3607 (1999). [CrossRef]

10.

M. Albota, D. Beljonne, J. L. Brédas, J. E. Ehrlich, J. Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X. L. Wu, and C. Xu, “Design of organic molecules with large two-photon absorption cross sections,” Science 281(5383), 1653–1656 (1998). [CrossRef] [PubMed]

11.

M. Kauert, P. C. Stoller, M. Frenz, and J. Ricka, “Absolute measurement of molecular two-photon absorption cross-sections using a fluorescence saturation technique,” Opt. Express 14(18), 8434–8447 (2006). [CrossRef] [PubMed]

12.

N. S. Makarov, M. Drobizhev, and A. Rebane, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range,” Opt. Express 16(6), 4029–4047 (2008). [CrossRef] [PubMed]

13.

A. Nag and D. Goswami, “Solvent effect on two-photon absorption and fluorescence of rhodamine dyes,” J. Photochem. Photobiol. Chem. 206(2-3), 188–197 (2009). [CrossRef] [PubMed]

14.

C. V. Bindhu, S. S. Harilal, G. K. Varier, R. C. Issac, V. P. N. Nampoori, and C. P. G. Vallabhan, “Measurement of the absolute fluorescence quantum yield of rhodamine B solution using a dual-beam thermal lens technique,” J. Phys. D 29(4), 1074–1079 (1996). [CrossRef]

15.

C. V. Bindhu and S. S. Harilal, “Effect of the excitation source on the quantum-yield measurements of rhodamine B laser dye studied using thermal-lens technique,” Anal. Sci. 17(1), 141–144 (2001). [CrossRef] [PubMed]

16.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd Edition (Springer, 2010).

17.

M. A. Montenegro, M. A. Nazareno, E. N. Durantini, and C. D. Borsarelli, “Singlet molecular oxygen quenching ability of carotenoids in a reverse-micelle membrane mimetic system,” Photochem. Photobiol. 75(4), 353–361 (2002). [CrossRef] [PubMed]

18.

A. Penzkofer, A. Beidoun, and M. Daiber, “Intersystem-crossing and excited-state absorption in eosin Y solutions determined by picosecond double pulse transient absorption measurements,” J. Lumin. 51(6), 297–314 (1992). [CrossRef]

19.

W.-S. Kuo, C.-H. Lien, K.-C. Cho, C.-Y. Chang, C.-Y. Lin, L. L. H. Huang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Multiphoton fabrication of freeform polymer microstructures with gold nanorods,” Opt. Express 18(26), 27550–27559 (2010). [CrossRef] [PubMed]

20.

M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J. 88(2), 1224–1237 (2005). [CrossRef] [PubMed]

21.

L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, K.-C. Cho, W.-C. Yen, N.-S. Chang, C. Xu, C. Y. Dong, and S.-J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express 20(8), 8939–8948 (2012). [CrossRef] [PubMed]

OCIS Codes
(160.2540) Materials : Fluorescent and luminescent materials
(190.4180) Nonlinear optics : Multiphoton processes
(220.4000) Optical design and fabrication : Microstructure fabrication

ToC Category:
Materials

History
Original Manuscript: March 27, 2012
Revised Manuscript: April 23, 2012
Manuscript Accepted: April 24, 2012
Published: June 4, 2012

Virtual Issues
Vol. 7, Iss. 8 Virtual Journal for Biomedical Optics

Citation
Chun-Yu Lin, Chi-Hsiang Lien, Keng-Chi Cho, Chia-Yuan Chang, Nan-Shan Chang, Paul J. Campagnola, Chen Yuan Dong, and Shean-Jen Chen, "Investigation of two-photon excited fluorescence increment via crosslinked bovine serum albumin," Opt. Express 20, 13669-13676 (2012)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-20-13-13669


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References

  1. S. Kawata, H. B. Sun, T. Tanaka, and K. Takada, “Finer features for functional microdevices,” Nature412(6848), 697–698 (2001). [CrossRef] [PubMed]
  2. P. Galajda and P. Ormos, “Complex micromachines produced and driven by light,” Appl. Phys. Lett.78(2), 249–251 (2001). [CrossRef]
  3. T. Tanaka, H. B. Sun, and S. Kawata, “Rapid sub-diffraction-limit laser micro/nanoprocessing in a threshold material system,” Appl. Phys. Lett.80(2), 312–314 (2002). [CrossRef]
  4. M. Miwa, S. Juodkazis, T. Kawakami, S. Matsuo, and H. Misawa, “Femtosecond two-photon stereo-lithography,” Appl. Phys., A Mater. Sci. Process.73(5), 561–566 (2001). [CrossRef]
  5. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
  6. D. C. Neckers, “Rose Bengal,” J. Photochem. Photobiol. A47(1), 1–29 (1989). [CrossRef]
  7. K.-C. Cho, C.-H. Lien, C.-Y. Lin, C.-Y. Chang, L. L. H. Huang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Enhanced two-photon excited fluorescence in three-dimensionally crosslinked bovine serum albumin microstructures,” Opt. Express19(12), 11732–11739 (2011). [CrossRef] [PubMed]
  8. C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B13(3), 481–491 (1996). [CrossRef]
  9. J. Y. Ye, M. Ishikawa, Y. Yamane, N. Tsurumachi, and H. Nakatsuka, “Enhancement of two-photon excited fluorescence using one-dimensional photonic crystals,” Appl. Phys. Lett.75(23), 3605–3607 (1999). [CrossRef]
  10. M. Albota, D. Beljonne, J. L. Brédas, J. E. Ehrlich, J. Y. Fu, A. A. Heikal, S. E. Hess, T. Kogej, M. D. Levin, S. R. Marder, D. McCord-Maughon, J. W. Perry, H. Röckel, M. Rumi, G. Subramaniam, W. W. Webb, X. L. Wu, and C. Xu, “Design of organic molecules with large two-photon absorption cross sections,” Science281(5383), 1653–1656 (1998). [CrossRef] [PubMed]
  11. M. Kauert, P. C. Stoller, M. Frenz, and J. Ricka, “Absolute measurement of molecular two-photon absorption cross-sections using a fluorescence saturation technique,” Opt. Express14(18), 8434–8447 (2006). [CrossRef] [PubMed]
  12. N. S. Makarov, M. Drobizhev, and A. Rebane, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range,” Opt. Express16(6), 4029–4047 (2008). [CrossRef] [PubMed]
  13. A. Nag and D. Goswami, “Solvent effect on two-photon absorption and fluorescence of rhodamine dyes,” J. Photochem. Photobiol. Chem.206(2-3), 188–197 (2009). [CrossRef] [PubMed]
  14. C. V. Bindhu, S. S. Harilal, G. K. Varier, R. C. Issac, V. P. N. Nampoori, and C. P. G. Vallabhan, “Measurement of the absolute fluorescence quantum yield of rhodamine B solution using a dual-beam thermal lens technique,” J. Phys. D29(4), 1074–1079 (1996). [CrossRef]
  15. C. V. Bindhu and S. S. Harilal, “Effect of the excitation source on the quantum-yield measurements of rhodamine B laser dye studied using thermal-lens technique,” Anal. Sci.17(1), 141–144 (2001). [CrossRef] [PubMed]
  16. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd Edition (Springer, 2010).
  17. M. A. Montenegro, M. A. Nazareno, E. N. Durantini, and C. D. Borsarelli, “Singlet molecular oxygen quenching ability of carotenoids in a reverse-micelle membrane mimetic system,” Photochem. Photobiol.75(4), 353–361 (2002). [CrossRef] [PubMed]
  18. A. Penzkofer, A. Beidoun, and M. Daiber, “Intersystem-crossing and excited-state absorption in eosin Y solutions determined by picosecond double pulse transient absorption measurements,” J. Lumin.51(6), 297–314 (1992). [CrossRef]
  19. W.-S. Kuo, C.-H. Lien, K.-C. Cho, C.-Y. Chang, C.-Y. Lin, L. L. H. Huang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Multiphoton fabrication of freeform polymer microstructures with gold nanorods,” Opt. Express18(26), 27550–27559 (2010). [CrossRef] [PubMed]
  20. M. Peter, S. M. Ameer-Beg, M. K. Y. Hughes, M. D. Keppler, S. Prag, M. Marsh, B. Vojnovic, and T. Ng, “Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions,” Biophys. J.88(2), 1224–1237 (2005). [CrossRef] [PubMed]
  21. L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, K.-C. Cho, W.-C. Yen, N.-S. Chang, C. Xu, C. Y. Dong, and S.-J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express20(8), 8939–8948 (2012). [CrossRef] [PubMed]

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