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Virtual Journal for Biomedical Optics

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  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 7, Iss. 7 — Jun. 25, 2012
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Long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay

Po-Yen Lin, Shin-Shian Lee, Chia-Seng Chang, and Fu-Jen Kao  »View Author Affiliations


Optics Express, Vol. 20, Issue 10, pp. 11445-11450 (2012)
http://dx.doi.org/10.1364/OE.20.011445


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Abstract

In this work, long working distance fluorescence lifetime imaging is realized with stimulated emission in combination with electronic time delay control. Spatial coherence, as a result of stimulated emission, supports unattenuated fluorescence detection at extended distance, using low NA optics. An electronic time delayed trigger provides an advantageous way in adjusting the pulse separation and probing the fluorescence lifetime in the nanosecond ranges. The fluorescence lifetime of selected fluorophores is accurately determined through the pump-probe configuration. The characteristics and applications in fluorescence lifetime measurement of stimulated emission are investigated and summarized succinctly here.

© 2012 OSA

1. Introduction

Fluorescence labeling and detection is a powerful method in numerous applications, due to its high sensitivity and specificity [1

1. R. Y. Tsien, “Imagining imaging’s future,” Nat. Rev. Mol. Cell Biol. 4(Suppl), SS16–SS21 (2003). [PubMed]

5

5. G. McConnell, “Confocal laser scanning fluorescence microscopy with a visible continuum source,” Opt. Express 12(13), 2844–2850 (2004). [CrossRef] [PubMed]

]. Fluorescence emission is a spontaneous process and the emitted photons are distributed among all solid angles (4Pi). Therefore, fluorescence imaging is commonly performed with high numerical aperture (NA) optics, to achieve both a high spatial resolution and effective collection of emitted photons. To detect weak fluorescence signals from dark fluorophores, a highly sensitive detector with a large gain is frequently used. Stimulated emission (SE) was demonstrated as an advantageous method in detecting dark fluorophores [6

6. W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009). [CrossRef] [PubMed]

]. The working principle of SE detection is to induce the electronic transition of an excited fluorophore into SE before its energy dissipates into other radiative or non-radiative decay processes. Therefore, the SE signals depend on both the population of excited state and the intensity of the stimulation beam. Without any saturation effects, the SE signal scales linearly with both excitation and stimulation beams, which renders its overall quadratic power dependence and the 3-D optical sectioning in imaging as a two-photon process. Additionally, spatial coherence of the stimulated fluorescence emission is maintained, leading to the emission in a narrow cone in the forward direction, similar to the case of second harmonic generation or other coherent optical processes. We have exploited the resulting spatial coherence for long working distance imaging of fluorescenctly labeled specimens [7

7. T. Dellwig, M. R. Foreman, and F.-J. Kao, “Coherent long-distance signal detection using stimulated emission: a feasibility study,” Chin. J. Physiol. 48, 873–884 (2010).

,8

8. T. Dellwig, P.-Y. Lin, and F.-J Kao, “Long-distance fluorescence lifetime imaging using stimulated emission,” J. Biomed. Opt. 17, (in press)

].

The use of high NA optics for efficient collection of fluorescence would, however, limit the working distance and the depth of focus accordingly, which would, in turn, limit the size and the depth of the targeted objects. Separately, fluorescence lifetime microscopy is an effective tool to extract critical information on molecular dynamics, such as molecular conformation and the changes in the immediate nano-environment of a biological sample [9

9. E. B. van Munster and T. W. J. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol. 95, 143–175 (2005). [PubMed]

12

12. D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett. 33(20), 2365–2367 (2008). [CrossRef] [PubMed]

]. It is widely used in mapping pertinent cellular parameters and tissue characteristics, such as ion concentration [13

13. C. Hille, M. Lahn, H.-G. Löhmannsröben, and C. Dosche, “Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands,” Photochem. Photobiol. Sci. 8(3), 319–327 (2009). [CrossRef] [PubMed]

], pH of the environment [14

14. M. Y. Berezin, J. Kao, and S. Achilefu, “pH-Dependent Optical Properties of Synthetic Fluorescent Imidazoles,” Chemistry 15(14), 3560–3566 (2009). [CrossRef] [PubMed]

], and carious dental tissue identification [15

15. P.-Y. Lin, H.-C. Lyu, C.-Y. S. Hsu, C.-S. Chang, and F.-J. Kao, “Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy,” Biomed. Opt. Express 2(1), 149–158 (2011). [CrossRef] [PubMed]

]. Measuring fluorescence lifetime of targeted molecules is usually conducted by detecting the emission of spontaneously emitted photons, by methods of time- or frequency domains. In this work, we are demonstrating fluorescence lifetime measurement by the pump-probe scheme [16

16. C.-Y. Dong, P. T. C. So, T. French, and E. Gratton, “Fluorescence lifetime imaging by asynchronous pump-probe microscopy,” Biophys. J. 69(6), 2234–2242 (1995). [CrossRef] [PubMed]

]. The temporal resolution of this method is typically determined by the pulse width of the lasers or the delay control between the pump-probe laser pair. We use electronic delay control and low NA optics to measure fluorescence lifetime of ATTO 647N dye and to achieve long distance detection. The fluorescence lifetime of ATTO 647N is approximately 3 ns, which would require an optical path length around a few meters to map out the delay. The relatively long length presents a substantial challenge in optical alignment and beam shaping if a mechanical translation stage is used. Electronics delay is used instead to overcome the above difficulties. Additionally, the dependences of SE on sample concentration and laser intensity are also characterized.

2. Materials and methods

The schematic of fluorescence lifetime detection with SE is shown in Fig. 1
Fig. 1 Schematic of the SE enabled long working distance fluorescence lifetime imaging setup. EOM: electro optical modulator. BP: band pass filter. DM: dichroic mirror. PD: photodiode.
. A pulsed diode laser (Picoquant, Berlin, Germany) that is operating at a wavelength of 635 nm acts as the excitation source and is synchronized with the mode-locked Ti:Sapphire laser (Mira900, Coherent, USA) through a fast photodiode (TDA 200, Picoquant, Berlin, Germany). The Ti:Sapphire laser is operated at 740 nm with a repetition rate of 76 MHz. The selected wavelengths closely match the absorption and emission spectra of the ATTO647N dye (ATTO-TEC, Germany), respectively.

Depending on the fluorescence lifetime of the targeted fluorophores, various time delay schemes may be used. For very short fluorescence lifetime fluorophores (in the order of picoseconds or shorter), using precision mechanical translation stage for path length adjustment, as in the conventional pump-probe experiments, is a preferred choice, with the temporal resolution limited by the pulse width. For longer fluorescence lifetime species (in the order of nanoseconds or longer), electronics delay is preferred. The electronics delay can be generated by adjusting the length of the cable that connects the fast photodiode to the trigger of the pulsed diode laser driver, which would allow a delay step increment of approximately 0.12 ns with the temporal resolution limited by the timing jittering in electronics (~100 ps), instead of the pulse width [9

9. E. B. van Munster and T. W. J. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol. 95, 143–175 (2005). [PubMed]

]. Alternatively, high speed timing circuits can also be used for time delay control after adequate calibration. In this work, the cable length adjustment is adopted for its simplicity and stability.

The wavelength of the stimulation beam is chosen to match the red-shift region of the ATTO 647N’s emission spectrum to prevent its re-excitation. The excitation and stimulation beams were coupled collinearly through a dichroic mirror (720SP, Semrock) and directed into a commercially available galvono-mirror scanning unit (FV300, Olympus) for scanning imaging. A pair of low NA objectives (10X, NA 0.3) was used for focusing and collecting the transmitted stimulation signals, respectively. For longer distance imaging, a doublet lens with a focal length of 40 mm is used to focus the laser beam. The transmitted beam that carried the SE signal was detected using a biased Si detector (DET36A, Thorlab) with a large detection area to compensate for the beam movement as a result of galvo-mirror scanning. Additionally, the apertureless detection scheme also prevents the possible artifact attributed to the thermal lensing effect in pump-probe measurements [17

17. S. Lu, W. Min, S. Chong, G. R. Holtom, and X. S. Xie, “Label-free imaging of heme proteins with two-photon excited photothermal lens microscopy,” Appl. Phys. Lett. 96(11), 113701 (2010). [CrossRef]

]. Bandpass filters (747/33, Semrock) and a neutral density filter are used jointly to transmit the stimulation beam only, and to reduce the intensity of the stimulation beam to prevent saturation of the photodiode. To detect the SE signal, lock-in detection was used to recover the SE photons that were carried in the stimulation beam. To do so, the excitation beam was first modulated using an electro-optical modulator (EOM, ConOptics) at 30 KHz, which was driven by a function generator. The SE beam is then demodulated by a lock-in amplifier (SR830, Stanford Research USA) to recover the signal. The output of the lock-in amplifier was connected to the A/D converter of the galvono-mirror based laser scanning system (FV300) to reconstruct the image. For imaging, 256 by 256 pixels are acquired. Frame acquisition time is 10 min, which is currently limited by modulation frequency. To obtain time-resolved images the acquisitions are taken at various time delays with an interval of 0.5 ns. Since the SE signal is proportional to the population of excited molecules, the fluorescence decay curves can be obtained by correlating the image intensity as a function of time delay. The time constants of the lock-in amplifier are set to 1 ms and 300 μs for image acquisition and fluorescence lifetime measurement, respectively.

3. Results and discussion

The SE signal as a function of pulse separation is shown in Fig. 3
Fig. 3 Fluorescence lifetime imaging. SE signal as a function of the relative time delay between the excitation and the stimulation pulses. The decay curve is obtained from a single scan position of the sample. Shown with the decaying curve are the SE images of ATTO 647 N prepared in Y-shape microfluidic channel. The image size is 600 μm × 600 μm.
. The gain of the stimulated beam is consistent with the quenching of spontaneous fluorescence. In the light quenching experiment, the intensity of fluorescence as a function of relative delay is described by Eq. (1),
I0II0=qexp(tdaτ),
(1)
where I0 and I are the fluorescence intensities in the absence and the presence of the stimulation pulses, respectively [23

23. I. Gryczynski, S. W. Hell, and J. R. Lakowicz, “Light quenching of pyridine2 fluorescence with time-delayed pulses,” Biophys. Chem. 66(1), 13–24 (1997). [CrossRef] [PubMed]

]. Therefore, SE signal (ΔIs) as a function of the delay between excitation and stimulation pulses should be descripted by Eq. (2),
ΔIsexp(tdτ),
(2)
with td and τ as the pulse delay and fluorescence lifetime of the fluorophore, respectively. By fitting the curve with the exponential decay function, a fluorescence lifetime of 3.0 ns is obtained. For comparison, the fluorescence lifetime of ATTO 647 N has been reported to be 1.8 ns and 3.4 ns [24

24. J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011). [CrossRef] [PubMed]

, 25

25. K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010). [CrossRef] [PubMed]

]. As discussed previously, the relatively long lifetime (of a few nanosecond) is difficult to measure by conventional pump-probe configuration using mechanical translation stage to adjust the relative pulse delay. The electronic delay used here provides a highly advantageous alternative in interrogating the changing population of excited states. The SE images at various time delays are shown in the Fig. 3. The ATTO 647 N sample was prepared in gel solution and injected into microfluidic channel with Y-shape topography for imaging. As expected, the maximum intensity image is obtained at zero time delay. The image intensity decreased as the time delay increased from 0 ns to 6 ns. Specifically, the low NA objective used here allows long working distance SE imaging.

4. Conclusion

We have shown the feasibility of long working distance fluorescence lifetime imaging by utilizing electronic trigger control with pump-probe configuration. The scheme is simple, highly accurate, and cost-effective, which also prevents optical distortion by removing mechanical movement. Further reduction in size and cost can be easily achieved with all semiconductor light sources, while improvement the robustness simultaneously. By taking advantage of SE’s inherent spatial coherence, the long distance imaging capacity is also demonstrated. This capacity is projected to accommodate a broad range of application environment.

Acknowledgment

The authors would like to thank the National Science Council, Taiwan (NSC99-2627-M-010-002-, NSC98-2627-M-010-006-, NSC97-2112-M-010-002-MY3, and NSC98-2112-M-010-001-MY3), as well as the Ministry of Education, Taiwan under the “Aim for Top University“ project for the generous support of the reported work.

References and links

1.

R. Y. Tsien, “Imagining imaging’s future,” Nat. Rev. Mol. Cell Biol. 4(Suppl), SS16–SS21 (2003). [PubMed]

2.

A. Miyawaki, A. Sawano, and T. Kogure, “Lighting up cells: labeling proteins with fluorophores,” Nat. Cell Biol. 5, S1–S7 (2003).

3.

A. Periasamy and R. M. Clegg, “FLIM applications in the biomedical sciences,” in FLIM Microscopy in Biology and Medicine, eds. A. Periasamy and R. M Clegg (CRC Press 2009), p. 385.

4.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992). [CrossRef] [PubMed]

5.

G. McConnell, “Confocal laser scanning fluorescence microscopy with a visible continuum source,” Opt. Express 12(13), 2844–2850 (2004). [CrossRef] [PubMed]

6.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009). [CrossRef] [PubMed]

7.

T. Dellwig, M. R. Foreman, and F.-J. Kao, “Coherent long-distance signal detection using stimulated emission: a feasibility study,” Chin. J. Physiol. 48, 873–884 (2010).

8.

T. Dellwig, P.-Y. Lin, and F.-J Kao, “Long-distance fluorescence lifetime imaging using stimulated emission,” J. Biomed. Opt. 17, (in press)

9.

E. B. van Munster and T. W. J. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol. 95, 143–175 (2005). [PubMed]

10.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (New.York: Plenum Press, 2006)

11.

H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol. 16(1), 19–27 (2005). [CrossRef] [PubMed]

12.

D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett. 33(20), 2365–2367 (2008). [CrossRef] [PubMed]

13.

C. Hille, M. Lahn, H.-G. Löhmannsröben, and C. Dosche, “Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands,” Photochem. Photobiol. Sci. 8(3), 319–327 (2009). [CrossRef] [PubMed]

14.

M. Y. Berezin, J. Kao, and S. Achilefu, “pH-Dependent Optical Properties of Synthetic Fluorescent Imidazoles,” Chemistry 15(14), 3560–3566 (2009). [CrossRef] [PubMed]

15.

P.-Y. Lin, H.-C. Lyu, C.-Y. S. Hsu, C.-S. Chang, and F.-J. Kao, “Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy,” Biomed. Opt. Express 2(1), 149–158 (2011). [CrossRef] [PubMed]

16.

C.-Y. Dong, P. T. C. So, T. French, and E. Gratton, “Fluorescence lifetime imaging by asynchronous pump-probe microscopy,” Biophys. J. 69(6), 2234–2242 (1995). [CrossRef] [PubMed]

17.

S. Lu, W. Min, S. Chong, G. R. Holtom, and X. S. Xie, “Label-free imaging of heme proteins with two-photon excited photothermal lens microscopy,” Appl. Phys. Lett. 96(11), 113701 (2010). [CrossRef]

18.

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007). [CrossRef]

19.

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005). [CrossRef] [PubMed]

20.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007). [CrossRef] [PubMed]

21.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008). [CrossRef] [PubMed]

22.

J. Lin, F. Lu, W. Zheng, S. Xu, D. Tai, H. Yu, and Z. Huang, “Assessment of liver steatosis and fibrosis in a bile duct ligation rat model using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique,” J. Biomed. Opt. 16, 116024 (2011). [CrossRef] [PubMed]

23.

I. Gryczynski, S. W. Hell, and J. R. Lakowicz, “Light quenching of pyridine2 fluorescence with time-delayed pulses,” Biophys. Chem. 66(1), 13–24 (1997). [CrossRef] [PubMed]

24.

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011). [CrossRef] [PubMed]

25.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010). [CrossRef] [PubMed]

OCIS Codes
(170.0110) Medical optics and biotechnology : Imaging systems
(170.1650) Medical optics and biotechnology : Coherence imaging
(170.6920) Medical optics and biotechnology : Time-resolved imaging

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: February 14, 2012
Revised Manuscript: April 19, 2012
Manuscript Accepted: April 27, 2012
Published: May 4, 2012

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

Citation
Po-Yen Lin, Shin-Shian Lee, Chia-Seng Chang, and Fu-Jen Kao, "Long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay," Opt. Express 20, 11445-11450 (2012)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-20-10-11445


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References

  1. R. Y. Tsien, “Imagining imaging’s future,” Nat. Rev. Mol. Cell Biol.4(Suppl), SS16–SS21 (2003). [PubMed]
  2. A. Miyawaki, A. Sawano, and T. Kogure, “Lighting up cells: labeling proteins with fluorophores,” Nat. Cell Biol.5, S1–S7 (2003).
  3. A. Periasamy and R. M. Clegg, “FLIM applications in the biomedical sciences,” in FLIM Microscopy in Biology and Medicine, eds. A. Periasamy and R. M Clegg (CRC Press 2009), p. 385.
  4. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A.89(4), 1271–1275 (1992). [CrossRef] [PubMed]
  5. G. McConnell, “Confocal laser scanning fluorescence microscopy with a visible continuum source,” Opt. Express12(13), 2844–2850 (2004). [CrossRef] [PubMed]
  6. W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature461(7267), 1105–1109 (2009). [CrossRef] [PubMed]
  7. T. Dellwig, M. R. Foreman, and F.-J. Kao, “Coherent long-distance signal detection using stimulated emission: a feasibility study,” Chin. J. Physiol.48, 873–884 (2010).
  8. T. Dellwig, P.-Y. Lin, and F.-J Kao, “Long-distance fluorescence lifetime imaging using stimulated emission,” J. Biomed. Opt. 17, (in press)
  9. E. B. van Munster and T. W. J. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol.95, 143–175 (2005). [PubMed]
  10. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (New.York: Plenum Press, 2006)
  11. H. Wallrabe and A. Periasamy, “Imaging protein molecules using FRET and FLIM microscopy,” Curr. Opin. Biotechnol.16(1), 19–27 (2005). [CrossRef] [PubMed]
  12. D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett.33(20), 2365–2367 (2008). [CrossRef] [PubMed]
  13. C. Hille, M. Lahn, H.-G. Löhmannsröben, and C. Dosche, “Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands,” Photochem. Photobiol. Sci.8(3), 319–327 (2009). [CrossRef] [PubMed]
  14. M. Y. Berezin, J. Kao, and S. Achilefu, “pH-Dependent Optical Properties of Synthetic Fluorescent Imidazoles,” Chemistry15(14), 3560–3566 (2009). [CrossRef] [PubMed]
  15. P.-Y. Lin, H.-C. Lyu, C.-Y. S. Hsu, C.-S. Chang, and F.-J. Kao, “Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy,” Biomed. Opt. Express2(1), 149–158 (2011). [CrossRef] [PubMed]
  16. C.-Y. Dong, P. T. C. So, T. French, and E. Gratton, “Fluorescence lifetime imaging by asynchronous pump-probe microscopy,” Biophys. J.69(6), 2234–2242 (1995). [CrossRef] [PubMed]
  17. S. Lu, W. Min, S. Chong, G. R. Holtom, and X. S. Xie, “Label-free imaging of heme proteins with two-photon excited photothermal lens microscopy,” Appl. Phys. Lett.96(11), 113701 (2010). [CrossRef]
  18. E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett.442(4-6), 483–487 (2007). [CrossRef]
  19. V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett.94(14), 143903 (2005). [CrossRef] [PubMed]
  20. K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett.99(22), 228105 (2007). [CrossRef] [PubMed]
  21. C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science322(5909), 1857–1861 (2008). [CrossRef] [PubMed]
  22. J. Lin, F. Lu, W. Zheng, S. Xu, D. Tai, H. Yu, and Z. Huang, “Assessment of liver steatosis and fibrosis in a bile duct ligation rat model using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique,” J. Biomed. Opt.16, 116024 (2011). [CrossRef] [PubMed]
  23. I. Gryczynski, S. W. Hell, and J. R. Lakowicz, “Light quenching of pyridine2 fluorescence with time-delayed pulses,” Biophys. Chem.66(1), 13–24 (1997). [CrossRef] [PubMed]
  24. J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express19(4), 3130–3143 (2011). [CrossRef] [PubMed]
  25. K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry16(1), 158–166 (2010). [CrossRef] [PubMed]

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