Abstract

We present a novel scheme to simultaneously detect coherent anti-Stokes Raman scattering (CARS) microscopy signals in the forward (F) and backward (epi - E) direction with a single avalanche photodiode (APD) detector using time-correlated single photon counting (TCSPC). By installing a mirror at a well-defined distance above the sample the forward-scattered F-CARS signal is reflected back into the microscope objective leading to spatial overlap of the F and E-CARS signals. Due to traveling an additional distance the F-CARS signal is time delayed relative to the E-CARS signal. TCSPC then allows for the two signals to be resolved in the time domain. This results in an efficient, simple, and compact method of CARS signal detection. We demonstrate this technique by analyzing forward and backward CARS signals obtained by imaging living adipocyte cells derived from human mesenchymal stem cells.

© 2008 Optical Society of America

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  1. M. D. Duncan, J. Reintjes, and T. J. Manuccia, "Scanning Coherent Anti-Stokes Raman Microscope," Opt. Lett. 7, 350-352 (1982).
    [CrossRef] [PubMed]
  2. A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-dimensional vibrational imaging by Coherent Anti- Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
    [CrossRef]
  3. J. X. Cheng, Y. K. Jia, G. F. Zheng, and X. S. Xie, "Laser-scanning coherent anti-stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
    [CrossRef] [PubMed]
  4. M. Müller and A. Zumbusch, "Coherent Anti-Stokes Raman Scattering Microscopy," ChemPhysChem 8, 2156-2170 (2007).
    [CrossRef] [PubMed]
  5. C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate Coherent Anti-Stokes Raman Scattering Microscopy," Proc. Natl. Acad. Sci. 102, 16807- 16812 (2005).
    [CrossRef] [PubMed]
  6. T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in caenorhabditis elegans using Coherent Anti-Stokes Raman Scattering (CARS) Microscopy," Proc Natl Acad Sci USA 104, 14658-14663 (2007).
    [CrossRef] [PubMed]
  7. J. X. Cheng, A. Volkmer, and X. S. Xie, "Theoretical and experimental characterization of Coherent Anti- Stokes Raman Scattering Microscopy," J. Opt. Soc. Am. B 19, 1363-1375 (2002).
    [CrossRef]
  8. J. X. Cheng and X. S. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications," J. Phys. Chem. B 108, 827-840 (2004).
    [CrossRef]
  9. A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent Anti-Stokes Raman Scattering Microscopy," Phys. Rev. Lett. 8702, 4 (2001).
  10. S. O. Konorov, C. H. Glover, J. M. Piret, J. Bryan, H. G. Schulze, M. W. Blades, and R. F. B. Turner, "In situ analysis of living embryonic stem cells by Coherent Anti-Stokes Raman Microscopy," Anal. Chem. 79, 7221-7225 (2007).
    [CrossRef] [PubMed]
  11. B. Kraemer, F. Koberling, U. Ortmann, M. Wahl, P. Kapusta, A. Buelter, and R. Erdmann, "Time-resolved laser scanning microscopy with FLIM and advanced FCS capability," Proc. SPIE 5700, 138-143 (2005).
    [CrossRef]
  12. D. Elson, J. Siegel, S. Webbb, S. Leveque-Fort, M. Lever, P. French, K. Lauritsen, M. Wahl, and R. Erdmann, "Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser," Opt. Lett. 27, 1409-1411 (2002).
    [CrossRef]
  13. M. Wahl, H. Rahn, I. Gregor, R. Erdmann, and J. Enderlein, "Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels " Rev. Sci. Instrum. 78, 033106 (2007).
    [CrossRef] [PubMed]
  14. S. Ly, G. McNerney, S. Fore, J. Chan, and T. Huser, "Time-gated single photon counting enables separation of CARS microscopy data from multiphoton-excited tissue autofluorescence," Opt. Express 15, 16839- 16851 (2007).
    [CrossRef] [PubMed]
  15. E. O. Potma, C. L. Evans, and X. S. Xie, "Heterodyne coherent anti-Stokes Raman scattering (CARS) imaging," Opt. Lett. 31, 241-243 (2006).
    [CrossRef] [PubMed]

2007

M. Müller and A. Zumbusch, "Coherent Anti-Stokes Raman Scattering Microscopy," ChemPhysChem 8, 2156-2170 (2007).
[CrossRef] [PubMed]

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in caenorhabditis elegans using Coherent Anti-Stokes Raman Scattering (CARS) Microscopy," Proc Natl Acad Sci USA 104, 14658-14663 (2007).
[CrossRef] [PubMed]

S. O. Konorov, C. H. Glover, J. M. Piret, J. Bryan, H. G. Schulze, M. W. Blades, and R. F. B. Turner, "In situ analysis of living embryonic stem cells by Coherent Anti-Stokes Raman Microscopy," Anal. Chem. 79, 7221-7225 (2007).
[CrossRef] [PubMed]

M. Wahl, H. Rahn, I. Gregor, R. Erdmann, and J. Enderlein, "Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels " Rev. Sci. Instrum. 78, 033106 (2007).
[CrossRef] [PubMed]

S. Ly, G. McNerney, S. Fore, J. Chan, and T. Huser, "Time-gated single photon counting enables separation of CARS microscopy data from multiphoton-excited tissue autofluorescence," Opt. Express 15, 16839- 16851 (2007).
[CrossRef] [PubMed]

2006

2005

B. Kraemer, F. Koberling, U. Ortmann, M. Wahl, P. Kapusta, A. Buelter, and R. Erdmann, "Time-resolved laser scanning microscopy with FLIM and advanced FCS capability," Proc. SPIE 5700, 138-143 (2005).
[CrossRef]

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate Coherent Anti-Stokes Raman Scattering Microscopy," Proc. Natl. Acad. Sci. 102, 16807- 16812 (2005).
[CrossRef] [PubMed]

2004

J. X. Cheng and X. S. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

2002

J. X. Cheng, A. Volkmer, and X. S. Xie, "Theoretical and experimental characterization of Coherent Anti- Stokes Raman Scattering Microscopy," J. Opt. Soc. Am. B 19, 1363-1375 (2002).
[CrossRef]

D. Elson, J. Siegel, S. Webbb, S. Leveque-Fort, M. Lever, P. French, K. Lauritsen, M. Wahl, and R. Erdmann, "Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser," Opt. Lett. 27, 1409-1411 (2002).
[CrossRef]

J. X. Cheng, Y. K. Jia, G. F. Zheng, and X. S. Xie, "Laser-scanning coherent anti-stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
[CrossRef] [PubMed]

2001

A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent Anti-Stokes Raman Scattering Microscopy," Phys. Rev. Lett. 8702, 4 (2001).

1999

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-dimensional vibrational imaging by Coherent Anti- Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

1982

Anal. Chem.

S. O. Konorov, C. H. Glover, J. M. Piret, J. Bryan, H. G. Schulze, M. W. Blades, and R. F. B. Turner, "In situ analysis of living embryonic stem cells by Coherent Anti-Stokes Raman Microscopy," Anal. Chem. 79, 7221-7225 (2007).
[CrossRef] [PubMed]

Biophys. J.

J. X. Cheng, Y. K. Jia, G. F. Zheng, and X. S. Xie, "Laser-scanning coherent anti-stokes Raman scattering microscopy and applications to cell biology," Biophys. J. 83, 502-509 (2002).
[CrossRef] [PubMed]

ChemPhysChem

M. Müller and A. Zumbusch, "Coherent Anti-Stokes Raman Scattering Microscopy," ChemPhysChem 8, 2156-2170 (2007).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B

J. Phys. Chem. B

J. X. Cheng and X. S. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

Opt. Express

Opt. Lett.

E. O. Potma, C. L. Evans, and X. S. Xie, "Heterodyne coherent anti-Stokes Raman scattering (CARS) imaging," Opt. Lett. 31, 241-243 (2006).
[CrossRef] [PubMed]

D. Elson, J. Siegel, S. Webbb, S. Leveque-Fort, M. Lever, P. French, K. Lauritsen, M. Wahl, and R. Erdmann, "Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser," Opt. Lett. 27, 1409-1411 (2002).
[CrossRef]

M. D. Duncan, J. Reintjes, and T. J. Manuccia, "Scanning Coherent Anti-Stokes Raman Microscope," Opt. Lett. 7, 350-352 (1982).
[CrossRef] [PubMed]

Phys. Rev. Lett.

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-dimensional vibrational imaging by Coherent Anti- Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

A. Volkmer, J. X. Cheng, and X. S. Xie, "Vibrational imaging with high sensitivity via epidetected coherent Anti-Stokes Raman Scattering Microscopy," Phys. Rev. Lett. 8702, 4 (2001).

Proc Natl Acad Sci USA

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in caenorhabditis elegans using Coherent Anti-Stokes Raman Scattering (CARS) Microscopy," Proc Natl Acad Sci USA 104, 14658-14663 (2007).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate Coherent Anti-Stokes Raman Scattering Microscopy," Proc. Natl. Acad. Sci. 102, 16807- 16812 (2005).
[CrossRef] [PubMed]

Proc. SPIE

B. Kraemer, F. Koberling, U. Ortmann, M. Wahl, P. Kapusta, A. Buelter, and R. Erdmann, "Time-resolved laser scanning microscopy with FLIM and advanced FCS capability," Proc. SPIE 5700, 138-143 (2005).
[CrossRef]

Rev. Sci. Instrum.

M. Wahl, H. Rahn, I. Gregor, R. Erdmann, and J. Enderlein, "Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels " Rev. Sci. Instrum. 78, 033106 (2007).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1.
Fig. 1.

(a). Schematics of our CARS microscopy system. An optical parametric oscillator (OPO) is pumped by a portion of a Nd;YVO4 laser (split by a beam-splitter cube (BS)) with 7 ps pulse length resulting in a synchronized, tunable probe beam. The pump and probe beams are temporally overlapped by a delay line (DL), then recombined on a dichroic mirror (DM1), passed through an electro-optical pulse picker (PP), and focused into the microscope. An Olympus 40x air objective lens (OL) focuses both beams onto the sample, a 0.55 NA condenser lens (CL) collects the forward-scattered light and focuses it onto a flat mirror (M). The reflected light is refocused onto the sample by the condenser lens. A shortpass dichroic mirror (DM2) passes the CARS signal and rejects the pump and Stokes beams. A shortpass filter (SP) further removes residual laser photons. E-CARS and F-CARS signals are detected either by a CCD camera or an avalanche photodiode detector (APD) by use of a flip mirror (FM). b) Time distribution histogram of the photon arrival time of the spatially overlapped E-CARS and F-CARS signals. Note that the temporally delayed F-CARS signal is significantly more intense than the E-CARS signal.

Fig. 2.
Fig. 2.

CARS microscopy of living, human MSC-derived adipocytes in culture. a) CARS image encoding the photon arrival time of the individual photons by false color. b) Normalized photon arrival time histograms obtained by histogramming all photons of the regions of interest shown in a). The area surrounding region 1 is comprised of mostly back-scattered E-CARS photons, while region 2 contains mostly signals from back-reflected F-CARS photons (resulting in a fixed delay in the photon arrival time delay of ~2 ns. Region 3 was taken from an area outside the cells and characterizes the nonresonant background signal. It was offset downwards for clarity. Note that the slope of all decay curves is approximately the same, which indicates that the arrival time signals are limited by the instrument response function. The image consists of 512×512 pixels with a pixel dwell time of 0.2ms, and was acquired in 4.3 min. The laser power of the pump and Stokes beams at the sample was 20 mW and 15 mW, respectively.

Fig. 3.
Fig. 3.

Time-gated CARS images of MSC-derived adipocytes. a) Time-gated intensity image of the F-CARS signal, where only photons arriving within the time gates indicated by the pink box in Fig. 1(b) were used to construct the image. b) Time-gated E-CARS image of the adipocyte sample with gates represented by the blue box shown in Fig. 1(b). c) Overlay of the E-CARS and F-CARS image where contrast for the E-CARS image was scaled as indicated in Fig. 3(b). d) Line section of the CARS signals as indicated by the white line in Fig. 3(c). All images were acquired with 512×512 pixels, a pixel dwell time of 0.2ms, and a total image acquisition time of 4.3 min. The laser power of the pump and Stokes beams at the sample was 20 mW and 15 mW, respectively.

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