Abstract

Multi-wavelength microscopic imaging is essential to visualize a variety of nanoscale cellular components with high specificity and high spatial resolution. However, previous techniques are based on fluorescence, and thus cannot visualize nonfluorescent species, which are much less suffered from photodamage or photobleaching and hence are intrinsically useful in wider range of optical microscopy. Here, we show that simultaneous multi-wavelength imaging of nonfluorescent species can be achieved with the use of a photothermal microscope. Dual-wavelength subdiffraction imaging of biological tissues stained with hematoxylin and eosin is demonstrated. Three-dimensional label-free imaging of mouse melanoma tissue section is also presented to demonstrate the effectiveness of the enhanced spatial resolution. Our technique can be implemented using cost-effective and compact laser diodes and is applicable for various types of both fluorescent and nonfluorescent tissues.

© 2015 Optical Society of America

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References

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  1. L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
    [Crossref] [PubMed]
  2. S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B. Lounis, “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment,” Phys. Rev. B 73(4), 045424 (2006).
    [Crossref]
  3. V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
    [Crossref] [PubMed]
  4. R. Radünz, D. Rings, K. Kroy, and F. Cichos, “Hot Brownian particles and photothermal correlation spectroscopy,” J. Phys. Chem. A 113(9), 1674–1677 (2009).
    [Crossref] [PubMed]
  5. P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
    [Crossref]
  6. A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Detection limits in photothermal microscopy,” Chem. Sci. 1(3), 343–350 (2010).
    [Crossref]
  7. A. Gaiduk, M. Yorulmaz, P. V. Ruijgrok, and M. Orrit, “Room-temperature detection of a single molecule’s absorption by photothermal contrast,” Science 330(6002), 353–356 (2010).
    [Crossref] [PubMed]
  8. D. A. Nedosekin, E. V. Shashkov, E. I. Galanzha, L. Hennings, and V. P. Zharov, “Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues,” Cytometry A 77A(11), 1049–1058 (2010).
    [Crossref] [PubMed]
  9. 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]
  10. C. Leduc, J. M. Jung, R. P. Carney, F. Stellacci, and B. Lounis, “Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging,” ACS Nano 5(4), 2587–2592 (2011).
    [Crossref] [PubMed]
  11. W.-S. Chang and S. Link, “Enhancing the sensitivity of single-particle photothermal imaging with thermotropic liquid crystals,” J. Phys. Chem. Lett. 3(10), 1393–1399 (2012).
    [Crossref]
  12. C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
    [PubMed]
  13. M. Selmke and F. Cichos, “Photothermal single particle Rutherford scattering microscopy,” Phys. Rev. Lett. 110(10), 103901 (2013).
    [Crossref] [PubMed]
  14. J. Miyazaki, H. Tsurui, K. Kawasumi, and T. Kobayashi, “Optimal detection angle in sub-diffraction resolution photothermal microscopy: application for high sensitivity imaging of biological tissues,” Opt. Express 22(16), 18833–18842 (2014).
    [Crossref] [PubMed]
  15. M. Selmke, A. Heber, M. Braun, and F. Cichos, “Photothermal single particle microscopy using a single laser beam,” Appl. Phys. Lett. 105(1), 013511 (2014).
    [Crossref]
  16. P. Vermeulen, L. Cognet, and B. Lounis, “Photothermal microscopy: optical detection of small absorbers in scattering environments,” J. Microsc. 254(3), 115–121 (2014).
    [Crossref] [PubMed]
  17. Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
    [Crossref]
  18. J. Miyazaki, H. Tsurui, A. Hayashi-Takagi, H. Kasai, and T. Kobayashi, “Sub-diffraction resolution pump-probe microscopy with shot-noise limited sensitivity using laser diodes,” Opt. Express 22(8), 9024–9032 (2014).
    [Crossref] [PubMed]
  19. J. Miyazaki, K. Kawasumi, and T. Kobayashi, “Frequency domain approach for time-resolved pump-probe microscopy using intensity modulated laser diodes,” Rev. Sci. Instrum. 85(9), 093703 (2014).
    [Crossref] [PubMed]
  20. J. Miyazaki, K. Kawasumi, and T. Kobayashi, “Resolution improvement in laser diode-based pump-probe microscopy with an annular pupil filter,” Opt. Lett. 39(14), 4219–4222 (2014).
    [Crossref] [PubMed]
  21. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
    [Crossref] [PubMed]
  22. 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]
  23. A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Making gold nanoparticles fluorescent for simultaneous absorption and fluorescence detection on the single particle level,” Phys. Chem. Chem. Phys. 13(1), 149–153 (2010).
    [Crossref] [PubMed]

2014 (6)

J. Miyazaki, H. Tsurui, K. Kawasumi, and T. Kobayashi, “Optimal detection angle in sub-diffraction resolution photothermal microscopy: application for high sensitivity imaging of biological tissues,” Opt. Express 22(16), 18833–18842 (2014).
[Crossref] [PubMed]

M. Selmke, A. Heber, M. Braun, and F. Cichos, “Photothermal single particle microscopy using a single laser beam,” Appl. Phys. Lett. 105(1), 013511 (2014).
[Crossref]

P. Vermeulen, L. Cognet, and B. Lounis, “Photothermal microscopy: optical detection of small absorbers in scattering environments,” J. Microsc. 254(3), 115–121 (2014).
[Crossref] [PubMed]

J. Miyazaki, H. Tsurui, A. Hayashi-Takagi, H. Kasai, and T. Kobayashi, “Sub-diffraction resolution pump-probe microscopy with shot-noise limited sensitivity using laser diodes,” Opt. Express 22(8), 9024–9032 (2014).
[Crossref] [PubMed]

J. Miyazaki, K. Kawasumi, and T. Kobayashi, “Frequency domain approach for time-resolved pump-probe microscopy using intensity modulated laser diodes,” Rev. Sci. Instrum. 85(9), 093703 (2014).
[Crossref] [PubMed]

J. Miyazaki, K. Kawasumi, and T. Kobayashi, “Resolution improvement in laser diode-based pump-probe microscopy with an annular pupil filter,” Opt. Lett. 39(14), 4219–4222 (2014).
[Crossref] [PubMed]

2013 (2)

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

M. Selmke and F. Cichos, “Photothermal single particle Rutherford scattering microscopy,” Phys. Rev. Lett. 110(10), 103901 (2013).
[Crossref] [PubMed]

2012 (2)

W.-S. Chang and S. Link, “Enhancing the sensitivity of single-particle photothermal imaging with thermotropic liquid crystals,” J. Phys. Chem. Lett. 3(10), 1393–1399 (2012).
[Crossref]

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

2011 (1)

C. Leduc, J. M. Jung, R. P. Carney, F. Stellacci, and B. Lounis, “Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging,” ACS Nano 5(4), 2587–2592 (2011).
[Crossref] [PubMed]

2010 (5)

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Detection limits in photothermal microscopy,” Chem. Sci. 1(3), 343–350 (2010).
[Crossref]

A. Gaiduk, M. Yorulmaz, P. V. Ruijgrok, and M. Orrit, “Room-temperature detection of a single molecule’s absorption by photothermal contrast,” Science 330(6002), 353–356 (2010).
[Crossref] [PubMed]

D. A. Nedosekin, E. V. Shashkov, E. I. Galanzha, L. Hennings, and V. P. Zharov, “Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues,” Cytometry A 77A(11), 1049–1058 (2010).
[Crossref] [PubMed]

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]

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Making gold nanoparticles fluorescent for simultaneous absorption and fluorescence detection on the single particle level,” Phys. Chem. Chem. Phys. 13(1), 149–153 (2010).
[Crossref] [PubMed]

2009 (3)

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

R. Radünz, D. Rings, K. Kroy, and F. Cichos, “Hot Brownian particles and photothermal correlation spectroscopy,” J. Phys. Chem. A 113(9), 1674–1677 (2009).
[Crossref] [PubMed]

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

2006 (1)

S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B. Lounis, “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment,” Phys. Rev. B 73(4), 045424 (2006).
[Crossref]

2003 (1)

L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
[Crossref] [PubMed]

1992 (1)

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]

1990 (1)

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

Beidoun, A.

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]

Berciaud, S.

S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B. Lounis, “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment,” Phys. Rev. B 73(4), 045424 (2006).
[Crossref]

Blab, G. A.

S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B. Lounis, “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment,” Phys. Rev. B 73(4), 045424 (2006).
[Crossref]

Boyer, D.

L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
[Crossref] [PubMed]

Braun, M.

M. Selmke, A. Heber, M. Braun, and F. Cichos, “Photothermal single particle microscopy using a single laser beam,” Appl. Phys. Lett. 105(1), 013511 (2014).
[Crossref]

Carney, R. P.

C. Leduc, J. M. Jung, R. P. Carney, F. Stellacci, and B. Lounis, “Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging,” ACS Nano 5(4), 2587–2592 (2011).
[Crossref] [PubMed]

Chang, W.-S.

W.-S. Chang and S. Link, “Enhancing the sensitivity of single-particle photothermal imaging with thermotropic liquid crystals,” J. Phys. Chem. Lett. 3(10), 1393–1399 (2012).
[Crossref]

Chong, S.

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]

Choquet, D.

L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
[Crossref] [PubMed]

Cichos, F.

M. Selmke, A. Heber, M. Braun, and F. Cichos, “Photothermal single particle microscopy using a single laser beam,” Appl. Phys. Lett. 105(1), 013511 (2014).
[Crossref]

M. Selmke and F. Cichos, “Photothermal single particle Rutherford scattering microscopy,” Phys. Rev. Lett. 110(10), 103901 (2013).
[Crossref] [PubMed]

R. Radünz, D. Rings, K. Kroy, and F. Cichos, “Hot Brownian particles and photothermal correlation spectroscopy,” J. Phys. Chem. A 113(9), 1674–1677 (2009).
[Crossref] [PubMed]

Cognet, L.

P. Vermeulen, L. Cognet, and B. Lounis, “Photothermal microscopy: optical detection of small absorbers in scattering environments,” J. Microsc. 254(3), 115–121 (2014).
[Crossref] [PubMed]

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B. Lounis, “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment,” Phys. Rev. B 73(4), 045424 (2006).
[Crossref]

L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
[Crossref] [PubMed]

Daiber, M.

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]

Denk, W.

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

Duchesne, L.

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

Fernig, D. G.

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

Fukui, K.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Gaiduk, A.

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Detection limits in photothermal microscopy,” Chem. Sci. 1(3), 343–350 (2010).
[Crossref]

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Making gold nanoparticles fluorescent for simultaneous absorption and fluorescence detection on the single particle level,” Phys. Chem. Chem. Phys. 13(1), 149–153 (2010).
[Crossref] [PubMed]

A. Gaiduk, M. Yorulmaz, P. V. Ruijgrok, and M. Orrit, “Room-temperature detection of a single molecule’s absorption by photothermal contrast,” Science 330(6002), 353–356 (2010).
[Crossref] [PubMed]

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

Galanzha, E. I.

D. A. Nedosekin, E. V. Shashkov, E. I. Galanzha, L. Hennings, and V. P. Zharov, “Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues,” Cytometry A 77A(11), 1049–1058 (2010).
[Crossref] [PubMed]

Gautier, J.

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

Gautreau, A.

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

Giannone, G.

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

Hashimoto, H.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Hayashi-Takagi, A.

Heber, A.

M. Selmke, A. Heber, M. Braun, and F. Cichos, “Photothermal single particle microscopy using a single laser beam,” Appl. Phys. Lett. 105(1), 013511 (2014).
[Crossref]

Hennings, L.

D. A. Nedosekin, E. V. Shashkov, E. I. Galanzha, L. Hennings, and V. P. Zharov, “Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues,” Cytometry A 77A(11), 1049–1058 (2010).
[Crossref] [PubMed]

Holtom, G. R.

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]

Itoh, K.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Jung, J. M.

C. Leduc, J. M. Jung, R. P. Carney, F. Stellacci, and B. Lounis, “Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging,” ACS Nano 5(4), 2587–2592 (2011).
[Crossref] [PubMed]

Kasai, H.

Kawasumi, K.

Kobayashi, T.

Krens, S. F. G.

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

Kroy, K.

R. Radünz, D. Rings, K. Kroy, and F. Cichos, “Hot Brownian particles and photothermal correlation spectroscopy,” J. Phys. Chem. A 113(9), 1674–1677 (2009).
[Crossref] [PubMed]

Kulzer, F.

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

Lasne, D.

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B. Lounis, “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment,” Phys. Rev. B 73(4), 045424 (2006).
[Crossref]

Leduc, C.

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

C. Leduc, J. M. Jung, R. P. Carney, F. Stellacci, and B. Lounis, “Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging,” ACS Nano 5(4), 2587–2592 (2011).
[Crossref] [PubMed]

Link, S.

W.-S. Chang and S. Link, “Enhancing the sensitivity of single-particle photothermal imaging with thermotropic liquid crystals,” J. Phys. Chem. Lett. 3(10), 1393–1399 (2012).
[Crossref]

Lounis, B.

P. Vermeulen, L. Cognet, and B. Lounis, “Photothermal microscopy: optical detection of small absorbers in scattering environments,” J. Microsc. 254(3), 115–121 (2014).
[Crossref] [PubMed]

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

C. Leduc, J. M. Jung, R. P. Carney, F. Stellacci, and B. Lounis, “Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging,” ACS Nano 5(4), 2587–2592 (2011).
[Crossref] [PubMed]

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B. Lounis, “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment,” Phys. Rev. B 73(4), 045424 (2006).
[Crossref]

L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
[Crossref] [PubMed]

Lu, S.

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]

Min, W.

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]

Miyazaki, J.

Nedosekin, D. A.

D. A. Nedosekin, E. V. Shashkov, E. I. Galanzha, L. Hennings, and V. P. Zharov, “Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues,” Cytometry A 77A(11), 1049–1058 (2010).
[Crossref] [PubMed]

Nishizawa, N.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Octeau, V.

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

Orrit, M.

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Making gold nanoparticles fluorescent for simultaneous absorption and fluorescence detection on the single particle level,” Phys. Chem. Chem. Phys. 13(1), 149–153 (2010).
[Crossref] [PubMed]

A. Gaiduk, M. Yorulmaz, P. V. Ruijgrok, and M. Orrit, “Room-temperature detection of a single molecule’s absorption by photothermal contrast,” Science 330(6002), 353–356 (2010).
[Crossref] [PubMed]

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Detection limits in photothermal microscopy,” Chem. Sci. 1(3), 343–350 (2010).
[Crossref]

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

Otsuka, Y.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Ozeki, Y.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Paulo, P. M. R.

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

Penzkofer, A.

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]

Radünz, R.

R. Radünz, D. Rings, K. Kroy, and F. Cichos, “Hot Brownian particles and photothermal correlation spectroscopy,” J. Phys. Chem. A 113(9), 1674–1677 (2009).
[Crossref] [PubMed]

Rings, D.

R. Radünz, D. Rings, K. Kroy, and F. Cichos, “Hot Brownian particles and photothermal correlation spectroscopy,” J. Phys. Chem. A 113(9), 1674–1677 (2009).
[Crossref] [PubMed]

Ruijgrok, P. V.

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Detection limits in photothermal microscopy,” Chem. Sci. 1(3), 343–350 (2010).
[Crossref]

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Making gold nanoparticles fluorescent for simultaneous absorption and fluorescence detection on the single particle level,” Phys. Chem. Chem. Phys. 13(1), 149–153 (2010).
[Crossref] [PubMed]

A. Gaiduk, M. Yorulmaz, P. V. Ruijgrok, and M. Orrit, “Room-temperature detection of a single molecule’s absorption by photothermal contrast,” Science 330(6002), 353–356 (2010).
[Crossref] [PubMed]

Satoh, S.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Schaeffer, N.

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

Schmidt, T.

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

Selmke, M.

M. Selmke, A. Heber, M. Braun, and F. Cichos, “Photothermal single particle microscopy using a single laser beam,” Appl. Phys. Lett. 105(1), 013511 (2014).
[Crossref]

M. Selmke and F. Cichos, “Photothermal single particle Rutherford scattering microscopy,” Phys. Rev. Lett. 110(10), 103901 (2013).
[Crossref] [PubMed]

Shashkov, E. V.

D. A. Nedosekin, E. V. Shashkov, E. I. Galanzha, L. Hennings, and V. P. Zharov, “Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues,” Cytometry A 77A(11), 1049–1058 (2010).
[Crossref] [PubMed]

Si, S.

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

Soto-Ribeiro, M.

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

Spaink, H. P.

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

Stellacci, F.

C. Leduc, J. M. Jung, R. P. Carney, F. Stellacci, and B. Lounis, “Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging,” ACS Nano 5(4), 2587–2592 (2011).
[Crossref] [PubMed]

Strickler, J. H.

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

Sumimura, K.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Tamarat, P.

L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
[Crossref] [PubMed]

Tardin, C.

L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
[Crossref] [PubMed]

Tsurui, H.

Umemura, W.

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Vermeulen, P.

P. Vermeulen, L. Cognet, and B. Lounis, “Photothermal microscopy: optical detection of small absorbers in scattering environments,” J. Microsc. 254(3), 115–121 (2014).
[Crossref] [PubMed]

Webb, W. W.

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

Wehrle-Haller, B.

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

Xie, X. S.

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]

Yorulmaz, M.

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Detection limits in photothermal microscopy,” Chem. Sci. 1(3), 343–350 (2010).
[Crossref]

A. Gaiduk, M. Yorulmaz, P. V. Ruijgrok, and M. Orrit, “Room-temperature detection of a single molecule’s absorption by photothermal contrast,” Science 330(6002), 353–356 (2010).
[Crossref] [PubMed]

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Making gold nanoparticles fluorescent for simultaneous absorption and fluorescence detection on the single particle level,” Phys. Chem. Chem. Phys. 13(1), 149–153 (2010).
[Crossref] [PubMed]

Zharov, V. P.

D. A. Nedosekin, E. V. Shashkov, E. I. Galanzha, L. Hennings, and V. P. Zharov, “Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues,” Cytometry A 77A(11), 1049–1058 (2010).
[Crossref] [PubMed]

ACS Nano (2)

V. Octeau, L. Cognet, L. Duchesne, D. Lasne, N. Schaeffer, D. G. Fernig, and B. Lounis, “Photothermal absorption correlation spectroscopy,” ACS Nano 3(2), 345–350 (2009).
[Crossref] [PubMed]

C. Leduc, J. M. Jung, R. P. Carney, F. Stellacci, and B. Lounis, “Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging,” ACS Nano 5(4), 2587–2592 (2011).
[Crossref] [PubMed]

Appl. Phys. Lett. (2)

M. Selmke, A. Heber, M. Braun, and F. Cichos, “Photothermal single particle microscopy using a single laser beam,” Appl. Phys. Lett. 105(1), 013511 (2014).
[Crossref]

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]

Chem. Sci. (1)

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Detection limits in photothermal microscopy,” Chem. Sci. 1(3), 343–350 (2010).
[Crossref]

Cytometry A (1)

D. A. Nedosekin, E. V. Shashkov, E. I. Galanzha, L. Hennings, and V. P. Zharov, “Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues,” Cytometry A 77A(11), 1049–1058 (2010).
[Crossref] [PubMed]

J. Lumin. (1)

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]

J. Microsc. (1)

P. Vermeulen, L. Cognet, and B. Lounis, “Photothermal microscopy: optical detection of small absorbers in scattering environments,” J. Microsc. 254(3), 115–121 (2014).
[Crossref] [PubMed]

J. Phys. Chem. A (1)

R. Radünz, D. Rings, K. Kroy, and F. Cichos, “Hot Brownian particles and photothermal correlation spectroscopy,” J. Phys. Chem. A 113(9), 1674–1677 (2009).
[Crossref] [PubMed]

J. Phys. Chem. C (1)

P. M. R. Paulo, A. Gaiduk, F. Kulzer, S. F. G. Krens, H. P. Spaink, T. Schmidt, and M. Orrit, “Photothermal Correlation Spectroscopy of Gold Nanoparticles in Solution,” J. Phys. Chem. C 113(27), 11451–11457 (2009).
[Crossref]

J. Phys. Chem. Lett. (1)

W.-S. Chang and S. Link, “Enhancing the sensitivity of single-particle photothermal imaging with thermotropic liquid crystals,” J. Phys. Chem. Lett. 3(10), 1393–1399 (2012).
[Crossref]

Nano Lett. (1)

C. Leduc, S. Si, J. Gautier, M. Soto-Ribeiro, B. Wehrle-Haller, A. Gautreau, G. Giannone, L. Cognet, and B. Lounis, “A highly specific gold nanoprobe for live-cell single-molecule imaging,” Nano Lett. 13(4), 1489–1494 (2013).
[PubMed]

Nat. Photonics (1)

Y. Ozeki, W. Umemura, Y. Otsuka, S. Satoh, H. Hashimoto, K. Sumimura, N. Nishizawa, K. Fukui, and K. Itoh, “High-speed molecular spectral imaging of tissue with stimulated Raman scattering,” Nat. Photonics 6(12), 845–850 (2012).
[Crossref]

Opt. Express (2)

Opt. Lett. (1)

Phys. Chem. Chem. Phys. (1)

A. Gaiduk, P. V. Ruijgrok, M. Yorulmaz, and M. Orrit, “Making gold nanoparticles fluorescent for simultaneous absorption and fluorescence detection on the single particle level,” Phys. Chem. Chem. Phys. 13(1), 149–153 (2010).
[Crossref] [PubMed]

Phys. Rev. B (1)

S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B. Lounis, “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment,” Phys. Rev. B 73(4), 045424 (2006).
[Crossref]

Phys. Rev. Lett. (1)

M. Selmke and F. Cichos, “Photothermal single particle Rutherford scattering microscopy,” Phys. Rev. Lett. 110(10), 103901 (2013).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (1)

L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, and B. Lounis, “Single metallic nanoparticle imaging for protein detection in cells,” Proc. Natl. Acad. Sci. U.S.A. 100(20), 11350–11355 (2003).
[Crossref] [PubMed]

Rev. Sci. Instrum. (1)

J. Miyazaki, K. Kawasumi, and T. Kobayashi, “Frequency domain approach for time-resolved pump-probe microscopy using intensity modulated laser diodes,” Rev. Sci. Instrum. 85(9), 093703 (2014).
[Crossref] [PubMed]

Science (2)

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

A. Gaiduk, M. Yorulmaz, P. V. Ruijgrok, and M. Orrit, “Room-temperature detection of a single molecule’s absorption by photothermal contrast,” Science 330(6002), 353–356 (2010).
[Crossref] [PubMed]

Supplementary Material (1)

» Media 1: AVI (4691 KB)     

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Figures (5)

Fig. 1
Fig. 1 Experimental setup and principle of dual-wavelength photothermal microscopy using intensity-modulated lasers. (a), Schematic of the experimental setup. DM: dichroic mirror, BS: beam splitter, PBS: polarization beam splitter, OBL: objective lens. CL: condenser lens, F: band-pass filter, ID: iris diaphragm, PMT: photomultiplier tube, MMF: multimode fiber, ABD: auto-balanced detector, LPF: low-pass filter. (b), Principle of dual-wavelength photothermal microscopy using intensity-modulated continuous-wave lasers. The pump-beam intensities are modulated at ω1 and ω2, and the probe beam intensity is modulated at ω3. Beat signals are generated by the photothermal effect at the sample position, and they are detected using two lock-in amplifiers referenced at the beat frequencies.
Fig. 2
Fig. 2 Photothermal images of signal gold nanoparticle and label-free 3D imaging of mouse melanoma tissue section. (a, b), Combined images of a 20-nm gold nanoparticle embedded in polyvinyl alcohol with excitation at 488 nm (blue) and 532 nm (green). The wavelengths of the two pump beams overlapped the absorption spectrum of the gold nanoparticles due to plasmon resonance with a peak at 524 nm. The 488-nm and 532-nm pump beams were modulated at 1.07 and 1.1 MHz, respectively, and the probe beam was modulated at 1.0 MHz. The time constant of the lock-in amplifier was 1 ms, and the pixel dwell time was 2 ms. Scale bars, 500 nm. (c, d), Intensity profiles of the single nanoparticle with excitation at 488 nm (filled circles) and 532 nm (open squares) in the horizontal and vertical directions, respectively. For the 488-nm pump beam, fitting of a single particle with the Gaussian (solid curves) gives the full-width at half-maximum (FWHM) values of 234 nm and 249 nm in the horizontal and vertical directions, respectively. For the 532-nm pump beam, the FWHM values are 256 nm and 257 nm in the horizontal and vertical directions, respectively. (e), Intensity profiles along the axial direction. FWHM values are 525 nm and 510 nm for the 488-nm and 532-nm pump beams, respectively. (f, g), Photothermal images of a slice of mouse melanoma in the lateral and axial planes, respectively. Time constant of the lock-in amplifier was 0.5 ms, and pixel dwell time was 1 ms. The pump and probe beam powers incident on the sample were 50 and 150 μW, respectively. A set of 30 images of a slice of mouse melanoma is subsequently acquired by changing the sample position in the axial direction with a step size of 0.28 μm (see Media 1). Each image size is 19.6 x 19.6 μm at 200 x 200 pixels. (h) Bright field image of slice of mouse melanoma in the same area as in (f). (i,j) Photothermal images in the simultaneous measurement with excitation at (i) 488 nm and (j) 532 nm . Scale bars, 2 μm in (f-j) and 0.5 μm in the inset of (f, h).
Fig. 3
Fig. 3 Dual-wavelength photothermal images of hematoxylin and eosin (H&E) stained biological tissues (a), Absorption (red solid curve) and fluorescence (red dot-dashed curve) spectra of eosin and absorption spectrum of hematoxylin (black solid curve). Laser wavelengths of the two pump beams are also shown. (b, c), Photothermal images of H&E-stained rabbit ovary slice with excitation at 488 nm and 532 nm, respectively. Probe beam power was 300 μW. Image size was 19.6 x 19.6 mm with 300 x 300 pixels. The time constant of the lock-in amplifier was 0.5 ms, and the pixel dwell time was 1 ms. Image acquisition time was ~120 s. (b) Bright field image of the tissue in the same area as that in (c,d). The inset is the entire picture of the oocyte. (e, f), Unmixed images reflecting the distribution of eosin (red) and hematoxylin (blue), respectively. (g), Dual-wavelength image produced by combining images e and f. (h, i), Cross-sectional images of a slice of rabbit ovary with excitation at 488 nm and 532 nm, respectively. Image size was 19.6 x 9.8 mm with 200 x 100 pixels, Image acquisition time was ~30 s. (j), Unmixed image reflecting the distribution of eosin and hematoxylin. (k), Combined image of photothermal (blue) and fluorescence (red) signals. Scale bars, 2 μm.
Fig. 4
Fig. 4 Relation between the signal intensity and collection angle of the probe beam. (a), Images of a slice of rabbit kidney stained with hematoxylin and eosin with various values of the numerical aperture of the condenser lens system (NAc). Scale bar, 5 μm. (b), Integrated intensity as a function of the NAc. The theoretical curve (solid curve) is calculated using Eq. (15) in Ref [14]. with rc = 0.2 μm.
Fig. 5
Fig. 5 Modulation frequency dependence in photothermal microscopy. (a), Intensity (upper) and phase (lower) images of a slice of rabbit kidney stained with hematoxylin and eosin with excitation at 488 nm. Modulation frequencies of the pump beam are 0.5, 1.13, and 2.53 MHz (from left to right), respectively. Scale bars, 5 μm. (b), Integrated intensity and averaged phase as a function of modulation frequency of the pump beam.

Equations (3)

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γ h/e = S h ( x,y)/ S e (x,y)dxdy,
S i j 2 I i σ j ( λ i ) c j ,
Δn~ NσP sfCρ r c 3 n T ,

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