Abstract

We demonstrate a novel multifocal, multiphoton microscope that is capable of simultaneous dynamic imaging of multiple focal planes. We show for the first time that multimodal, multiphoton images excited with orthogonal polarizations can be acquired simultaneously in both the transmission and epi directions.

© 2008 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, "Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions," IEEE Trans. Nanobiosci. 3, 237-242 (2004).
    [CrossRef]
  2. B. Rózsa, G. Katona, E. S. Vizi, Z. Várallyay, A. Sághy, L. Valenta, P. Maák, J. Fekete, A. Bányász, and R. Szipöcs, "Random access three-dimensional two-photon microscopy," Appl. Opt. 46, 1860-1865 (2007).
    [CrossRef] [PubMed]
  3. W. Amir, R. Carriles, E. E. Hoover, T. A. Planchon, C. G. Durfee, and J. A. Squier, "Simultaneous imaging of multiple focal planes using a two-photon scanning microscope," Opt. Lett. 32, 1731-1733 (2007).
    [CrossRef] [PubMed]
  4. J. O. Hamblen, T. S. Hall, and M. D. Furman, Rapid Prototyping of Digital Systems: Quartus II Edition (Springer, 2006).
  5. R. Carriles, E. E. Hoover, W. Amir, and J. A. Squier, "Simultaneous imaging of multiple focal planes in scanning two-photon absorption microscope by photon counting," Proc. SPIE 6771, 67710C-2 - 67710C-8 (2007).
  6. J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
    [CrossRef]
  7. A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
    [CrossRef]
  8. M. Fricke, and T. Nielsen, "Two-dimensional imaging without scanning by multifocal multiphoton microscopy," Appl. Opt. 44, 2984-2988 (2005).
    [CrossRef] [PubMed]
  9. D. J. Gallant, B. Bouchet, and P. M. Baldwin, "Microscopy of starch: evidence of a new level of granule organization," Carbohydr. Polym. 32, 177-191 (1997).
    [CrossRef]
  10. G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
    [CrossRef]
  11. S. W. Chu, I. H. Chen, T. M. Liu, P. C. Chen, C. K. Sun, and B. L. Lin, "Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser," Opt. Lett. 26, 1909-1911 (2001).
    [CrossRef]
  12. K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
    [CrossRef]
  13. R. Cisek, N. Prent, C. Greenhalgh, D. Sandkuijl, A. Tuer, A. Major, and V. Barzda, "Multicontrast nonlinear imaging microscopy," in Biochemical Applications of Nonlinear Optical Spectroscopy, V. V. Yakovlev, ed. (CRC Press, in press).

2008 (1)

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

2007 (2)

2005 (1)

2004 (1)

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, "Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions," IEEE Trans. Nanobiosci. 3, 237-242 (2004).
[CrossRef]

2001 (1)

2000 (1)

G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
[CrossRef]

1998 (2)

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
[CrossRef]

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

1997 (1)

D. J. Gallant, B. Bouchet, and P. M. Baldwin, "Microscopy of starch: evidence of a new level of granule organization," Carbohydr. Polym. 32, 177-191 (1997).
[CrossRef]

Amat-Roldán, I.

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

Amir, W.

Artigas, D.

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

Baldwin, P. M.

D. J. Gallant, B. Bouchet, and P. M. Baldwin, "Microscopy of starch: evidence of a new level of granule organization," Carbohydr. Polym. 32, 177-191 (1997).
[CrossRef]

Bányász, A.

Bewersdorf, J.

Bouchet, B.

D. J. Gallant, B. Bouchet, and P. M. Baldwin, "Microscopy of starch: evidence of a new level of granule organization," Carbohydr. Polym. 32, 177-191 (1997).
[CrossRef]

Brakenhoff, G. J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Buist, A. H.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Carriles, R.

Chen, I. H.

Chen, P. C.

Chu, S. W.

Cormack, I. G.

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

Durfee, C. G.

Fekete, J.

Fricke, M.

Gallant, D. J.

D. J. Gallant, B. Bouchet, and P. M. Baldwin, "Microscopy of starch: evidence of a new level of granule organization," Carbohydr. Polym. 32, 177-191 (1997).
[CrossRef]

Gualda, E. J.

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

Hell, S. W.

Hoover, E. E.

Katona, G.

Lin, B. L.

Liu, T. M.

Loza-Alvarez, P.

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

Maák, P.

Mathew, M.

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

Mizutani, G.

G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
[CrossRef]

Müller, M.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Nielsen, T.

Ober, R. J.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, "Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions," IEEE Trans. Nanobiosci. 3, 237-242 (2004).
[CrossRef]

Pick, R.

Planchon, T. A.

Prabhat, P.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, "Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions," IEEE Trans. Nanobiosci. 3, 237-242 (2004).
[CrossRef]

Psilodimitrakopoulos, S.

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

Ram, S.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, "Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions," IEEE Trans. Nanobiosci. 3, 237-242 (2004).
[CrossRef]

Rózsa, B.

Sághy, A.

Sakamoto, M.

G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
[CrossRef]

Sano, H.

G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
[CrossRef]

Sonoda, Y.

G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
[CrossRef]

Squier, J.

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Squier, J. A.

Sun, C. K.

Szipöcs, R.

Takahashi, T.

G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
[CrossRef]

Thayil, K. N. A.

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

Ushioda, S.

G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
[CrossRef]

Valenta, L.

Várallyay, Z.

Vizi, E. S.

Ward, E. S.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, "Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions," IEEE Trans. Nanobiosci. 3, 237-242 (2004).
[CrossRef]

Appl. Opt. (2)

Carbohydr. Polym. (1)

D. J. Gallant, B. Bouchet, and P. M. Baldwin, "Microscopy of starch: evidence of a new level of granule organization," Carbohydr. Polym. 32, 177-191 (1997).
[CrossRef]

IEEE Trans. Nanobiosci. (1)

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, "Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions," IEEE Trans. Nanobiosci. 3, 237-242 (2004).
[CrossRef]

J. Lumin. (1)

G. Mizutani, Y. Sonoda, H. Sano, M. Sakamoto, T. Takahashi, and S. Ushioda, "Detection of starch granules in a living plant by optical second harmonic microscopy," J. Lumin. 87-89, 824-826 (2000).
[CrossRef]

J. Microsc. (2)

K. N. A. Thayil, E. J. Gualda, S. Psilodimitrakopoulos, I. G. Cormack, I. Amat-Roldán, M. Mathew, D. Artigas, and P. Loza-Alvarez, "Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy," J. Microsc. 230, 70-75 (2008).
[CrossRef]

A. H. Buist, M. Müller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Opt. Lett. (3)

Other (3)

J. O. Hamblen, T. S. Hall, and M. D. Furman, Rapid Prototyping of Digital Systems: Quartus II Edition (Springer, 2006).

R. Carriles, E. E. Hoover, W. Amir, and J. A. Squier, "Simultaneous imaging of multiple focal planes in scanning two-photon absorption microscope by photon counting," Proc. SPIE 6771, 67710C-2 - 67710C-8 (2007).

R. Cisek, N. Prent, C. Greenhalgh, D. Sandkuijl, A. Tuer, A. Major, and V. Barzda, "Multicontrast nonlinear imaging microscopy," in Biochemical Applications of Nonlinear Optical Spectroscopy, V. V. Yakovlev, ed. (CRC Press, in press).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (6)

Fig. 1.
Fig. 1.

Schematic representation of the experimental setup (not to scale): D, dichroic mirror; DM, deformable mirror; F, interference and/or color glass filters; FM, flipper mirrors; HWP, half wave plate; L, lenses (L1=200 mm; L2, L5, L6=100 mm; L3, L4=160 mm); PBS, polarizing beam splitter; PD, photodiode; PMT, photomultiplier tube; QWP, quarter wave plate. The CCD camera is a Hamamatsu chilled CCD model C5985. The pulse train is divided in two parts at the beamsplitter, one delayed by one-half the laser period and the other one reflected from a deformable mirror to change its divergence properties. Both parts are recombined after double passing quarter wave plates to form a 46MHz pulse train. The photodiode is used to provide a laser clock for demultiplexing. Note the alternating polarizations of the pulses after the beamsplitter. By means of the flipper mirrors we can obtain white light images of the region of interest.

Fig. 2.
Fig. 2.

Series of two-images taken simultaneously in the epi configuration using a 0.65 NA objective. The images show Euglena swimming from the top focal plane and crossing into the lower plane. The two depths are separated by 8 µm. Each set of depth 1-depth 2 images was taken simultaneously with a single detector, the time separation between adjacent frames is ~650 ms.

Fig. 3.
Fig. 3.

Second harmonic generation (SHG) images of cornstarch in water taken simultaneously with different linear excitation polarizations of the fundamental light. (a) White light image of the region of interest. Panels (b) and (c) show SHG images of the same area and same depth taken simultaneously with two linear polarizations perpendicular to each other. Images are 128×128 pixels, 0.65 NA objectives were used for excitation and collection; the acquisition time was 12 s.

Fig. 4.
Fig. 4.

Second harmonic generation (SHG) images of cornstarch in water taken with different linear polarizations of the fundamental light. (a) White light image. Panels (b) and (c) show SHG images taken simultaneously with two linear polarizations perpendicular to each other. Images are 128×128 pixels, a 1.25 NA objective was used for excitation and a 0.65 NA objective was used for collection; the acquisition time was 12 s.

Fig. 5.
Fig. 5.

Simultaneous SHG images of cornstarch in water. Focal planes are the same only the polarization differs. Panels (a) and (c) were taken in the epi configuration (orthogonal polarizations); (b) and (d) were taken in transmission (orthogonal polarizations). Images are 128×128 pixels, 0.65 NA objectives were used for excitation and collection; the acquisition time was 12 s.

Fig. 6.
Fig. 6.

Simultaneous TPA fluorescence and SHG images from a mixture of cornstarch and fluorescent microspheres in water. Panel (a) shows a white light image of the region of interest. (b) and (d) are TPA fluorescence images obtained in epi configuration (orthogonal polarizations). (d) and (e) are SHG images obtained in transmission (orthogonal polarizations). Images are 128×128 pixels, 0.65 NA objectives were used for excitation and collection; the acquisition time was 8 s.

Metrics