Abstract

Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x,y,z,t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8×8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity.

© 2007 Optical Society of America

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References

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2007 (1)

K. Steenkeste, S. Lécart, A. Deniset, P. Pernot, P. Eschwège, S. Ferlicot, S. Lévêque-Fort, R. Briandet, and M. P. Fontaine-Aupart, "Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis," Photochem. Photobiol. 83, 1157-1166 (2007).
[CrossRef] [PubMed]

2006 (3)

L. Delanaye, M. A. Bahri, F. Tfibel, M. P. Fontaine-Aupart, A. Mouithys-Mickalad, B. Heine, J. Piette, and M. Hoebecke, "Physical and chemical properties of pyropheophorbide-a methylester in ethanol, phosphate buffer and aqueous dispersion of small unilamellar dimyristoyl-L-α-phosphatidylcholine vesicles," Photochem. Photobiol. Sci. 5, 317-325 (2006).
[CrossRef] [PubMed]

L. Liu, J. Qu, Z. Lin, L. Wang, Z. Fu, B. Guo, and H. Niu, "Simultaneous time- and spectrum-resolved multifocal multiphoton microscopy," Appl. Phys. B 84, 379-383 (2006).
[CrossRef]

J. E. Jureller, H. Y. Kim, and N. F. Scherer, "Stochastic scanning multiphoton multifocal microscopy," Opt. Express 14, 3406-3414 (2006).
[CrossRef] [PubMed]

2005 (3)

M. Fricke and T. Nielsen, "Two-dimensional imaging scanning by multifocal multiphoton microscopy," Appl. Opt. 44, 2984-2988 (2005).
[CrossRef] [PubMed]

A. Deniset, S. Lévêque-Fort, M. P. Fontaine-Aupart, G. Roger, and P. Georges, "Multifocal multiphoton fluorescence lifetime microscopy for biomedical applications," Proc. SPIE 5860, 59-66 (2005).

R. Yuste, "Fluorescence microscopy today," Nat. Methods 2, 902-904 (2005).
[CrossRef] [PubMed]

2004 (2)

2003 (2)

2001 (3)

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

J. Y. Matroule, C. M. Carthy, D. J. Granville, O. Jolois, D. W. Hunt, and J. Piette, "Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization," Oncogene 20, 4070-4084 (2001).
[CrossRef] [PubMed]

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

2000 (3)

D. Fittinghoff, P. Wiseman, and J. Squier, "Wide-field multiphoton and temporally decorrelated multifocal multiphoton microscopy," Opt. Express 7, 273-279 (2000).
[CrossRef] [PubMed]

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, S. Kawata, and T. Takamatsu, "Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays," Opt. Commun. 174, 7-12 (2000).
[CrossRef]

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, "Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier," J. Microsc. 194, 528-531 (2000).
[CrossRef] [PubMed]

1998 (3)

M. Straub and S. H. Hell, "Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope," Appl. Phys. Lett. 73, 1769-1771 (1998).
[CrossRef]

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

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

1993 (1)

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

Anal. Chem. (1)

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Phys. B (1)

L. Liu, J. Qu, Z. Lin, L. Wang, Z. Fu, B. Guo, and H. Niu, "Simultaneous time- and spectrum-resolved multifocal multiphoton microscopy," Appl. Phys. B 84, 379-383 (2006).
[CrossRef]

Appl. Phys. Lett. (1)

M. Straub and S. H. Hell, "Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope," Appl. Phys. Lett. 73, 1769-1771 (1998).
[CrossRef]

J. Microsc. (4)

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

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, T. Takamatsu, and S. Kawata, "Real-time imaging of two-photon-induced fluorescence with a microlens-array scanner and a regenerative amplifier," J. Microsc. 194, 528-531 (2000).
[CrossRef] [PubMed]

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index," J. Microsc. 169, 391-405 (1993).
[CrossRef]

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Nat. Methods (1)

R. Yuste, "Fluorescence microscopy today," Nat. Methods 2, 902-904 (2005).
[CrossRef] [PubMed]

Oncogene (1)

J. Y. Matroule, C. M. Carthy, D. J. Granville, O. Jolois, D. W. Hunt, and J. Piette, "Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization," Oncogene 20, 4070-4084 (2001).
[CrossRef] [PubMed]

Opt. Commun. (1)

K. Fujita, O. Nakamura, T. Kaneko, M. Oyamada, S. Kawata, and T. Takamatsu, "Confocal multipoint multiphoton excitation microscope with microlens and pinhole arrays," Opt. Commun. 174, 7-12 (2000).
[CrossRef]

Opt. Express (2)

Opt. Lett. (4)

Photochem. Photobiol. (1)

K. Steenkeste, S. Lécart, A. Deniset, P. Pernot, P. Eschwège, S. Ferlicot, S. Lévêque-Fort, R. Briandet, and M. P. Fontaine-Aupart, "Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis," Photochem. Photobiol. 83, 1157-1166 (2007).
[CrossRef] [PubMed]

Photochem. Photobiol. Sci. (1)

L. Delanaye, M. A. Bahri, F. Tfibel, M. P. Fontaine-Aupart, A. Mouithys-Mickalad, B. Heine, J. Piette, and M. Hoebecke, "Physical and chemical properties of pyropheophorbide-a methylester in ethanol, phosphate buffer and aqueous dispersion of small unilamellar dimyristoyl-L-α-phosphatidylcholine vesicles," Photochem. Photobiol. Sci. 5, 317-325 (2006).
[CrossRef] [PubMed]

Proc. SPIE (1)

A. Deniset, S. Lévêque-Fort, M. P. Fontaine-Aupart, G. Roger, and P. Georges, "Multifocal multiphoton fluorescence lifetime microscopy for biomedical applications," Proc. SPIE 5860, 59-66 (2005).

Other (1)

J. Bewersdorf, A. Egner, and S. W. Hell, Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Springer, 2006), pp. 550-560.
[CrossRef]

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Figures (10)

Fig. 1
Fig. 1

Beam splitters principle: each beam splitter is composed of 50% dichroic mirror (BS, BS′) associated to four mirrors (M1–M4, M1′–M4′). The first system generates a line of eight points, rotated by using a periscope into a column, which entered the second beam splitter creating 8 × 8 excitation beams.

Fig. 2
Fig. 2

(Color online) (a) Direct insight of the fluorescence localization with the 64 foci fixed on a cluster of three urothelial cells, (b) intensity image obtained after beams scanning for the same sample area as in (a).

Fig. 3
Fig. 3

(Color online) Schematic of the 3D time-resolved MMM and of the time-gated detection. In this case, n time-gated images have been recorded at t 1 , t 2 , t 3 , and t n after the excitation pulse. Consequently, we can thus reconstruct the lifetime map of the sample after fitting each pixel with a monoexponential.

Fig. 4
Fig. 4

Typical intensity image of the 64 foci on a fluorescein layer using 63 × objective (NA 1.4) and only a CCD camera for detection (OrcaER Hamamatsu).

Fig. 5
Fig. 5

Lateral resolution measured upon 800   nm excitation of a fluorescein layer ( 63 × objective, NA 1.4), using a CCD camera only or coupled to a high-rate imager (HRI) for time-gated detection.

Fig. 6
Fig. 6

(Color online) Axial response for a single beam on a 10 μ m diameter fluorescent bead, z r = 1.0 μ m .

Fig. 7
Fig. 7

Fluorescence decay of the photosensitizer PPME in an ethanol solution for TCSPC and time-gating methods.

Fig. 8
Fig. 8

(Color online) Fluorescence image of multilabeled ox pulmonary artery endothelial cells. (a) Overlay of intensity image obtained with 780   nm excitation (in light gray, green online) and 850   nm excitation (in dark gray, red online). (b)–(d) Images under 780   nm excitation mainly efficient for nuclei labeling: (b) Intensity image; (c), (d) lifetime images for two different z planes. (e)–(g) Images under 850   nm excitation mainly efficient for microtubule labeling: (e) intensity image; (f), (g) lifetime images for two different z planes.

Fig. 9
Fig. 9

(Color online) Results obtained with a classical two-photon microscope (excitation at 860   nm ) using TCSPC method as detection system on HCT 116 cells incubated with PPME ( 5 μ M , overnight). (a) Transmittance image, (b) fluorescence intensity image ( 50 μ m × 50   μ m ) , and (c) lifetime measurements ( 2 μ m × 10 μ m , time exposure: 1 s per point).

Fig. 10
Fig. 10

(Color online) 4D imaging of PPME inside HCT 116 cells acquired with the MMM system: (a) transmittance image; (b), (c) fluorescence intensity images of HCT-116 cells after overnight incubation with PPME at 5 μ M at different z positions extracted from a stack of 40 images (steps of 0.5 μ m ); (d), (e) lifetime image associated; (f) PPME decay in solution compared to cellular medium (HCT 116) for the two techniques.

Equations (1)

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I edge = A π ( π 2 + arctan ( z - z 0 z R ) + 1 z R z - z 0 + z - z 0 z R ) ,

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