Abstract

We report the design and implementation of spectroscopic and multicolor imaging capabilities into a fibered confocal fluorescence microscope (FCFM) already capable of in vivo imaging. The real time imaging device and the high resolution fiber probe make this system the first reported capable of performing multi color detection in the field of FCFM. The advantages of the system will allow in vivo morphological and functional imaging. Preliminary experiments were carried out in tissue samples to demonstrate the potential of the technique. The quality of the axial sectioning achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and applications to multicolor imaging are shown.

© 2007 Optical Society of America

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  1. E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
    [CrossRef] [PubMed]
  2. A. R. Rouse, A. Kano, J. A. Udovich, S. M. Kroto and A. F. Gmitro, "Design and demonstration of a miniature catheter for a confocal microendoscope," Appl. Opt. 43, 5763-5771 (2004).
    [CrossRef] [PubMed]
  3. K. Carlson, M. Chidley, K. B. Sung, M. Descour, A. Gillenwater, M. Follen and R. Richards-Kortum, "In vivo fiber-optic confocal reflectance microscope with an injection-molded plastic miniature objective lens," Appl. Opt. 44, 1792-1797 (2005).
    [CrossRef] [PubMed]
  4. P. M. Lane, A. Dlugan, R. Richards-Kortum and C. E. MacCaulay, "Fiber-optic confocal microscopy using a spatial light modulator," Opt. Lett. 25,1 780-1782 (2000).
    [CrossRef]
  5. T. D. Wang, M. J. Mandella, C. H. Contag and G. S. Kino, "Dual-axis confocal microscope for high-resolution in vivo imaging," Opt. Lett. 28, 414-416 (2003).
    [CrossRef] [PubMed]
  6. P. M. Delaney, M. R. Harris and R. G. King, "Fibre-optic laser scanning confocal microscope suitable for fluorescence imaging," Appl. Opt. 33, 573-577 (1994).
    [CrossRef] [PubMed]
  7. D. L. Dickensheets and G. S. Kino, "Micromachined scanning confocal optical microscope," Opt. Lett. 21, 764- 766 (1996).
    [CrossRef] [PubMed]
  8. H. Miyajima, K. Murakami and M. Katashiro, "MEMS Optical scanners for microscopes," IEEE J. Quantum Electron. 10, 514-527 (2004).
    [CrossRef]
  9. K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
    [PubMed]
  10. A. Perchant, G. Le Goualher, M. Genet, B. Viellerobe and F. Berier, "An integrated fibered confocal microscopy system for in vivo and in situ fluorescence imaging - applications to endoscopy in small animal imaging," in Proceedings of the IEEE International Symposium on Biomedical Imaging: From Nano to Macro, 2004.
  11. A. R. Rouse and A. F. Gmitro, "Multispectral imaging with a confocal microendoscope," Opt. Lett. 25, 1708-1710 (2000).
    [CrossRef]
  12. G. Le Goualher, A. Perchant, M. Genet, C. Cave, B. Viellerobe, F. Berier, B. Abrat and N. Ayache, "Towards optical biopsies with an integrated fibered confocal fluorescence microscope," Lecture Notes in Computer Science 3217(II):761-768, Springer (Medical Image Computing and Computer Assisted Intervention), 2004.
    [CrossRef]
  13. P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
    [CrossRef] [PubMed]
  14. L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

2007 (1)

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

2006 (1)

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

2005 (1)

2004 (3)

E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
[CrossRef] [PubMed]

A. R. Rouse, A. Kano, J. A. Udovich, S. M. Kroto and A. F. Gmitro, "Design and demonstration of a miniature catheter for a confocal microendoscope," Appl. Opt. 43, 5763-5771 (2004).
[CrossRef] [PubMed]

H. Miyajima, K. Murakami and M. Katashiro, "MEMS Optical scanners for microscopes," IEEE J. Quantum Electron. 10, 514-527 (2004).
[CrossRef]

2003 (2)

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

T. D. Wang, M. J. Mandella, C. H. Contag and G. S. Kino, "Dual-axis confocal microscope for high-resolution in vivo imaging," Opt. Lett. 28, 414-416 (2003).
[CrossRef] [PubMed]

2000 (2)

1996 (1)

1994 (1)

Aaron, J.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Adler-Storthz, K.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Arap, W.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Bourgeais, L.

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

Bourg-Heckly, G.

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

Carlson, K.

Cave, C.

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

Changeux, J-P.

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

Charvet, I.

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

Chidley, M.

Contag, C. H.

Delaney, P. M.

Descour, M.

K. Carlson, M. Chidley, K. B. Sung, M. Descour, A. Gillenwater, M. Follen and R. Richards-Kortum, "In vivo fiber-optic confocal reflectance microscope with an injection-molded plastic miniature objective lens," Appl. Opt. 44, 1792-1797 (2005).
[CrossRef] [PubMed]

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Dickensheets, D. L.

Dlugan, A.

Follen, M.

K. Carlson, M. Chidley, K. B. Sung, M. Descour, A. Gillenwater, M. Follen and R. Richards-Kortum, "In vivo fiber-optic confocal reflectance microscope with an injection-molded plastic miniature objective lens," Appl. Opt. 44, 1792-1797 (2005).
[CrossRef] [PubMed]

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Genet, M.

E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
[CrossRef] [PubMed]

Gillenwater, A.

K. Carlson, M. Chidley, K. B. Sung, M. Descour, A. Gillenwater, M. Follen and R. Richards-Kortum, "In vivo fiber-optic confocal reflectance microscope with an injection-molded plastic miniature objective lens," Appl. Opt. 44, 1792-1797 (2005).
[CrossRef] [PubMed]

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Gmitro, A. F.

Harris, M. R.

Hsu, B.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Kano, A.

Katashiro, M.

H. Miyajima, K. Murakami and M. Katashiro, "MEMS Optical scanners for microscopes," IEEE J. Quantum Electron. 10, 514-527 (2004).
[CrossRef]

King, R. G.

Kino, G. S.

Korgel, B.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Kroto, S. M.

Laemmel, E.

E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
[CrossRef] [PubMed]

Lam, W.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Lambert, R.

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

Lane, P. M.

Le Gargasson, J. F.

E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
[CrossRef] [PubMed]

Le Goualher, G.

E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
[CrossRef] [PubMed]

Leresche, N.

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

MacAulay, C.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

MacCaulay, C. E.

Mandella, M. J.

Meda, P.

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

Miyajima, H.

H. Miyajima, K. Murakami and M. Katashiro, "MEMS Optical scanners for microscopes," IEEE J. Quantum Electron. 10, 514-527 (2004).
[CrossRef]

Moreno-Swirc, S.

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

Murakami, K.

H. Miyajima, K. Murakami and M. Katashiro, "MEMS Optical scanners for microscopes," IEEE J. Quantum Electron. 10, 514-527 (2004).
[CrossRef]

Nida, D.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Pasqualini, R.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Peltier, E.

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

Perchant, A.

E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
[CrossRef] [PubMed]

Richards-Kortum, R.

Rouse, A. R.

Sokolov, K.

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Stoppini, L.

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

Sung, K. B.

Thiberville, L.

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

Udovich, J. A.

Vercauteren, T.

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

Vicaut, E.

E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
[CrossRef] [PubMed]

Vincent, P.

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

Wang, T. D.

Am. J. Respir. Crit. Care Med. (1)

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cave, G. Bourg-Heckly, "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy," Am. J. Respir. Crit. Care Med. 1, 175, 22-31 (2007).

Appl. Opt. (3)

EMBO Reports (1)

P. Vincent, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J-P. Changeux, R. Lambert, P. Meda, D. Paupardin- Tritsch, "Live imaging of neural structure and function by fibered fluorescence microscopy," EMBO Reports 7, 11, 1154-1161 (2006).
[CrossRef] [PubMed]

IEEE J. Quantum Electron. (1)

H. Miyajima, K. Murakami and M. Katashiro, "MEMS Optical scanners for microscopes," IEEE J. Quantum Electron. 10, 514-527 (2004).
[CrossRef]

J. Vasc. Res. (1)

E. Laemmel, M. Genet, G. Le Goualher, A. Perchant, J. F. Le Gargasson and E. Vicaut, "Fibered Confocal Fluorescence Microscopy (Cell-viZioTM) Facilitates Extended Imaging in the Field of Microcirculation: A Comparison with Intravital Microscopy," J. Vasc. Res. 41, 400-411 (2004).
[CrossRef] [PubMed]

Opt. Lett. (4)

Technol. Cancer Res. Treat. (1)

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer.," Technol. Cancer Res. Treat. 2, 491-504 (2003).
[PubMed]

Other (2)

A. Perchant, G. Le Goualher, M. Genet, B. Viellerobe and F. Berier, "An integrated fibered confocal microscopy system for in vivo and in situ fluorescence imaging - applications to endoscopy in small animal imaging," in Proceedings of the IEEE International Symposium on Biomedical Imaging: From Nano to Macro, 2004.

G. Le Goualher, A. Perchant, M. Genet, C. Cave, B. Viellerobe, F. Berier, B. Abrat and N. Ayache, "Towards optical biopsies with an integrated fibered confocal fluorescence microscope," Lecture Notes in Computer Science 3217(II):761-768, Springer (Medical Image Computing and Computer Assisted Intervention), 2004.
[CrossRef]

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Figures (7)

Fig. 1.
Fig. 1.

Lay-out of the fibered confocal fluorescence spectroscopy and multicolor imaging system.

Fig. 2.
Fig. 2.

Scheme of probe II.

Fig. 3.
Fig. 3.

Typical images obtained in vivo with the miniaturized version of probe II. Healthy colonic crypts stained with fluorescein can be seen. FOV is 240 μm. The working distance is 30 μm.

Fig. 4.
Fig. 4.

Images and spectra of a fixed human cervix sample stained with a 100μM solution of DiA (Invitrogen) (a,d) and with a 100μM solution of POPO-1 (Invitrogen) excited respectively at 488 nm and 405 nm (c,e). (b) is the fusion of these two images. (Probe II, FOV: 80μm×100μm). Signal losses visible on the spectra are due to filters spectral response in the case of 405/488nm architecture system.

Fig. 5.
Fig. 5.

Axial intensity profiles of a fluorescent bead showing the resolution of the spectroscopic channel for Probe I and Probe II at 488 nm. Axial resolution is determined by the FWHM of the curves.

Fig. 6.
Fig. 6.

Multilabeled images from fixed squamous and glandular human tissues with probe II. Cervix (a) and thyroid (b) samples are stained with 100μM solutions of DiA (red) and To-Pro-1 (Invitrogen) (blue), respectively excited at 488 nm and 638 nm. Multilabeled images (c,d,e) from fixed human cervix samples exhibiting different parakeratosis grades, confirmed by the histological analysis (FOV: 80μm×100μm).

Fig. 7.
Fig. 7.

Multilabeled images with probe I from fixed human cervix samples stained with DiA (blue) excited at 488 nm and To-Pro-1 (red) excited at 638 nm (FOV: 430μm×300μm). The density of cell nuclei (in red) indicates a high-grade parakeratosis. The lower resolution of the Probe I cannot give a detailed of view of the cellular membranes (in blue). The squamous epithelium pattern is clearly visible.

Tables (3)

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Table 1. Specifications of the probe II’s miniaturized version.

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Table 2. Dichroic and rejecting filters properties in the case of 488/635 nm architecture.

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Table 3. Lateral and axial resolution (LR, AR) measured at 488 and 638 nm, with probes I and II, for the spectroscopic and imaging channels (micrometers).

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