Abstract

Two-photon excitation spectroscopy is a powerful technique for the characterization of the optical properties of genetically encoded and synthetic fluorescent molecules. Excitation spectroscopy requires tuning the wavelength of the Ti:sapphire laser while carefully monitoring the delivered power. To assist laser tuning and the control of delivered power, we developed an Arduino Due based tool for the automatic acquisition of high quality spectra. This tool is portable, fast, affordable and precise. It allowed studying the impact of scattering and of blood absorption on two-photon excitation light. In this way, we determined the wavelength-dependent deformation of excitation spectra occurring in deep tissues in vivo.

© 2016 Optical Society of America

Full Article  |  PDF Article
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References

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  6. P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
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    [Crossref] [PubMed]
  20. R. Bizzarri, M. Serresi, S. Luin, and F. Beltram, “Green fluorescent protein based pH indicators for in vivo use: a review,” Anal. Bioanal. Chem. 393(4), 1107–1122 (2009).
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  21. Z. Liu, C. Zhang, Y. Chen, W. He, and Z. Guo, “An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations,” Chem. Commun. (Camb.) 48(67), 8365–8367 (2012).
    [Crossref] [PubMed]
  22. D. L. Wokosin, C. M. Loughrey, and G. L. Smith, “Characterization of a range of fura dyes with two-photon excitation,” Biophys. J. 86(3), 1726–1738 (2004).
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  23. A. Zhu, C. Ding, and Y. Tian, “A two-photon ratiometric fluorescence probe for Cupric Ions in Live Cells and Tissues,” Sci. Rep. 3, 2933 (2013).
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  24. J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
    [Crossref] [PubMed]
  25. N. Y. Baek, C. H. Heo, C. S. Lim, G. Masanta, B. R. Cho, and H. M. Kim, “A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue,” Chem. Commun. (Camb.) 48(38), 4546–4548 (2012).
    [Crossref] [PubMed]

2015 (2)

A. Sheinin, A. Lavi, and I. Michaelevski, “StimDuino: An Arduino-based electrophysiological stimulus isolator,” J. Neurosci. Methods 243, 8–17 (2015).
[Crossref] [PubMed]

C. Vinegoni, S. Lee, A. D. Aguirre, and R. Weissleder, “New techniques for motion-artifact-free in vivo cardiac microscopy,” Front. Physiol. 6, 147 (2015).
[Crossref] [PubMed]

2014 (1)

O. Pineño, “ArduiPod Box: a low-cost and open-source Skinner box using an iPod Touch and an Arduino microcontroller,” Behav. Res. Methods 46(1), 196–205 (2014).
[Crossref] [PubMed]

2013 (5)

T. W. Schubert, A. D’Ausilio, and R. Canto, “Using Arduino microcontroller boards to measure response latencies,” Behav. Res. Methods 45(4), 1332–1346 (2013).
[Crossref] [PubMed]

S. L. Jacques, “Optical properties of biological tissues: a review,” Phys. Med. Biol. 58(11), R37–R61 (2013).
[Crossref] [PubMed]

J. A. Kornuta, M. E. Nipper, and J. B. Dixon, “Low-cost microcontroller platform for studying lymphatic biomechanics in vitro,” J. Biomech. 46(1), 183–186 (2013).
[Crossref] [PubMed]

A. Zhu, C. Ding, and Y. Tian, “A two-photon ratiometric fluorescence probe for Cupric Ions in Live Cells and Tissues,” Sci. Rep. 3, 2933 (2013).
[Crossref] [PubMed]

J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
[Crossref] [PubMed]

2012 (4)

N. Y. Baek, C. H. Heo, C. S. Lim, G. Masanta, B. R. Cho, and H. M. Kim, “A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue,” Chem. Commun. (Camb.) 48(38), 4546–4548 (2012).
[Crossref] [PubMed]

Z. Liu, C. Zhang, Y. Chen, W. He, and Z. Guo, “An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations,” Chem. Commun. (Camb.) 48(67), 8365–8367 (2012).
[Crossref] [PubMed]

P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
[Crossref] [PubMed]

A. D’Ausilio, “Arduino: a low-cost multipurpose lab equipment,” Behav. Res. Methods 44(2), 305–313 (2012).
[Crossref] [PubMed]

2009 (1)

R. Bizzarri, M. Serresi, S. Luin, and F. Beltram, “Green fluorescent protein based pH indicators for in vivo use: a review,” Anal. Bioanal. Chem. 393(4), 1107–1122 (2009).
[Crossref] [PubMed]

2007 (1)

G. O. Clay, C. B. Schaffer, and D. Kleinfeld, “Large two-photon absorptivity of hemoglobin in the infrared range of 780-880 nm,” J. Chem. Phys. 126(2), 025102 (2007).
[Crossref] [PubMed]

2006 (2)

R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, “Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies,” Biophys. J. 90(9), 3300–3314 (2006).
[Crossref] [PubMed]

G. O. Clay, A. C. Millard, C. B. Schaffer, J. Aus-der-Au, P. S. Tsai, J. A. Squier, and D. Kleinfeld, “Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin,” J. Opt. Soc. Am. B 23(5), 932–950 (2006).
[Crossref]

2004 (1)

D. L. Wokosin, C. M. Loughrey, and G. L. Smith, “Characterization of a range of fura dyes with two-photon excitation,” Biophys. J. 86(3), 1726–1738 (2004).
[Crossref] [PubMed]

1997 (1)

V. Tuchin, “Light scattering study of tissues,” Phys. Uspekhi 40(5), 495–515 (1997).
[Crossref]

Aguirre, A. D.

C. Vinegoni, S. Lee, A. D. Aguirre, and R. Weissleder, “New techniques for motion-artifact-free in vivo cardiac microscopy,” Front. Physiol. 6, 147 (2015).
[Crossref] [PubMed]

Akerman, C. J.

J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
[Crossref] [PubMed]

Alves, A.

A. Alves, H. Silva, A. Lourenc, and A. Fried, “BITalino: a biosignal acquisition system based on Arduino,” Proceeding of the 6th Conference on Biometical Electronicsand Devices (BIODEVICES) (2013).

Arcangeli, C.

R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, “Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies,” Biophys. J. 90(9), 3300–3314 (2006).
[Crossref] [PubMed]

Arosio, D.

R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, “Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies,” Biophys. J. 90(9), 3300–3314 (2006).
[Crossref] [PubMed]

Aus-der-Au, J.

Baek, N. Y.

N. Y. Baek, C. H. Heo, C. S. Lim, G. Masanta, B. R. Cho, and H. M. Kim, “A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue,” Chem. Commun. (Camb.) 48(38), 4546–4548 (2012).
[Crossref] [PubMed]

Beltram, F.

R. Bizzarri, M. Serresi, S. Luin, and F. Beltram, “Green fluorescent protein based pH indicators for in vivo use: a review,” Anal. Bioanal. Chem. 393(4), 1107–1122 (2009).
[Crossref] [PubMed]

R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, “Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies,” Biophys. J. 90(9), 3300–3314 (2006).
[Crossref] [PubMed]

Bizzarri, R.

R. Bizzarri, M. Serresi, S. Luin, and F. Beltram, “Green fluorescent protein based pH indicators for in vivo use: a review,” Anal. Bioanal. Chem. 393(4), 1107–1122 (2009).
[Crossref] [PubMed]

R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, “Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies,” Biophys. J. 90(9), 3300–3314 (2006).
[Crossref] [PubMed]

Canto, R.

T. W. Schubert, A. D’Ausilio, and R. Canto, “Using Arduino microcontroller boards to measure response latencies,” Behav. Res. Methods 45(4), 1332–1346 (2013).
[Crossref] [PubMed]

Cardarelli, F.

R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, “Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies,” Biophys. J. 90(9), 3300–3314 (2006).
[Crossref] [PubMed]

Chen, Y.

Z. Liu, C. Zhang, Y. Chen, W. He, and Z. Guo, “An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations,” Chem. Commun. (Camb.) 48(67), 8365–8367 (2012).
[Crossref] [PubMed]

Cho, B. R.

N. Y. Baek, C. H. Heo, C. S. Lim, G. Masanta, B. R. Cho, and H. M. Kim, “A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue,” Chem. Commun. (Camb.) 48(38), 4546–4548 (2012).
[Crossref] [PubMed]

Clay, G. O.

Cooper, H. M.

P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
[Crossref] [PubMed]

D’Ausilio, A.

T. W. Schubert, A. D’Ausilio, and R. Canto, “Using Arduino microcontroller boards to measure response latencies,” Behav. Res. Methods 45(4), 1332–1346 (2013).
[Crossref] [PubMed]

A. D’Ausilio, “Arduino: a low-cost multipurpose lab equipment,” Behav. Res. Methods 44(2), 305–313 (2012).
[Crossref] [PubMed]

Ding, C.

A. Zhu, C. Ding, and Y. Tian, “A two-photon ratiometric fluorescence probe for Cupric Ions in Live Cells and Tissues,” Sci. Rep. 3, 2933 (2013).
[Crossref] [PubMed]

Dixon, J. B.

J. A. Kornuta, M. E. Nipper, and J. B. Dixon, “Low-cost microcontroller platform for studying lymphatic biomechanics in vitro,” J. Biomech. 46(1), 183–186 (2013).
[Crossref] [PubMed]

Dumortier, D.

P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
[Crossref] [PubMed]

Faraci, P.

R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, “Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies,” Biophys. J. 90(9), 3300–3314 (2006).
[Crossref] [PubMed]

Fried, A.

A. Alves, H. Silva, A. Lourenc, and A. Fried, “BITalino: a biosignal acquisition system based on Arduino,” Proceeding of the 6th Conference on Biometical Electronicsand Devices (BIODEVICES) (2013).

Gronfier, C.

P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
[Crossref] [PubMed]

Guo, Z.

Z. Liu, C. Zhang, Y. Chen, W. He, and Z. Guo, “An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations,” Chem. Commun. (Camb.) 48(67), 8365–8367 (2012).
[Crossref] [PubMed]

He, W.

Z. Liu, C. Zhang, Y. Chen, W. He, and Z. Guo, “An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations,” Chem. Commun. (Camb.) 48(67), 8365–8367 (2012).
[Crossref] [PubMed]

Heo, C. H.

N. Y. Baek, C. H. Heo, C. S. Lim, G. Masanta, B. R. Cho, and H. M. Kim, “A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue,” Chem. Commun. (Camb.) 48(38), 4546–4548 (2012).
[Crossref] [PubMed]

Jacques, S. L.

S. L. Jacques, “Optical properties of biological tissues: a review,” Phys. Med. Biol. 58(11), R37–R61 (2013).
[Crossref] [PubMed]

Joyce, B.

J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
[Crossref] [PubMed]

Kay, L.

J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
[Crossref] [PubMed]

Kim, H. M.

N. Y. Baek, C. H. Heo, C. S. Lim, G. Masanta, B. R. Cho, and H. M. Kim, “A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue,” Chem. Commun. (Camb.) 48(38), 4546–4548 (2012).
[Crossref] [PubMed]

Kleinfeld, D.

Knoblauch, K.

P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
[Crossref] [PubMed]

Kornuta, J. A.

J. A. Kornuta, M. E. Nipper, and J. B. Dixon, “Low-cost microcontroller platform for studying lymphatic biomechanics in vitro,” J. Biomech. 46(1), 183–186 (2013).
[Crossref] [PubMed]

Lavi, A.

A. Sheinin, A. Lavi, and I. Michaelevski, “StimDuino: An Arduino-based electrophysiological stimulus isolator,” J. Neurosci. Methods 243, 8–17 (2015).
[Crossref] [PubMed]

Lee, S.

C. Vinegoni, S. Lee, A. D. Aguirre, and R. Weissleder, “New techniques for motion-artifact-free in vivo cardiac microscopy,” Front. Physiol. 6, 147 (2015).
[Crossref] [PubMed]

Lim, C. S.

N. Y. Baek, C. H. Heo, C. S. Lim, G. Masanta, B. R. Cho, and H. M. Kim, “A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue,” Chem. Commun. (Camb.) 48(38), 4546–4548 (2012).
[Crossref] [PubMed]

Liu, Z.

Z. Liu, C. Zhang, Y. Chen, W. He, and Z. Guo, “An excitation ratiometric Zn2+ sensor with mitochondria-targetability for monitoring of mitochondrial Zn2+ release upon different stimulations,” Chem. Commun. (Camb.) 48(67), 8365–8367 (2012).
[Crossref] [PubMed]

Loughrey, C. M.

D. L. Wokosin, C. M. Loughrey, and G. L. Smith, “Characterization of a range of fura dyes with two-photon excitation,” Biophys. J. 86(3), 1726–1738 (2004).
[Crossref] [PubMed]

Lourenc, A.

A. Alves, H. Silva, A. Lourenc, and A. Fried, “BITalino: a biosignal acquisition system based on Arduino,” Proceeding of the 6th Conference on Biometical Electronicsand Devices (BIODEVICES) (2013).

Luin, S.

R. Bizzarri, M. Serresi, S. Luin, and F. Beltram, “Green fluorescent protein based pH indicators for in vivo use: a review,” Anal. Bioanal. Chem. 393(4), 1107–1122 (2009).
[Crossref] [PubMed]

Malkki, H.

P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
[Crossref] [PubMed]

Masanta, G.

N. Y. Baek, C. H. Heo, C. S. Lim, G. Masanta, B. R. Cho, and H. M. Kim, “A highly sensitive two-photon fluorescent probe for mitochondrial zinc ions in living tissue,” Chem. Commun. (Camb.) 48(38), 4546–4548 (2012).
[Crossref] [PubMed]

Michaelevski, I.

A. Sheinin, A. Lavi, and I. Michaelevski, “StimDuino: An Arduino-based electrophysiological stimulus isolator,” J. Neurosci. Methods 243, 8–17 (2015).
[Crossref] [PubMed]

Millard, A. C.

Najjar, R. P.

P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
[Crossref] [PubMed]

Newey, S. E.

J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
[Crossref] [PubMed]

Nipper, M. E.

J. A. Kornuta, M. E. Nipper, and J. B. Dixon, “Low-cost microcontroller platform for studying lymphatic biomechanics in vitro,” J. Biomech. 46(1), 183–186 (2013).
[Crossref] [PubMed]

Pineño, O.

O. Pineño, “ArduiPod Box: a low-cost and open-source Skinner box using an iPod Touch and an Arduino microcontroller,” Behav. Res. Methods 46(1), 196–205 (2014).
[Crossref] [PubMed]

Raimondo, J. V.

J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
[Crossref] [PubMed]

Ricci, F.

R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, “Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies,” Biophys. J. 90(9), 3300–3314 (2006).
[Crossref] [PubMed]

Schaffer, C. B.

Schlagheck, T.

J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
[Crossref] [PubMed]

Schubert, T. W.

T. W. Schubert, A. D’Ausilio, and R. Canto, “Using Arduino microcontroller boards to measure response latencies,” Behav. Res. Methods 45(4), 1332–1346 (2013).
[Crossref] [PubMed]

Serresi, M.

R. Bizzarri, M. Serresi, S. Luin, and F. Beltram, “Green fluorescent protein based pH indicators for in vivo use: a review,” Anal. Bioanal. Chem. 393(4), 1107–1122 (2009).
[Crossref] [PubMed]

Sheinin, A.

A. Sheinin, A. Lavi, and I. Michaelevski, “StimDuino: An Arduino-based electrophysiological stimulus isolator,” J. Neurosci. Methods 243, 8–17 (2015).
[Crossref] [PubMed]

Silva, H.

A. Alves, H. Silva, A. Lourenc, and A. Fried, “BITalino: a biosignal acquisition system based on Arduino,” Proceeding of the 6th Conference on Biometical Electronicsand Devices (BIODEVICES) (2013).

Smith, G. L.

D. L. Wokosin, C. M. Loughrey, and G. L. Smith, “Characterization of a range of fura dyes with two-photon excitation,” Biophys. J. 86(3), 1726–1738 (2004).
[Crossref] [PubMed]

Squier, J. A.

Srinivas, S.

J. V. Raimondo, B. Joyce, L. Kay, T. Schlagheck, S. E. Newey, S. Srinivas, and C. J. Akerman, “A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system,” Front. Cell. Neurosci. 7, 202 (2013).
[Crossref] [PubMed]

Teikari, P.

P. Teikari, R. P. Najjar, H. Malkki, K. Knoblauch, D. Dumortier, C. Gronfier, and H. M. Cooper, “An inexpensive Arduino-based LED stimulator system for vision research,” J. Neurosci. Methods 211(2), 227–236 (2012).
[Crossref] [PubMed]

Tian, Y.

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Supplementary Material (1)

NameDescription
» Code 1       Spectrumino: an Arduino Due based tool for spectroscopy

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Figures (6)

Fig. 1
Fig. 1 (a) A sketch of both analog and digital connections between the Arduino Due and the other devices of a typical two-photon microscope. Dark red lines represent the laser beam (with intensity roughly proportional to their thickness), and double arrow represents its polarization direction (with horizontal direction actually meaning out of plane polarization). (b) A screenshot of the RS-232 text-based user interface, in which the parameters (first wavelength, step, number of wavelengths in the spectrum, power of the laser beam) are passed to the Arduino Due.
Fig. 2
Fig. 2 Electronic scheme of the digital connections between the Arduino Due and the RS-232 ports to pc and laser (pin 14-15-18-19 of the Arduino board), to the beam shutter (pin 8) and to the frame trigger of the microscope (pin 5). Analog ports are connected to the power meter (ADC 0) and to the Pockels cell (DAC 0). The latter uses a voltage divider and a low pass filter to avoid any damage on the Pockels cell electronics. All electrolytic capacitors connected to the Max 3232 ICs are 0.1 μF / 10V.
Fig. 3
Fig. 3 (a) Transfer function of the microscope. This function is proportional to the transmittance of the microscope optics. Data are obtained by measuring the intensity of the laser beam at the objective exit while the beam power on the optic bench downstream of the Pockels cell is kept constant. Data are normalized at 1 at 800 nm. (b) Time course of the measured laser beam power (left axis, black) and of the control voltage issued to the Pockels cell amplifier by the DAC port of the Arduino Due (right axis, red) in a typical 800 nm to 1000 nm spectrum, with a step size of 10 nm. Target power is fixed to 3 mW by the user, through the serial communication with the PC. The control voltage varies with the wavelength because of the laser cavity efficiency and the different Pockels cell response at different wavelengths. (c) Magnification of the first ramp of the data of panel a. This plot shows the 3 steps algorithm used to identify the correct setting for the Pockels cell control signal. The plot shows that the increment of control voltage (red data) decreases as the laser power converges to the target power (3mW). When the correct target power is reached, imaging is started.
Fig. 4
Fig. 4 (a) Complete ECG signal at the output of the amplifier (top panel) and after rectification (middle panel). The lower panel shows the TTL signal generated by the Arduino Due to trigger the acquisition on its rising edge. (b) Imaging in vivo of dendrites expressing YFP without and with the heartbeat triggering. Three consecutive frames have been represented with a red, a green and a blue lookup table, and then superimposed. (c) Standard deviation images calculated by a 20 frames-long continuous acquisition, without and with the heartbeat triggering.
Fig. 5
Fig. 5 (a) Two-photon images of YFP expressing HEK 293 cells at different excitation wavelengths and at fixed excitation power (3mW). Only 5 of the 21 images of the whole spectrum are shown. (b) two-photon microscopy of layer-5 neurons excited at 960 nm. Imaging is performed in acute slice at a depth of 20 µm from the slice surface. (c) In vivo two-photon microscopy of dendrites in layers 2-3 of the somatosensory cortex. Imaging was performed 10 µm under the surface. The dark areas are indicative of superficial blood vessels. (d) Imaging in a region immediately under that vessel, at a depth of 20 µm. The red rectangle indicates the area immediately underneath the blood vessel. (e) two-photon excitation spectra of YFP expressed in HEK 293 cells (black), in layer-5 neurons in slice (blue), and in vivo (layers 2-3) under a blood vessel (red) and far from it (green). The last two spectra (the red one and the green one) are subtly different (see Fig. 6(a)), the red spectrum superimposes partially onto the green one. Scale bars: 20 µm.
Fig. 6
Fig. 6 (a) Scaling of probability for two-photon excitation in scattering tissue as a function of wavelength, i.e. in slice (blue), in vivo under a blood vessel (red) and far from it (green). Data are obtained by dividing each YFP excitation spectrum by the reference spectrum obtained in scattering-free conditions (HEK 293), and by normalizing at 1 at 960nm. (b) Ratio between the in vivo YFP two-photon excitation spectra obtained far from the blood vessel (S) or directly underneath (Su). The error bars come from standard error propagation. This spectrum deformation is interpreted as caused by absorption phenomena and has been compared to the one-photon absorption of oxyhemoglobin (dashed gray line) and deoxyhemoglobin (solid grey line) peaked around 430 nm (top-right axes) (reproduced from Clay et al., 2007).

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