Abstract

We developed a new illumination method called the simultaneous illumination method. This method does not require synchronization between light sources and sensor signals, which drastically simplifies the instrumentation. As a proof-of-concept, we applied this method to an oceanographic fluorometer. In principle, using this method, one can easily increase the number of characterized emission wavelengths by mounting optical sensors for as many emission wavelengths as needed. Our fluorometer has two emission-wavelength channels and twelve excitation wavelengths. The aim of this prototype is to demonstrate a viable in situ N-channel emission fluorometer with multiple wavelengths of excitation, which has not been previously realized.

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References

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  1. G. C. Papageorgiou, “2.Fluorescence of photosynthetic pigments in vitro and in vivo,” in Chlorophyll a Fluorescence: A Signature of Photosynthesis, G. C. Papageorgiou and Govindjee, ed. (Springer, 2004).
  2. A. Grinvald, W. N. Ross, and I. Farber, “Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons,” Proc. Natl. Acad. Sci. U.S.A. 78(5), 3245–3249 (1981).
    [CrossRef] [PubMed]
  3. C. S. Yentsch and D. W. Menzel, “A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence,” Deep-Sea Res. Oceanogr. Abstr. 10(3), 221–231 (1963).
    [CrossRef]
  4. N. K. Højerslev, “Bio-optical measurements in the Southwest Florida shelf ecosystem,” J. Cons. Cons. Int. Explor. Mer 42, 65–82 (1985).
  5. N. K. Højerslev and T. Aarup, “Optical measurements on the Louisiana Shelf off the Mississippi River,” Estuar. Coast. Shelf Sci. 55(4), 599–611 (2002).
    [CrossRef]
  6. P. G. Falkowski and J. A. Raven, Aquatic Photosynthesis (Blackwell Science, 1997)
  7. BBE CO., Ltd., “Fluoro Probe,” http://www.bbe-moldaenke.de/chlorophyll/fluoroprobe/ .
  8. JFE Advantech CO, Ltd., “Multi-Exciter,” http://www.jfe-alec.co.jp/html/multi-exciter-e.htm .
  9. L. Zhou, S. Ye, H. Chen, J. Pan, and J. Yang, “The design of a low-power self-contained multi-EX-EM array deep sea in situ organism measurement device,” The 2nd International Conference on Bioinformatics and Biomedical Engineering, ICBBE 2008, 1196–1199 (2008).
  10. C. A. Parker, Photoluminescence of Solutions (Elsevier, 1968).
  11. T. Oishi, A. Tanaka, S. Yano, H. Ebata, Y. Takahashi, H. Kondo, H. Tan, and R. Doerffer, “Development of simultaneous multi-wavelength excitation fluorometer,” in Ocean Optics XIX, CD-ROM (2008).
  12. N. G. Jarlov, Marine Optics (Elsevier, 1976)
  13. C. V. I. Melles Griot, “Fundamental Optics Guide,” http://www.cvimellesgriot.com/products/Documents/TechnicalGuide/fundamental-Optics.pdf .
  14. T. Oishi, “Significant relationship between the backward scattering coefficient of sea water and the scatterance at 120 °,” Appl. Opt. 29(31), 4658–4665 (1990).
    [CrossRef] [PubMed]
  15. M. Jonasz, and G. Fournier, Light Scattering by Particles in Water: Theoretical and Experimental Foundations (Elsevier, 2007).

2002 (1)

N. K. Højerslev and T. Aarup, “Optical measurements on the Louisiana Shelf off the Mississippi River,” Estuar. Coast. Shelf Sci. 55(4), 599–611 (2002).
[CrossRef]

1990 (1)

1985 (1)

N. K. Højerslev, “Bio-optical measurements in the Southwest Florida shelf ecosystem,” J. Cons. Cons. Int. Explor. Mer 42, 65–82 (1985).

1981 (1)

A. Grinvald, W. N. Ross, and I. Farber, “Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons,” Proc. Natl. Acad. Sci. U.S.A. 78(5), 3245–3249 (1981).
[CrossRef] [PubMed]

1963 (1)

C. S. Yentsch and D. W. Menzel, “A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence,” Deep-Sea Res. Oceanogr. Abstr. 10(3), 221–231 (1963).
[CrossRef]

Aarup, T.

N. K. Højerslev and T. Aarup, “Optical measurements on the Louisiana Shelf off the Mississippi River,” Estuar. Coast. Shelf Sci. 55(4), 599–611 (2002).
[CrossRef]

Farber, I.

A. Grinvald, W. N. Ross, and I. Farber, “Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons,” Proc. Natl. Acad. Sci. U.S.A. 78(5), 3245–3249 (1981).
[CrossRef] [PubMed]

Grinvald, A.

A. Grinvald, W. N. Ross, and I. Farber, “Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons,” Proc. Natl. Acad. Sci. U.S.A. 78(5), 3245–3249 (1981).
[CrossRef] [PubMed]

Højerslev, N. K.

N. K. Højerslev and T. Aarup, “Optical measurements on the Louisiana Shelf off the Mississippi River,” Estuar. Coast. Shelf Sci. 55(4), 599–611 (2002).
[CrossRef]

N. K. Højerslev, “Bio-optical measurements in the Southwest Florida shelf ecosystem,” J. Cons. Cons. Int. Explor. Mer 42, 65–82 (1985).

Menzel, D. W.

C. S. Yentsch and D. W. Menzel, “A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence,” Deep-Sea Res. Oceanogr. Abstr. 10(3), 221–231 (1963).
[CrossRef]

Oishi, T.

Ross, W. N.

A. Grinvald, W. N. Ross, and I. Farber, “Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons,” Proc. Natl. Acad. Sci. U.S.A. 78(5), 3245–3249 (1981).
[CrossRef] [PubMed]

Yentsch, C. S.

C. S. Yentsch and D. W. Menzel, “A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence,” Deep-Sea Res. Oceanogr. Abstr. 10(3), 221–231 (1963).
[CrossRef]

Appl. Opt. (1)

Deep-Sea Res. Oceanogr. Abstr. (1)

C. S. Yentsch and D. W. Menzel, “A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence,” Deep-Sea Res. Oceanogr. Abstr. 10(3), 221–231 (1963).
[CrossRef]

Estuar. Coast. Shelf Sci. (1)

N. K. Højerslev and T. Aarup, “Optical measurements on the Louisiana Shelf off the Mississippi River,” Estuar. Coast. Shelf Sci. 55(4), 599–611 (2002).
[CrossRef]

J. Cons. Cons. Int. Explor. Mer (1)

N. K. Højerslev, “Bio-optical measurements in the Southwest Florida shelf ecosystem,” J. Cons. Cons. Int. Explor. Mer 42, 65–82 (1985).

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

A. Grinvald, W. N. Ross, and I. Farber, “Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons,” Proc. Natl. Acad. Sci. U.S.A. 78(5), 3245–3249 (1981).
[CrossRef] [PubMed]

Other (10)

G. C. Papageorgiou, “2.Fluorescence of photosynthetic pigments in vitro and in vivo,” in Chlorophyll a Fluorescence: A Signature of Photosynthesis, G. C. Papageorgiou and Govindjee, ed. (Springer, 2004).

M. Jonasz, and G. Fournier, Light Scattering by Particles in Water: Theoretical and Experimental Foundations (Elsevier, 2007).

P. G. Falkowski and J. A. Raven, Aquatic Photosynthesis (Blackwell Science, 1997)

BBE CO., Ltd., “Fluoro Probe,” http://www.bbe-moldaenke.de/chlorophyll/fluoroprobe/ .

JFE Advantech CO, Ltd., “Multi-Exciter,” http://www.jfe-alec.co.jp/html/multi-exciter-e.htm .

L. Zhou, S. Ye, H. Chen, J. Pan, and J. Yang, “The design of a low-power self-contained multi-EX-EM array deep sea in situ organism measurement device,” The 2nd International Conference on Bioinformatics and Biomedical Engineering, ICBBE 2008, 1196–1199 (2008).

C. A. Parker, Photoluminescence of Solutions (Elsevier, 1968).

T. Oishi, A. Tanaka, S. Yano, H. Ebata, Y. Takahashi, H. Kondo, H. Tan, and R. Doerffer, “Development of simultaneous multi-wavelength excitation fluorometer,” in Ocean Optics XIX, CD-ROM (2008).

N. G. Jarlov, Marine Optics (Elsevier, 1976)

C. V. I. Melles Griot, “Fundamental Optics Guide,” http://www.cvimellesgriot.com/products/Documents/TechnicalGuide/fundamental-Optics.pdf .

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Figures (7)

Fig. 1
Fig. 1

Schematic of the simultaneous illumination method (SIM).

Fig. 2
Fig. 2

Relationship between the transmittance obtained via a conventional method and that via the amplitudes derived using FFT.

Fig. 3
Fig. 3

Schematic of a multi-channel emission fluorometer using multiple excitation wavelengths.

Fig. 4
Fig. 4

Photograph of our fluorometer. The height of the fluorometer is about 50 cm. Two Si photodiodes are mounted at the sides of the cylindrical cell and face each other.

Fig. 5
Fig. 5

Spectral distributions of the LEDs used as the excitation light sources.

Fig. 6
Fig. 6

Data processing procedure. (a) FFT-analyzed signal at emission wavelengths of 500 nm, (b) 600 nm and (c) 675 nm. The arrowheads show the signal from elastic scattering. (d) Fluorescence excitation–emission contour map. The emission wavelengths are on the X axis and excitation wavelengths are on the Y axis.

Fig. 7
Fig. 7

Fluorescence excitation–emission contour maps for cultured phytoplankton measured using our two-channel emission fluorometer with multi-wavelength excitation based on the SIM. ((a) Synechococcus sp., (b) Porphyridium purpureum, (c) Chlorella kessleri, (d) Tetraselmis tetrathele, (e) Nannochloropsis oculata, (f) Chaetoceros ceratosporum and (g) Phaeodactylum tricornutum).

Tables (2)

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Table 1 Specifications of the Interference Filters

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Table 2 Specifications of the LEDs and their Corresponding Modulation Frequencies

Equations (4)

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P s ( λ ) = K T s ( λ ) I ( λ ) D ( λ ) ,
P r ( λ ) = K T r ( λ ) I ( λ ) D ( λ ) ,
P s ( λ ) P r ( λ ) = K T s ( λ ) I ( λ ) D ( λ ) K T r ( λ ) I ( λ ) D ( λ ) = T s ( λ ) T r ( λ ) .
P s ( λ ) P r ( λ ) = K T s ( λ ) I ( λ ) D ( λ ) K T r ( λ ) I ( λ ) D ( λ ) = T s ( λ ) T r ( λ )

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