Abstract

We present the use and characterization of a photon-counting detector for increased sensitivity at low signal levels in fluorescence laser scanning microscopy (LSM). Conventional LSM photomultiplier tube detectors utilize analog current integration and thus suffer from excessive noise at low signal levels, introduced during current measurement. In this Letter we describe the implementation of a fast single-photon-counting (SPC) detector on a conventional two-photon laser scanning microscope and detail its use in imaging low fluorescence intensities. We show that for a low photon flux, the SPC detector is shot-noise limited and thus provides increased detection sensitivity compared with analog current integration.

© 2008 Optical Society of America

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References

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  1. J. B. Pawley, ed., Handbook of Biological Confocal Microscopy (Plenum Press, 1995).
  2. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  3. R. K. P. Benninger, M. Hao, and D. W. Piston, Rev. Physiol. Biochem. Pharmacol. 160, 71 (2008).
    [CrossRef] [PubMed]
  4. B. D. Bennett, T. L. Jetton, G. T. Ying, M. A. Magnuson, and D. W. Piston, J. Biol. Chem. 271, 3647 (1996).
    [CrossRef] [PubMed]
  5. B. Chance and H. Baltscheffsky, J. Biol. Chem. 233, 736 (1958).
    [PubMed]
  6. D. W. Piston and S. M. Knobel, Methods Enzymol. 307, 351 (1999).
    [CrossRef] [PubMed]
  7. G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, Proc. Natl. Acad. Sci. U.S.A. 97, 5203 (2000).
    [CrossRef] [PubMed]
  8. C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, J. Fluoresc. 15, 41 (2005).
    [CrossRef] [PubMed]

2008 (1)

R. K. P. Benninger, M. Hao, and D. W. Piston, Rev. Physiol. Biochem. Pharmacol. 160, 71 (2008).
[CrossRef] [PubMed]

2005 (1)

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, J. Fluoresc. 15, 41 (2005).
[CrossRef] [PubMed]

2000 (1)

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, Proc. Natl. Acad. Sci. U.S.A. 97, 5203 (2000).
[CrossRef] [PubMed]

1999 (1)

D. W. Piston and S. M. Knobel, Methods Enzymol. 307, 351 (1999).
[CrossRef] [PubMed]

1996 (1)

B. D. Bennett, T. L. Jetton, G. T. Ying, M. A. Magnuson, and D. W. Piston, J. Biol. Chem. 271, 3647 (1996).
[CrossRef] [PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1958 (1)

B. Chance and H. Baltscheffsky, J. Biol. Chem. 233, 736 (1958).
[PubMed]

Arkhammar, P.

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, Proc. Natl. Acad. Sci. U.S.A. 97, 5203 (2000).
[CrossRef] [PubMed]

Baltscheffsky, H.

B. Chance and H. Baltscheffsky, J. Biol. Chem. 233, 736 (1958).
[PubMed]

Bennett, B. D.

B. D. Bennett, T. L. Jetton, G. T. Ying, M. A. Magnuson, and D. W. Piston, J. Biol. Chem. 271, 3647 (1996).
[CrossRef] [PubMed]

Benninger, R. K. P.

R. K. P. Benninger, M. Hao, and D. W. Piston, Rev. Physiol. Biochem. Pharmacol. 160, 71 (2008).
[CrossRef] [PubMed]

Buehler, C.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, J. Fluoresc. 15, 41 (2005).
[CrossRef] [PubMed]

Chance, B.

B. Chance and H. Baltscheffsky, J. Biol. Chem. 233, 736 (1958).
[PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Greuter, U.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, J. Fluoresc. 15, 41 (2005).
[CrossRef] [PubMed]

Hao, M.

R. K. P. Benninger, M. Hao, and D. W. Piston, Rev. Physiol. Biochem. Pharmacol. 160, 71 (2008).
[CrossRef] [PubMed]

Jetton, T. L.

B. D. Bennett, T. L. Jetton, G. T. Ying, M. A. Magnuson, and D. W. Piston, J. Biol. Chem. 271, 3647 (1996).
[CrossRef] [PubMed]

Kim, K. H.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, J. Fluoresc. 15, 41 (2005).
[CrossRef] [PubMed]

Knobel, S. M.

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, Proc. Natl. Acad. Sci. U.S.A. 97, 5203 (2000).
[CrossRef] [PubMed]

D. W. Piston and S. M. Knobel, Methods Enzymol. 307, 351 (1999).
[CrossRef] [PubMed]

Magnuson, M. A.

B. D. Bennett, T. L. Jetton, G. T. Ying, M. A. Magnuson, and D. W. Piston, J. Biol. Chem. 271, 3647 (1996).
[CrossRef] [PubMed]

Patterson, G. H.

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, Proc. Natl. Acad. Sci. U.S.A. 97, 5203 (2000).
[CrossRef] [PubMed]

Pawley, J. B.

J. B. Pawley, ed., Handbook of Biological Confocal Microscopy (Plenum Press, 1995).

Piston, D. W.

R. K. P. Benninger, M. Hao, and D. W. Piston, Rev. Physiol. Biochem. Pharmacol. 160, 71 (2008).
[CrossRef] [PubMed]

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, Proc. Natl. Acad. Sci. U.S.A. 97, 5203 (2000).
[CrossRef] [PubMed]

D. W. Piston and S. M. Knobel, Methods Enzymol. 307, 351 (1999).
[CrossRef] [PubMed]

B. D. Bennett, T. L. Jetton, G. T. Ying, M. A. Magnuson, and D. W. Piston, J. Biol. Chem. 271, 3647 (1996).
[CrossRef] [PubMed]

Schlumpf, N.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, J. Fluoresc. 15, 41 (2005).
[CrossRef] [PubMed]

So, P. T. C.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, J. Fluoresc. 15, 41 (2005).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Thastrup, O.

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, Proc. Natl. Acad. Sci. U.S.A. 97, 5203 (2000).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Ying, G. T.

B. D. Bennett, T. L. Jetton, G. T. Ying, M. A. Magnuson, and D. W. Piston, J. Biol. Chem. 271, 3647 (1996).
[CrossRef] [PubMed]

J. Biol. Chem. (2)

B. D. Bennett, T. L. Jetton, G. T. Ying, M. A. Magnuson, and D. W. Piston, J. Biol. Chem. 271, 3647 (1996).
[CrossRef] [PubMed]

B. Chance and H. Baltscheffsky, J. Biol. Chem. 233, 736 (1958).
[PubMed]

J. Fluoresc. (1)

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, J. Fluoresc. 15, 41 (2005).
[CrossRef] [PubMed]

Methods Enzymol. (1)

D. W. Piston and S. M. Knobel, Methods Enzymol. 307, 351 (1999).
[CrossRef] [PubMed]

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

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, Proc. Natl. Acad. Sci. U.S.A. 97, 5203 (2000).
[CrossRef] [PubMed]

Rev. Physiol. Biochem. Pharmacol. (1)

R. K. P. Benninger, M. Hao, and D. W. Piston, Rev. Physiol. Biochem. Pharmacol. 160, 71 (2008).
[CrossRef] [PubMed]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Other (1)

J. B. Pawley, ed., Handbook of Biological Confocal Microscopy (Plenum Press, 1995).

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Figures (3)

Fig. 1
Fig. 1

Schematic of the SPC detector system. The photon-counting head (PCH) relays the single-photon event TTL pulse via the pulse delay generator (PDG) to the field programmable gate array (FPGA). Line synchronization (sync) and pixel clock signals from the confocal scan head are also sent to the FPGA. On the FPGA, after every pixel clock tick, the photon event pulses are counted until the following pixel clock tick. This count ( Σ ) is then input into the preallocated memory array, at a position defined by the line sync and pixel clock signal (i, j).

Fig. 2
Fig. 2

Gray-scale images of islet NAD(P)H detected using (a) the conventional NDD detector and (b) the SPC detector. The NAD(P)H distribution within the islet can be resolved using both forms of detection, yielding approximately equivalent image quality. Scale bar in (a) represents 50 μ m . Note, color scale in (a) is to an 8 - bit DN (scaled between 0 and 180), whereas in (b) it is to the number of photons collected (ph’tn, scaled between 0 and 50).

Fig. 3
Fig. 3

(a) Comparison of the S/N ratio obtained using the SPC detector (triangles) and conventional NDD (circles). Using equivalent settings to Fig. 2, for low concentrations of free NADH the SPC detector gives a superior S/N ratio. At higher concentrations > 400 μ M ( 13   photons pixel ) , the S/N ratio obtained using the NDD exceeds that obtained using the SPC detector. (b) The noise on the signal from the SPC detector, when varying the excitation power and concentrations of NADH (triangles), is at the shot-noise limit (dotted line).

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