Abstract

We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.

© 2007 Optical Society of America

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References

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  1. J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).
    [CrossRef]
  2. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  3. E. Meyer, H. J. Hug, and R. Bennewitz, Scanning Probe Microscopy: the Lab on a Tip (Springer, 2003).
  4. R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, Nat. Biotechnol. 25, 249 (2007).
    [CrossRef] [PubMed]
  5. G. H. Patterson and D. W. Piston, Biophys. J. 78, 2159 (2000).
    [CrossRef] [PubMed]

2007 (1)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[CrossRef] [PubMed]

2000 (1)

G. H. Patterson and D. W. Piston, Biophys. J. 78, 2159 (2000).
[CrossRef] [PubMed]

1990 (1)

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

Biophys. J. (1)

G. H. Patterson and D. W. Piston, Biophys. J. 78, 2159 (2000).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[CrossRef] [PubMed]

Science (1)

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

Other (2)

E. Meyer, H. J. Hug, and R. Bennewitz, Scanning Probe Microscopy: the Lab on a Tip (Springer, 2003).

J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

AI microscope layout. Signal power S from a sample X is detected, producing a voltage V out that is maintained at a set point V set by analog feedback to an EOM that controls the illumination power P.

Fig. 2
Fig. 2

Relative sensitivity dependence on fluorophore concentration (lower is better). The dashed curve represents conventional TPEF microscopy; the discontinuity at the right indicates saturation. The solid curve denotes AI-TPEF microscopy; the discontinuity towards the left occurs when the illumination power reaches maximum and the system crosses into the PL regime. P max > P 0 in this plot.

Fig. 3
Fig. 3

AI-TPEF image reconstruction of prepared bovine pulmonary artery endothelial (BPAE) cells, actin labeled with Texas Red-X phalloidin. Scale bar 20 μ m . a, Image of S (12-bit), held by the feedback circuit to a set point. b, Image of P (12-bit), output of the feedback circuit to the EOM, corresponding to the illumination power used. c, Reconstructed X image (float). d, Conventional TPEF image (12 bit). e and f, Enlargements of the insets from c and d, respectively, both with applied gamma correction of 0.65.

Fig. 4
Fig. 4

Logarithmic gray-scale map of Invitrogen Fluocells 2 fibroblast images. Scale bar 20 μ m . a, Conventional TPEF image (12-bit). b, AI-TPEF reconstructed image (float). Images a and b are normalized to the same maximum pixel value. c, Histograms of images a (black) and b (green or gray) on a log y-axis. Inset, zoomed-in portion of the histogram with a linear y-axis.

Equations (3)

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X = α S P 2 .
Σ F A = 1 X X P = 2 X α S set ,
Σ P L = 1 X X S = α P max 2 X .

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