Abstract

Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. We describe and demonstrate a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. A theoretical and computational analysis of the influence of noise on active illumination feedback is presented, along with imaging examples illustrating the benefits of AIM. While AIM is applicable to any type of scanning microscope, we apply it here specifically to two-photon microscopy.

© 2010 Optical Society of America

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References

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  1. J. B. Pawley, Handbook of biological confocal microscopy (Springer, New York, 2006).
  2. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluuorescence microscopy,” Science 248, 73–76 (1990).
  3. R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).
  4. K. K. Chu, D. Lim, and J. Mertz, “Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination,” Opt. Lett. 32, 2846–2848 (2007).
  5. G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).
  6. M. Steinbauer, A. G. Harris, C. Abels, and K. Messmer, “Charactierization and prevention of phototoxic effects intravital fluorescence microscopy in the hamster dorsal skinfold model,” Langenbeck’s Arch. Surg. 385, 290–298 (2000).
  7. I. Navarro-Quiroga, R. Chittajalu, V. Gallo, and T. F. Haydar, “Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation,” J. Neurosci. 27, 5007–5011 (2007).

2007 (3)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).

I. Navarro-Quiroga, R. Chittajalu, V. Gallo, and T. F. Haydar, “Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation,” J. Neurosci. 27, 5007–5011 (2007).

K. K. Chu, D. Lim, and J. Mertz, “Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination,” Opt. Lett. 32, 2846–2848 (2007).

2000 (2)

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).

M. Steinbauer, A. G. Harris, C. Abels, and K. Messmer, “Charactierization and prevention of phototoxic effects intravital fluorescence microscopy in the hamster dorsal skinfold model,” Langenbeck’s Arch. Surg. 385, 290–298 (2000).

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluuorescence microscopy,” Science 248, 73–76 (1990).

Abels, C.

M. Steinbauer, A. G. Harris, C. Abels, and K. Messmer, “Charactierization and prevention of phototoxic effects intravital fluorescence microscopy in the hamster dorsal skinfold model,” Langenbeck’s Arch. Surg. 385, 290–298 (2000).

Chittajalu, R.

I. Navarro-Quiroga, R. Chittajalu, V. Gallo, and T. F. Haydar, “Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation,” J. Neurosci. 27, 5007–5011 (2007).

Chu, K. K.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluuorescence microscopy,” Science 248, 73–76 (1990).

Dhonukshe, P. B.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).

Gadella, T. W. J.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).

Gallo, V.

I. Navarro-Quiroga, R. Chittajalu, V. Gallo, and T. F. Haydar, “Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation,” J. Neurosci. 27, 5007–5011 (2007).

Harris, A. G.

M. Steinbauer, A. G. Harris, C. Abels, and K. Messmer, “Charactierization and prevention of phototoxic effects intravital fluorescence microscopy in the hamster dorsal skinfold model,” Langenbeck’s Arch. Surg. 385, 290–298 (2000).

Haydar, T. F.

I. Navarro-Quiroga, R. Chittajalu, V. Gallo, and T. F. Haydar, “Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation,” J. Neurosci. 27, 5007–5011 (2007).

Hoebe, R. A.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).

Lim, D.

Manders, E. M. M.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).

Mertz, J.

Messmer, K.

M. Steinbauer, A. G. Harris, C. Abels, and K. Messmer, “Charactierization and prevention of phototoxic effects intravital fluorescence microscopy in the hamster dorsal skinfold model,” Langenbeck’s Arch. Surg. 385, 290–298 (2000).

Navarro-Quiroga, I.

I. Navarro-Quiroga, R. Chittajalu, V. Gallo, and T. F. Haydar, “Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation,” J. Neurosci. 27, 5007–5011 (2007).

Patterson, G. H.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).

Pawley, J. B.

J. B. Pawley, Handbook of biological confocal microscopy (Springer, New York, 2006).

Piston, D. W.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).

Steinbauer, M.

M. Steinbauer, A. G. Harris, C. Abels, and K. Messmer, “Charactierization and prevention of phototoxic effects intravital fluorescence microscopy in the hamster dorsal skinfold model,” Langenbeck’s Arch. Surg. 385, 290–298 (2000).

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluuorescence microscopy,” Science 248, 73–76 (1990).

Van Noorden, C. J. F.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).

Van Oven, C. H.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluuorescence microscopy,” Science 248, 73–76 (1990).

Biophys. J. (1)

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78, 2159–2162 (2000).

J. Neurosci. (1)

I. Navarro-Quiroga, R. Chittajalu, V. Gallo, and T. F. Haydar, “Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation,” J. Neurosci. 27, 5007–5011 (2007).

Langenbeck’s Arch. Surg. (1)

M. Steinbauer, A. G. Harris, C. Abels, and K. Messmer, “Charactierization and prevention of phototoxic effects intravital fluorescence microscopy in the hamster dorsal skinfold model,” Langenbeck’s Arch. Surg. 385, 290–298 (2000).

Nature Biotechnology (1)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nature Biotechnology 25, 249–253 (2007).

Opt. Lett. (1)

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluuorescence microscopy,” Science 248, 73–76 (1990).

Other (1)

J. B. Pawley, Handbook of biological confocal microscopy (Springer, New York, 2006).

Supplementary Material (1)

» Media 1: AVI (2344 KB)     

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Figures (6)

Fig. 1.
Fig. 1.

AIM layout for two-photon microscopy. Dashed line represents the integrated AIM instrument. Fluorescent output XP 2 from a sample X is detected by a photomultiplier tube (PMT), producing a signal S that is maintained at a set point Sset by analog feedback to an EOM that controls the illumination power P. X is reconstructed and output on a log scale.

Fig. 2.
Fig. 2.

Signal to noise ratio (SNR) vs. fluorescent sample strength (X) under varying AIM settings. SNR follows standard shot noise model in the power-limited regime but is capped to a constant maximum once the system reaches Sset .

Fig. 3.
Fig. 3.

Block diagram of feedback circuit components with noise introduced.

Fig. 4.
Fig. 4.

Image of mouse brain labeled with GFP (scale bar 20 μm). Conventional TPEF image, linear scale (a) and log scale (c). AIM image, linearized in software (b) and as acquired on log scale (d). Magnified insets of (c) and (d) shown respectively in (e) and (f). Video online of (a) to (d) showing a depth scan through 98 μm at a 2 μm spacing between slices.

Fig. 5.
Fig. 5.

Line profile of power spectral density of images from Fig. 4a (conventional image, blue trace) and 4b (AIM image, black trace). Power density shown on a log scale against kx on x-axis and ky = 0. The noise floor for AIM (black) is lower than for normal imaging (blue), reflecting improved SNR.

Fig. 6.
Fig. 6.

Image of GFP-labeled mouse neurons; conventional TPEF (top) cannot properly quantify what should be a bright neuron body indicated by arrow due to saturation. Saturation is avoided and the fluorescence of the neuron is properly captured in the linearized AIM image (bottom) without sacrificing SNR for dim objects. Arbitrary units of fluorescence are represented by a color look-up table (right) to highlight this effect; the bottom of the bar is zero fluorescence. Scale bar is 20 μm.

Equations (13)

Equations on this page are rendered with MathJax. Learn more.

Y = log a ( X ) = log a ( S ) 2 log a ( P )
P = H ( S ̂ S set ) = H ( ( GX ( P + δP ) 2 + δS S set )
GX ( P 2 + 2 PδP ) + δS S set = 0
P = S set δS GX δP
X ̂ = S ̂ G P 2 GX ( P 2 + 2 PδP ) + δS G P 2
X ̂ = X ( 1 + 2 δP P + δS GX P 2 )
X ̂ = X ( 1 + 2 δP GX S set + δS S set )
SNR AI = S set 2 σ P GXS set + σ S
SNR 0 = GXP 0 2 2 GXP 0 σ PL + σ S
SNR AI SNR 0 = S set GXP 0 2 ξ
ξ = 2 GXP 0 σ PL + σ S 2 ( σ PL + σ PM ) GXS set + σ S
DR 0 = S max σ S
DR AI = S set P max 2 σ S σ P 2 = DR 0 ( P max σ P ) 2 S set S max

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