Abstract

Excitation laser spatial and temporal characteristics at the objective focal point are critical to the performance of two-photon scanning microscopes. Optical aberrations in scanning systems increase the microscope objective focal spot area and introduce pulse time broadening in the deflected beam, resulting in degradation of two-photon-induced fluorescence across the scan field. The geometrical pulse broadening is investigated for what is believed to be the first time and then combined with a focused spot area to provide a normalized two-photon fluorescence intensity correction factor. This factor, calculated using OSLO optical software, is compared for four reflective scan engines and allows compensation of the detected signal with position across the scan field. This new metric highlights that a parabolic mirror afocal relay exhibits superior performance as a reflective scan engine for two-photon scanning microscopy.

© 2010 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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2009 (2)

2008 (2)

B. Furlong and Sh. Motakef, “Scanning lenses and systems,” Photonik Int. 3(2), 20-23 (2008).

A. T. Yeh, H. Gibbs, J.-J. Hu, and A. M. Larson, “Advances in nonlinear optical microscopy for visualizing dynamic tissue properties in culture,” Tissue Eng. Part B 14, 119-131(2008).
[CrossRef]

2006 (1)

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50, 823-839 (2006).
[CrossRef] [PubMed]

2003 (1)

W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95, 335-342 (2003).
[CrossRef] [PubMed]

2002 (1)

M. D. Cahalan, I. Parker, S. H. Wei, and M. J. Miller, “Two-photon tissue imaging: seeing the immune system in a fresh light,” Nat. Rev. Immun. 2, 872-880 (2002).
[CrossRef]

1996 (1)

C. Soeller and M. B. Cannell, “Construction of a two-photon microscope and optimisation of illumination pulse duration,” Pflügers Arch. Eur. J. Physiol. 432, 555-561 (1996).
[CrossRef]

1995 (1)

D. W. Piston, B. D. Bennett, and G. Ying, “Imaging of cellular dynamics by two-photon excitation microscopy,” Microsc. Microanal. 1, 25-34 (1995).
[CrossRef]

1984 (1)

Achtner, B.

G. Gross, F. Blechinger, and B. Achtner, Handbook of Optical Systems: Survey of Optical Instruments (Wiley-VCH, 2008), Vol. 4.

Amos, W. B.

W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95, 335-342 (2003).
[CrossRef] [PubMed]

W. B. Amos, “Achromatic scanning system,” U.S. patent 4,997,242 (5 March 1991).

Bennett, B. D.

D. W. Piston, B. D. Bennett, and G. Ying, “Imaging of cellular dynamics by two-photon excitation microscopy,” Microsc. Microanal. 1, 25-34 (1995).
[CrossRef]

Blechinger, F.

G. Gross, F. Blechinger, and B. Achtner, Handbook of Optical Systems: Survey of Optical Instruments (Wiley-VCH, 2008), Vol. 4.

Brown, D.

D. Brown, “Rapid scanning applications drive mirror design,” Laser Focus World 45(3), 45-50 (2009).

Cahalan, M. D.

M. D. Cahalan, I. Parker, S. H. Wei, and M. J. Miller, “Two-photon tissue imaging: seeing the immune system in a fresh light,” Nat. Rev. Immun. 2, 872-880 (2002).
[CrossRef]

Cannell, M. B.

C. Soeller and M. B. Cannell, “Construction of a two-photon microscope and optimisation of illumination pulse duration,” Pflügers Arch. Eur. J. Physiol. 432, 555-561 (1996).
[CrossRef]

Cox, G.

G. Cox, Optical Imaging Techniquues in Cell Biology (CRS Press, 2007), p. 268.

Davidson, M. W.

K. R. Spring, T. J. Fellers, and M. W. Davidson, “Confocal microscope scanning systems,” (Olympus Corporation, 2009), retrieved 26 August 2009, http://www.olympusconfocal.com/theory/confocalscanningsystems.html.

Denk, W.

W. Denk, J. P. Strickler, and W. W. Webb, “Two-photon laser microscopy,” U.S. patent 5,034,613 (23 July 1991).

Diaspro, A.

A. Diaspro and C. Sheppard, “Two-photon excitation fluorescence microscopy,” in Confocal and Two-Photon Microscopy: Foundations, Applications and Advances, 1st ed., A. Diaspro, ed. (Wiley-Liss, 2002), p. 567.

Diels, J.-C.

J.-C. Diels and W. Rudolph, “Ultrashort laser pulse phenomena: fundamentals, techniques, and applications on a femtosecond time scale,” in Optics and Photonics (Academic, 1996), p. 581.

Drexler, W.

G. Tempeat, T. Le, M. Hofer, A. Stingl, and W. Drexler, “Dispersion management for microscope objectives,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2005), paper CThBB6

Fellers, T. J.

K. R. Spring, T. J. Fellers, and M. W. Davidson, “Confocal microscope scanning systems,” (Olympus Corporation, 2009), retrieved 26 August 2009, http://www.olympusconfocal.com/theory/confocalscanningsystems.html.

Fork, R. L.

Furlong, B.

B. Furlong and Sh. Motakef, “Scanning lenses and systems,” Photonik Int. 3(2), 20-23 (2008).

Gibbs, H.

A. T. Yeh, H. Gibbs, J.-J. Hu, and A. M. Larson, “Advances in nonlinear optical microscopy for visualizing dynamic tissue properties in culture,” Tissue Eng. Part B 14, 119-131(2008).
[CrossRef]

Gordon, J. P.

Gross, G.

G. Gross, F. Blechinger, and B. Achtner, Handbook of Optical Systems: Survey of Optical Instruments (Wiley-VCH, 2008), Vol. 4.

Hofer, M.

G. Tempeat, T. Le, M. Hofer, A. Stingl, and W. Drexler, “Dispersion management for microscope objectives,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2005), paper CThBB6

Holdsworth, J.

Hu, J.-J.

A. T. Yeh, H. Gibbs, J.-J. Hu, and A. M. Larson, “Advances in nonlinear optical microscopy for visualizing dynamic tissue properties in culture,” Tissue Eng. Part B 14, 119-131(2008).
[CrossRef]

Laikin, M.

M. Laikin, “Optical science and engineering,” Lens Design, 4th ed. (CRC Press, 2007), p. 479.

Larson, A. M.

A. T. Yeh, H. Gibbs, J.-J. Hu, and A. M. Larson, “Advances in nonlinear optical microscopy for visualizing dynamic tissue properties in culture,” Tissue Eng. Part B 14, 119-131(2008).
[CrossRef]

Le, T.

G. Tempeat, T. Le, M. Hofer, A. Stingl, and W. Drexler, “Dispersion management for microscope objectives,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2005), paper CThBB6

Martinez, O. E.

Miller, M. J.

M. D. Cahalan, I. Parker, S. H. Wei, and M. J. Miller, “Two-photon tissue imaging: seeing the immune system in a fresh light,” Nat. Rev. Immun. 2, 872-880 (2002).
[CrossRef]

Montagu, J.

J. Montagu, “Galvanometric and resonant scanners,” in Handbook of Optical and Laser Scanning, G. F. Marshall, ed. (Marcell Dekker, 2004), pp. 417-476.
[CrossRef]

Motakef, Sh.

B. Furlong and Sh. Motakef, “Scanning lenses and systems,” Photonik Int. 3(2), 20-23 (2008).

Parker, I.

M. D. Cahalan, I. Parker, S. H. Wei, and M. J. Miller, “Two-photon tissue imaging: seeing the immune system in a fresh light,” Nat. Rev. Immun. 2, 872-880 (2002).
[CrossRef]

Pawley, J. B.

J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, 1995), p. 632.

Piston, W.

D. W. Piston, B. D. Bennett, and G. Ying, “Imaging of cellular dynamics by two-photon excitation microscopy,” Microsc. Microanal. 1, 25-34 (1995).
[CrossRef]

Rudolph, W.

J.-C. Diels and W. Rudolph, “Ultrashort laser pulse phenomena: fundamentals, techniques, and applications on a femtosecond time scale,” in Optics and Photonics (Academic, 1996), p. 581.

Sagan, S. F.

S. F. Sagan, “Optical systems for laser scanners,” in Handbook of Optical and Laser Scanning, G. F. Marshall, ed. (Marcell Dekker, 2004), pp. 71-137.
[CrossRef]

Sharafutdinova, G.

Sheppard, C.

A. Diaspro and C. Sheppard, “Two-photon excitation fluorescence microscopy,” in Confocal and Two-Photon Microscopy: Foundations, Applications and Advances, 1st ed., A. Diaspro, ed. (Wiley-Liss, 2002), p. 567.

So, P. T. C.

P. T. C. So, Two-Photon Fluorescence Light Microscopy (Wiley, 2002).

Soeller, C.

C. Soeller and M. B. Cannell, “Construction of a two-photon microscope and optimisation of illumination pulse duration,” Pflügers Arch. Eur. J. Physiol. 432, 555-561 (1996).
[CrossRef]

Spring, K. R.

K. R. Spring, T. J. Fellers, and M. W. Davidson, “Confocal microscope scanning systems,” (Olympus Corporation, 2009), retrieved 26 August 2009, http://www.olympusconfocal.com/theory/confocalscanningsystems.html.

Stingl, A.

G. Tempeat, T. Le, M. Hofer, A. Stingl, and W. Drexler, “Dispersion management for microscope objectives,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2005), paper CThBB6

Strickler, J. P.

W. Denk, J. P. Strickler, and W. W. Webb, “Two-photon laser microscopy,” U.S. patent 5,034,613 (23 July 1991).

Svoboda, K.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50, 823-839 (2006).
[CrossRef] [PubMed]

Tempeat, G.

G. Tempeat, T. Le, M. Hofer, A. Stingl, and W. Drexler, “Dispersion management for microscope objectives,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2005), paper CThBB6

van Helden, D.

Webb, W. W.

W. Denk, J. P. Strickler, and W. W. Webb, “Two-photon laser microscopy,” U.S. patent 5,034,613 (23 July 1991).

Wei, S. H.

M. D. Cahalan, I. Parker, S. H. Wei, and M. J. Miller, “Two-photon tissue imaging: seeing the immune system in a fresh light,” Nat. Rev. Immun. 2, 872-880 (2002).
[CrossRef]

White, J. G.

W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95, 335-342 (2003).
[CrossRef] [PubMed]

Yasuda, R.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50, 823-839 (2006).
[CrossRef] [PubMed]

Yeh, A. T.

A. T. Yeh, H. Gibbs, J.-J. Hu, and A. M. Larson, “Advances in nonlinear optical microscopy for visualizing dynamic tissue properties in culture,” Tissue Eng. Part B 14, 119-131(2008).
[CrossRef]

Ying, G.

D. W. Piston, B. D. Bennett, and G. Ying, “Imaging of cellular dynamics by two-photon excitation microscopy,” Microsc. Microanal. 1, 25-34 (1995).
[CrossRef]

Appl. Opt. (1)

Biol. Cell (1)

W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95, 335-342 (2003).
[CrossRef] [PubMed]

Laser Focus World (1)

D. Brown, “Rapid scanning applications drive mirror design,” Laser Focus World 45(3), 45-50 (2009).

Microsc. Microanal. (1)

D. W. Piston, B. D. Bennett, and G. Ying, “Imaging of cellular dynamics by two-photon excitation microscopy,” Microsc. Microanal. 1, 25-34 (1995).
[CrossRef]

Nat. Rev. Immun. (1)

M. D. Cahalan, I. Parker, S. H. Wei, and M. J. Miller, “Two-photon tissue imaging: seeing the immune system in a fresh light,” Nat. Rev. Immun. 2, 872-880 (2002).
[CrossRef]

Neuron (1)

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50, 823-839 (2006).
[CrossRef] [PubMed]

Opt. Lett. (1)

Pflügers Arch. Eur. J. Physiol. (1)

C. Soeller and M. B. Cannell, “Construction of a two-photon microscope and optimisation of illumination pulse duration,” Pflügers Arch. Eur. J. Physiol. 432, 555-561 (1996).
[CrossRef]

Photonik Int. (1)

B. Furlong and Sh. Motakef, “Scanning lenses and systems,” Photonik Int. 3(2), 20-23 (2008).

Tissue Eng. Part B (1)

A. T. Yeh, H. Gibbs, J.-J. Hu, and A. M. Larson, “Advances in nonlinear optical microscopy for visualizing dynamic tissue properties in culture,” Tissue Eng. Part B 14, 119-131(2008).
[CrossRef]

Other (13)

W. Denk, J. P. Strickler, and W. W. Webb, “Two-photon laser microscopy,” U.S. patent 5,034,613 (23 July 1991).

P. T. C. So, Two-Photon Fluorescence Light Microscopy (Wiley, 2002).

A. Diaspro and C. Sheppard, “Two-photon excitation fluorescence microscopy,” in Confocal and Two-Photon Microscopy: Foundations, Applications and Advances, 1st ed., A. Diaspro, ed. (Wiley-Liss, 2002), p. 567.

J.-C. Diels and W. Rudolph, “Ultrashort laser pulse phenomena: fundamentals, techniques, and applications on a femtosecond time scale,” in Optics and Photonics (Academic, 1996), p. 581.

M. Laikin, “Optical science and engineering,” Lens Design, 4th ed. (CRC Press, 2007), p. 479.

S. F. Sagan, “Optical systems for laser scanners,” in Handbook of Optical and Laser Scanning, G. F. Marshall, ed. (Marcell Dekker, 2004), pp. 71-137.
[CrossRef]

G. Gross, F. Blechinger, and B. Achtner, Handbook of Optical Systems: Survey of Optical Instruments (Wiley-VCH, 2008), Vol. 4.

K. R. Spring, T. J. Fellers, and M. W. Davidson, “Confocal microscope scanning systems,” (Olympus Corporation, 2009), retrieved 26 August 2009, http://www.olympusconfocal.com/theory/confocalscanningsystems.html.

J. Montagu, “Galvanometric and resonant scanners,” in Handbook of Optical and Laser Scanning, G. F. Marshall, ed. (Marcell Dekker, 2004), pp. 417-476.
[CrossRef]

G. Tempeat, T. Le, M. Hofer, A. Stingl, and W. Drexler, “Dispersion management for microscope objectives,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2005), paper CThBB6

J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, 1995), p. 632.

G. Cox, Optical Imaging Techniquues in Cell Biology (CRS Press, 2007), p. 268.

W. B. Amos, “Achromatic scanning system,” U.S. patent 4,997,242 (5 March 1991).

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Figures (11)

Fig. 1
Fig. 1

(a) Incident angle dependence of focusing lens optical performance. (b) Four spot diagrams and (c) four intensity PSFs represent incident beam angles at the lens of (1) 0 ° , (2) 10 ° in the horizontal direction, (3) 10 ° in the vertical direction, and (4) 10 ° simultaneously in both directions relative to the optical axis. The scale on the spot diagrams is different, and relative size may be gauged by the black Airy disk circle on the spot diagrams.

Fig. 2
Fig. 2

Four simulated scanning designs: (a) single mirror engine used as the reference design RS, (b) close coupled engine CCE, (c) spherical reflector engine SSE, and (d) parabolic reflector engine PSE. (e) Scan pattern points analyzed.

Fig. 3
Fig. 3

(a) PSE optical relay. (b) and (d) Circular arc traced on surfaces 4 and 6, respectively. (c) Scan angle dependent parallel ray paths between the first and second scan mirrors.

Fig. 4
Fig. 4

Scan engine position in a typical two-photon scanning microscope arrangement. The scan lens used in this work was an optimized scan lens design from the OSLO database as shown in the expanded detail.

Fig. 5
Fig. 5

Center of mass points for 25 configurations of the four scan engines. The RS system scan pattern is symmetrical and shows symmetrical pincushion distortion. The PSE engine creates straight scan lines with pincushion distortion in both sides, similar to the RS system. The CCE system produced elongated scan lines with pincushion distortions on the sides. The SSE created scan lines have different curvatures, which increase down the scan area. The created scan lines are straight only for the PSE system.

Fig. 6
Fig. 6

(a) Spot diagrams and (b) intensity PSFs for four scan engines in 25 scan configurations. Scan angle range in both directions is from 10 ° to + 10 ° . (c) and (d) PSF maximum values as contour and 2D maps across the whole scanning field at a resolution of 0.1 ° . The color scale bar is common for (d) and (c) of each column.

Fig. 7
Fig. 7

Wavefront aberrations for four scan engines. (a) Wavefront contours, (b) ln (PV-OPD), (c) scaled 2-D maps of central ( ± 5 ° ) scan field.

Fig. 8
Fig. 8

Spot areas at the intermediate image plane for (a) RS, (b) PSE, (c) CCE, and (d) SSE systems.

Fig. 9
Fig. 9

Time delay introduced by scan engines for (a) RS, (b) PSE, (c) CCE, and (d) SSE.

Fig. 10
Fig. 10

Relative fluorescence intensity factor for (a) PSE, (b) CCE, and (c) SSE across the investigated area.

Fig. 11
Fig. 11

Calculating optical path difference between peripheral rays for any scan point.

Tables (1)

Tables Icon

Table 1 Minimal and Maximum Relative Fluorescence Intensity Factors for PSE, CCE, and SSE Compared to the RS Range of Values for the Central Section of the Investigation Area

Equations (3)

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

I fl ( t ) = δ 2 P ave 2 τ p f p [ π NA 2 h c λ ] 2 ,
I fl 1 Area 2 τ p = F ,
OPD = B C = A C * sin a .

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