Abstract

In-phase focal modulation microscopy (IPFMM) with single photon excited fluorescence is presented. Optical transfer functions and images of thin and thick fluorescent edges in IPFMM are investigated. The results show that, compared with confocal microscopy, using IPFMM can result in a sharper image of the edge, and the edge gradient can be increased up to 75.4% and 58.9% for a thick edge and a thin edge, respectively. Signal level is also discussed, and the results show that, to obtain high transverse resolution with IPFMM, the normalized detector pinhole radius should not exceed 2.8.

© 2009 Optical Society of America

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  1. C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
    [CrossRef] [PubMed]
  2. J. P. Yuan, W. Zeng, M. R. Dorwart, Y.-J. Choi, P. F. Worley, and S. Muallem, “SOAR and the polybasic STIM1 domains gate and regulate Orai channels,” Nature Cell Biol. 11, 337-343(2009).
    [CrossRef] [PubMed]
  3. X. Deng and M. Gu, “Penetration depth of single-, two-, and three-photon fluorescence microscopic imaging through human cortex structures: Monte Carlo simulation,” Appl. Opt. 42, 3321-3329 (2003).
    [CrossRef] [PubMed]
  4. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28, 1022-1024 (2003).
    [CrossRef] [PubMed]
  5. J. G. Fujimoto, “Optical coherence tomography for ultrahigh resolution in vivo imaging,” Nat. Biotechnol. 21, 1361-1367(2003).
    [CrossRef] [PubMed]
  6. K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99, 228105(2007).
    [CrossRef]
  7. M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13, 050507 (2008).
    [CrossRef] [PubMed]
  8. N. G. Chen, C. H. Wong, and C. J. R. Sheppard, “Focal modulation microscopy,” Opt. Express 16, 18764-18769 (2008).
    [CrossRef]
  9. C. J. R. Sheppard, “Edge-setting criterion in confocal microscopy,” Appl. Opt. 31, 4575-4577 (1992).
    [CrossRef] [PubMed]
  10. C. J. Koester, “Comparison of optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Confocal Microscopy, J. Pawley, ed. (Plenum, 1990).
  11. C. J. R. Sheppard, W. Gong, and K. Si, “The divided aperture technique for microscopy through scattering media,” Opt. Express 16, 17031-17038 (2008).
    [CrossRef] [PubMed]
  12. K. Si, W. Gong, and C. J. R. Sheppard, “Three-dimensional coherent transfer function for a confocal microscope with two D-shaped pupils,” Appl. Opt. 48, 810-817 (2009).
    [CrossRef] [PubMed]
  13. C. H. Wong, S. P. Chong, C. J. R. Sheppard, and N. Chen, “Simple spatial phase modulator for focal modulation microscopy,” Appl. Opt. 48, 3237-3242 (2009).
    [CrossRef] [PubMed]
  14. M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, 1996), p. 141.
  15. W. Gong, K. Si, and C. J. R. Sheppard, “Improved spatial resolution in fluorescence focal modulation microscopy,” Opt. Lett. 34 (2009), to be published.
    [PubMed]

2009

2008

2007

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99, 228105(2007).
[CrossRef]

2003

1992

Chang, K. C.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Chen, N.

Chen, N. G.

Choi, Y.-J.

J. P. Yuan, W. Zeng, M. R. Dorwart, Y.-J. Choi, P. F. Worley, and S. Muallem, “SOAR and the polybasic STIM1 domains gate and regulate Orai channels,” Nature Cell Biol. 11, 337-343(2009).
[CrossRef] [PubMed]

Chong, S. P.

Deng, X.

Denk, W.

Fujimoto, J. G.

J. G. Fujimoto, “Optical coherence tomography for ultrahigh resolution in vivo imaging,” Nat. Biotechnol. 21, 1361-1367(2003).
[CrossRef] [PubMed]

Fujita, K.

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99, 228105(2007).
[CrossRef]

Gong, W.

Gu, M.

Hasan, M. T.

Hotchkiss, R. S.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Kawano, S.

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99, 228105(2007).
[CrossRef]

Kawata, S.

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99, 228105(2007).
[CrossRef]

Kobayashi, M.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99, 228105(2007).
[CrossRef]

Koester, C. J.

C. J. Koester, “Comparison of optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Confocal Microscopy, J. Pawley, ed. (Plenum, 1990).

Kung, H. F.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Mach, R. H.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Muallem, S.

J. P. Yuan, W. Zeng, M. R. Dorwart, Y.-J. Choi, P. F. Worley, and S. Muallem, “SOAR and the polybasic STIM1 domains gate and regulate Orai channels,” Nature Cell Biol. 11, 337-343(2009).
[CrossRef] [PubMed]

Shen, D.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Sheppard, C. J. R.

Si, K.

Smith, N. I.

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

Theer, P.

Vangveravong, S.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Wheeler, K. T.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Wong, C. H.

Xu, J.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Yamanaka, M.

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99, 228105(2007).
[CrossRef]

Yuan, J. P.

J. P. Yuan, W. Zeng, M. R. Dorwart, Y.-J. Choi, P. F. Worley, and S. Muallem, “SOAR and the polybasic STIM1 domains gate and regulate Orai channels,” Nature Cell Biol. 11, 337-343(2009).
[CrossRef] [PubMed]

Zeng, C.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Zeng, W.

J. P. Yuan, W. Zeng, M. R. Dorwart, Y.-J. Choi, P. F. Worley, and S. Muallem, “SOAR and the polybasic STIM1 domains gate and regulate Orai channels,” Nature Cell Biol. 11, 337-343(2009).
[CrossRef] [PubMed]

Zhuang, Z.-P.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

Appl. Opt.

Cancer Res.

C. Zeng, S. Vangveravong, J. Xu, K. C. Chang, R. S. Hotchkiss, K. T. Wheeler, D. Shen, Z.-P. Zhuang, H. F. Kung, and R. H. Mach, “Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy,” Cancer Res. 67, 6708-6716 (2007).
[CrossRef] [PubMed]

J. Biomed. Opt.

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13, 050507 (2008).
[CrossRef] [PubMed]

Nat. Biotechnol.

J. G. Fujimoto, “Optical coherence tomography for ultrahigh resolution in vivo imaging,” Nat. Biotechnol. 21, 1361-1367(2003).
[CrossRef] [PubMed]

Nature Cell Biol.

J. P. Yuan, W. Zeng, M. R. Dorwart, Y.-J. Choi, P. F. Worley, and S. Muallem, “SOAR and the polybasic STIM1 domains gate and regulate Orai channels,” Nature Cell Biol. 11, 337-343(2009).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

W. Gong, K. Si, and C. J. R. Sheppard, “Improved spatial resolution in fluorescence focal modulation microscopy,” Opt. Lett. 34 (2009), to be published.
[PubMed]

P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28, 1022-1024 (2003).
[CrossRef] [PubMed]

Phys. Rev. Lett.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99, 228105(2007).
[CrossRef]

Other

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, 1996), p. 141.

C. J. Koester, “Comparison of optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Confocal Microscopy, J. Pawley, ed. (Plenum, 1990).

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Figures (7)

Fig. 1
Fig. 1

Schematic diagram of FMM: BE, beam expander; SPM, spatial phase modulator; DM, dichroic mirror; FSM, fast steering mirror; L 1 , objective lens; LPF, long pass filter; L 2 , achromat; PMT, photomultiplier.

Fig. 2
Fig. 2

Three-dimensional optical transfer functions (a) of confocal microscope, v d = 0 ; (b)  C ( m , n = 0 , s ) of IPFMM, v d = 0 ; (c)  C ( m = 0 , n , s ) of IPFMM, v d = 0 ; (d) of confocal microscope, v d = 4 ; (e)  C ( m , n = 0 , s ) of IPFMM, v d = 4 ; (f)  C ( m = 0 , n , s ) of IPFMM, v d = 4 .

Fig. 3
Fig. 3

One-photon fluorescence images of the thick edge in IPFMM compared with confocal microscopy (CM) for (a) point detector; (b) detector pinhole radius v d = 2.8 .

Fig. 4
Fig. 4

Intensity gradient of the image ( I / v x , y ) v x , y = 0 of the thick edge in IPFMM compared with confocal microscopy (CM).

Fig. 5
Fig. 5

One-photon fluorescence images of the thin edge in IPFMM compared with confocal microscopy (CM) for (a) point detector; (b) detector pinhole radius v d = 2.8 .

Fig. 6
Fig. 6

Intensity gradient of the image ( I / v x , y ) v x , y = 0 of the thick edge in IPFMM compared with confocal microscopy (CM).

Fig. 7
Fig. 7

Signal level η as a function of detector pinhole radius v d in IPFMM.

Equations (9)

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I ( v x , v y , u ) = | h 1 a ( v x , v y , u ) + h 1 b ( v x , v y , u ) e i 2 π f t | 2 ( | h 2 ( v x , v y , u ) | 2 2 D ( v x , v y ) ) ,
I 2 π f in phase = ( h 1 a h 1 b * + h 1 a * h 1 b ) ( | h 2 | 2 2 D ) ,
C ( m , n , s ) = F 3 [ I 2 π f in phase ] ,
C ( m , n , s ) = F 3 [ ( h 1 a h 1 b * + h 1 a * h 1 b ) | h 2 | 2 ] .
{ o f ( x , y , z ) = { 1 x 0 0 x < 0 o f ( x , y , z ) = { 1 y 0 0 y < 0 .
{ I ( v x ) = I 2 π f in phase 3 o f = 1 2 + 1 π 0 l c C ( m , n = 0 , s = 0 ) sin ( v x m ) m d m I ( v y ) = I 2 π f in phase 3 o f = 1 2 + 1 π 0 l c C ( m = 0 , n , s = 0 ) sin ( v y n ) n d n .
{ o f ( x , y , z ) = δ ( z ) { 1 x 0 0 x < 0 o f ( x , y , z ) = δ ( z ) { 1 y 0 0 y < 0 .
{ I ( v x ) = I 2 π f in phase 3 o f = 1 2 + 1 π 0 l c C 2 ( m , n = 0 ) sin ( v x m ) m d m I ( v y ) = I 2 π f in phase 3 o f = 1 2 + 1 π 0 l c C 2 ( m = 0 , n ) sin ( v y n ) n d n ,
η ( v d ) = 0 s c C ( m = 0 , n = 0 , s ) d s ,

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