Abstract

Focal modulation microscopy (FMM) is a simple, yet efficient, method to preserve image quality in terms of signal-to-background ratio by selecting ballistic photons for image formation. The aim of this paper is to investigate the effect of the various aperture configurations of the spatial phase modulator on the modulation depth of the FMM signal. The definition of modulation depth in FMM and its calculation method are introduced. According to two brief principles of choosing aperture configuration, three types of configurations with different numbers of zones ranging from two to six (totaling eight aperture configurations) are selected, and their corresponding modulation depths and attainable spatial resolutions are simulated. The results show that the modulation depth increases significantly when the number of zones varies from two to six, with a slight or no sacrifice in resolution. In summary, the annular configuration is superior to the fan- and stripe-shaped configurations in modulation depth and spatial resolution.

© 2011 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. M. Minsky, “Microscopy apparatus,” U. S. patent 3013467, (19 December 1961).
  2. C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81, 31–44 (1978).
    [PubMed]
  3. N. Chen, C.-H. Wong, and C. J. R. Sheppard, “Focal modulation microscopy,” Opt. Express 16, 18764–18769 (2008).
    [CrossRef]
  4. 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]
  5. S. P. Chong, C.-H. Wong, C. J. R. Sheppard, and N. Chen, “Focal modulation microscopy: atheoretical study,” Opt. Lett. 35, 1804–1806 (2010).
    [CrossRef] [PubMed]
  6. O. Haeberle and B. Simon, “Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams,” Opt. Commun. 259, 400–408(2006).
    [CrossRef]
  7. P. P. Mondal and A. Diaspro, “Lateral resolution improvement in two-photon excitation microscopy by aperture engineering,” Opt. Commun. 281, 1855–1859 (2008).
    [CrossRef]
  8. O. Schwartz and D. Oron, “Using variable pupil filters to optimize the resolution in multiphoton and saturable fluorescence confocal microscopy,” Opt. Lett. 34, 464–466 (2009).
    [CrossRef] [PubMed]
  9. J. Keller, A. Schonle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15, 3361–3371 (2007).
    [CrossRef] [PubMed]
  10. W. Gong, K. Si, N. Chen, and C. J. R. Sheppard, “Improved spatial resolution in fluorescence focal modulation microscopy,” Opt. Lett. 34, 3508–3510 (2009).
    [CrossRef] [PubMed]
  11. W. Gong, K. Si, and C. J. R. Sheppard, “Optimization of axial resolution in a confocal microscope with D-shaped apertures,” Appl. Opt. 48, 3998–4002 (2009).
    [CrossRef] [PubMed]
  12. C. J. R. Sheppard and M. Gu, “Three-dimensional optical transfer function for an annular lens,” Opt. Commun. 81, 276–280 (1991).
    [CrossRef]
  13. M. Gu and C. Sheppard, “Three-dimensional imaging in confocal fluorescent microscopy with annular lenses,” J. Mod. Opt. 38, 2247–2263 (1991).
    [CrossRef]

2010 (1)

2009 (4)

2008 (2)

P. P. Mondal and A. Diaspro, “Lateral resolution improvement in two-photon excitation microscopy by aperture engineering,” Opt. Commun. 281, 1855–1859 (2008).
[CrossRef]

N. Chen, C.-H. Wong, and C. J. R. Sheppard, “Focal modulation microscopy,” Opt. Express 16, 18764–18769 (2008).
[CrossRef]

2007 (1)

2006 (1)

O. Haeberle and B. Simon, “Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams,” Opt. Commun. 259, 400–408(2006).
[CrossRef]

1991 (2)

C. J. R. Sheppard and M. Gu, “Three-dimensional optical transfer function for an annular lens,” Opt. Commun. 81, 276–280 (1991).
[CrossRef]

M. Gu and C. Sheppard, “Three-dimensional imaging in confocal fluorescent microscopy with annular lenses,” J. Mod. Opt. 38, 2247–2263 (1991).
[CrossRef]

1978 (1)

C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81, 31–44 (1978).
[PubMed]

Chen, N.

Chong, S. P.

Cremer, C.

C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81, 31–44 (1978).
[PubMed]

Cremer, T.

C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81, 31–44 (1978).
[PubMed]

Diaspro, A.

P. P. Mondal and A. Diaspro, “Lateral resolution improvement in two-photon excitation microscopy by aperture engineering,” Opt. Commun. 281, 1855–1859 (2008).
[CrossRef]

Gong, W.

Gu, M.

C. J. R. Sheppard and M. Gu, “Three-dimensional optical transfer function for an annular lens,” Opt. Commun. 81, 276–280 (1991).
[CrossRef]

M. Gu and C. Sheppard, “Three-dimensional imaging in confocal fluorescent microscopy with annular lenses,” J. Mod. Opt. 38, 2247–2263 (1991).
[CrossRef]

Haeberle, O.

O. Haeberle and B. Simon, “Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams,” Opt. Commun. 259, 400–408(2006).
[CrossRef]

Hell, S. W.

Keller, J.

Minsky, M.

M. Minsky, “Microscopy apparatus,” U. S. patent 3013467, (19 December 1961).

Mondal, P. P.

P. P. Mondal and A. Diaspro, “Lateral resolution improvement in two-photon excitation microscopy by aperture engineering,” Opt. Commun. 281, 1855–1859 (2008).
[CrossRef]

Oron, D.

Schonle, A.

Schwartz, O.

Sheppard, C.

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]

M. Gu and C. Sheppard, “Three-dimensional imaging in confocal fluorescent microscopy with annular lenses,” J. Mod. Opt. 38, 2247–2263 (1991).
[CrossRef]

Sheppard, C. J. R.

Si, K.

Simon, B.

O. Haeberle and B. Simon, “Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams,” Opt. Commun. 259, 400–408(2006).
[CrossRef]

Wong, C.-H.

Appl. Opt. (2)

J. Mod. Opt. (1)

M. Gu and C. Sheppard, “Three-dimensional imaging in confocal fluorescent microscopy with annular lenses,” J. Mod. Opt. 38, 2247–2263 (1991).
[CrossRef]

Microsc. Acta (1)

C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81, 31–44 (1978).
[PubMed]

Opt. Commun. (3)

O. Haeberle and B. Simon, “Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams,” Opt. Commun. 259, 400–408(2006).
[CrossRef]

P. P. Mondal and A. Diaspro, “Lateral resolution improvement in two-photon excitation microscopy by aperture engineering,” Opt. Commun. 281, 1855–1859 (2008).
[CrossRef]

C. J. R. Sheppard and M. Gu, “Three-dimensional optical transfer function for an annular lens,” Opt. Commun. 81, 276–280 (1991).
[CrossRef]

Opt. Express (2)

Opt. Lett. (3)

Other (1)

M. Minsky, “Microscopy apparatus,” U. S. patent 3013467, (19 December 1961).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (5)

Fig. 1
Fig. 1

Schematic of the FMM system. L 1 is the objective lens and L 2 is the collection lens. The excitation beam (green) is modulated by SPM. The fluorescence emission (red) from the sample is collected by the detector behind the pinhole. The image signal can be subtracted by the signal processor and displayed on the computer.

Fig. 2
Fig. 2

Configurations of SPM apertures that are being examined. The green areas are nonmodulated; those in blue are modulated. According to the first principle of aperture selection, the internal radii of annular apertures are r / 2 for two-zone annular apertures; r / 2 , r / 2 , 3 r / 2 for four-zone annular apertures; and r / 6 , r / 3 , r / 2 , 6 r / 3 , 30 r / 6 for six-zone annular apertures. Similarly, the widths for stripe apertures are a 0.404 r for a four-zone stripe aperture and b 1 0.215 r , b 2 0.338 r for a six-zone stripe aperture. r is the external radius of aperture.

Fig. 3
Fig. 3

IPSFs for (a) annular, (b) fan-shaped, and (c) stripe-shaped apertures. In (a) and (b), the first, second, and third columns correspond to two, four, and six zones. In (c), the result of two zones is omitted because it is the same as the two-zone fan-shaped aperture. The icon on the top right of each graph is the corresponding aperture shape.

Fig. 4
Fig. 4

Modulation depth for various aperture patterns.

Fig. 5
Fig. 5

(a) Lateral and (b) axial resolutions of apertures. The lateral resolution of all apertures are actually same when the noises and optical aberrations of the practical system are considered; annular apertures have slightly better axial resolution than the fan- and stripe-shaped apertures and, with the increase of zones number, its axial resolution degrades slightly.

Equations (8)

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

M = ( I max I min ) / 2 ( I max + I min ) / 2 = I max I min I max + I min ,
E ( x , y , z , t ) = | A P ( x , y , t ) exp { i k f ( x 2 + y 2 ) i k 2 z [ ( x x ) 2 + ( y y ) 2 ] } d x d y | 2 ,
I ( t ) = E ( x , y , z , t ) × [ | h D ( x , y , z ) | 2 2 D ( x , y ) ] d x d y d z ,
E CM ( x , y , z ) = | A exp { i k f ( x 2 + y 2 ) i k 2 z [ ( x x ) 2 + ( y y ) 2 ] } d x d y | 2 .
I max = E CM ( x , y , z ) × [ | h D ( x , y , z ) | 2 2 D ( x , y ) ] d x d y d z .
E FMM ( x , y , z ) = E CM ( x , y , z ) | A P ( x , y , t ) | φ ( t ) = π exp { i k f ( x 2 + y 2 ) i k 2 z [ ( x x ) 2 + ( y y ) 2 ] } d x d y | 2 ,
I max I min = E FMM ( x , y , z ) × [ | h D ( x , y , z ) | 2 2 D ( x , y ) ] d x d y d z .
I Total = I non + I mod + 2 I non I mod cos ( Δ ϕ mod ) ,

Metrics