Abstract

Image inversion interferometers have the potential to significantly enhance the lateral resolution and light efficiency of scanning fluorescence microscopes. Self-interference of a point source’s coherent point spread function with its inverted copy leads to a reduction in the integrated signal for off-axis sources compared to sources on the inversion axis. This can be used to enhance the resolution in a confocal laser scanning microscope. We present a simple image inversion interferometer relying solely on reflections off planar surfaces. Measurements of the detection point spread function for several types of light sources confirm the predicted performance and suggest its usability for scanning confocal fluorescence microscopy.

©2009 Optical Society of America

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References

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  1. W. B. Amos, J. G. White, and M. Fordham, “Use of confocal imaging in the study of biological structures,” Appl. Opt. 26, 3239–3243 (1987).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref]
  5. N. Sandeau and H. Giovannini, “Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation,” Opt. Commun. 264, 123–129 (2006).
    [Crossref]
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    [Crossref] [PubMed]
  7. M. V. Berry, “Interpreting the anholonomy of coiled light,” Nature 326, 277–278 (1987).
    [Crossref]

2007 (1)

2006 (2)

N. Sandeau and H. Giovannini, “Increasing the lateral resolution of 4Pi fluorescence microscopes,” J. Opt. Soc. Am. A 23, 1089–1095 (2006).
[Crossref]

N. Sandeau and H. Giovannini, “Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation,” Opt. Commun. 264, 123–129 (2006).
[Crossref]

2005 (1)

2003 (1)

1987 (2)

Amos, W. B.

Berry, M. V.

M. V. Berry, “Interpreting the anholonomy of coiled light,” Nature 326, 277–278 (1987).
[Crossref]

Fordham, M.

Giovannini, H.

N. Sandeau and H. Giovannini, “Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation,” Opt. Commun. 264, 123–129 (2006).
[Crossref]

N. Sandeau and H. Giovannini, “Increasing the lateral resolution of 4Pi fluorescence microscopes,” J. Opt. Soc. Am. A 23, 1089–1095 (2006).
[Crossref]

Gweon, D.

Heintzmann, R.

Kang, D.

Sandeau, N.

N. Sandeau and H. Giovannini, “Increasing the lateral resolution of 4Pi fluorescence microscopes,” J. Opt. Soc. Am. A 23, 1089–1095 (2006).
[Crossref]

N. Sandeau and H. Giovannini, “Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation,” Opt. Commun. 264, 123–129 (2006).
[Crossref]

White, J. G.

Wicker, K.

Appl. Opt. (1)

J. Opt. Soc. Am. A (1)

Nature (1)

M. V. Berry, “Interpreting the anholonomy of coiled light,” Nature 326, 277–278 (1987).
[Crossref]

Opt. Commun. (1)

N. Sandeau and H. Giovannini, “Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation,” Opt. Commun. 264, 123–129 (2006).
[Crossref]

Opt. Express (1)

Opt. Lett. (2)

Supplementary Material (1)

» Media 1: AVI (1022 KB)     

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Figures (5)

Fig. 1.
Fig. 1. Image inversion in a UZ-Interferometer, named after the U- and Z-shaped beam paths. Light coming from the microscope is split at the first beam splitter BS 1. Two mirrors then reflect the light in each arm to form a Z-shaped path (M Z;1, M Z;2) and a U-shaped path (M U;1, M U;2), which stand perpendicularly on each other. The two paths are then recombined at the second beam splitter BS 2. Lenses LC and LD re-focus the light, generating image planes at the (optional) pinholes PHC and PHD , before it is detected in the detectors DC and DD . The double arrows indicates the translational movement of mirrors M U;1 and M U;2 to form an optical trombone for adjusting the path length of the interferometer, and the translational movement of mirror M U;2 to adjust the relative phase of the interferometer arms.

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Fig. 2.
Fig. 2. Single frames from fibre scan across the inversion axis (Media 1). (a) For a distance of d=7.7µm from the inversion axis there is hardly any interference and the intensity is divided equally over the two channels. (b) At d=1.5µm there is interference, leading to more light being detected in the constructive channel (right). (c) When the fibre is located on the inversion axis, interference results in practically all light being detected in the constructive channel, whereas the destructive channel (left) remains dark.

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Fig. 3.
Fig. 3. Fibre scan across the inversion axis. (a) shows the normalised integrated intensity of the constructive (blue, solid) and the destructive (red, dashed) output. The constructive signal reaches a maximum of 99.3%, the destructive signal drops to 0.7%. The difference in intensity for off-axis points is due to non-perfect 50/50 splitting of the beam splitters. (b) shows the difference signal (the scaled subtraction of constructive and destructive output; blue, solid) and the widefield signal (red, dashed). The interference signal exhibits hardly any sidelobes. This is due to the Gaussian mode profile of the light coming from the fibre.

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Fig. 4.
Fig. 4. CCD images of the normalised interferometer output. (a)–(c) show the measurements for the sodium vapour lamp, (d)–(f) the white light incandescent lamp. (a) and (d) show the constructive, (b) and (e) the destructive measurement, where the intensity values were normalised so that the non-interfering regions are at I=.5. The false colour images (c) and (f) show the weighted difference signal. These PSF measurements were done by recording the image of an incoherent source of illumination. This is mathematically equivalent to the integrated signal of a point source with respect to the source position, i.e. the detection PSF.

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Fig. 5.
Fig. 5. Circular averages of the difference signals as shown in Fig. 4 (c) and (f). (a) shows measurements for the sodium vapour discharge lamp, and the theoretically predicted detection PSF (solid line). (b) shows the white light measurements. Due to spectral smearing this curve shows much reduced side lobes compared to the sodium vapour data. A theoretical curve would require precise knowledge of the spectral composition of the light and was therefore omitted.

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Equations (2)

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Idif (r→xy)=[a⊗a*](2r→xy)
I± (r→xy,s→xy)=∣a(r→xy−s→xy)±a(−r→xy+s→xy)∣2,

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