Abstract

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called “CINCH”.

© 2014 Optical Society of America

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References

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  3. J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photon. 2(3), 190–195 (2008).
    [Crossref]
  4. G. Brooker, N. Siegel, V. Wang, and J. Rosen, “Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy,” Opt. Express 19(6), 5047–5062 (2011).
    [Crossref] [PubMed]
  5. J. Rosen, N. Siegel, and G. Brooker, “Theoretical and experimental demonstration of resolution beyond the Rayleigh limit by FINCH fluorescence microscopic imaging,” Opt. Express 19(27), 26249–26268 (2011).
    [Crossref] [PubMed]
  6. P. Bouchal, J. Kapitán, R. Chmelík, and Z. Bouchal, “Point spread function and two-point resolution in fresnel incoherent correlation holography,” Opt. Express 19(16), 15603–15620 (2011).
    [Crossref] [PubMed]
  7. X. Lai, Y. Zhao, X. Lv, Z. Zhou, and S. Zeng, “Fluorescence holography with improved signal-to-noise ratio by near image plane recording,” Opt. Lett. 37(13), 2445–2447 (2012).
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    [Crossref] [PubMed]
  9. N. Siegel, J. Rosen, and G. Brooker, “Reconstruction of objects above and below the objective focal plane with dimensional fidelity by FINCH fluorescence microscopy,” Opt. Express 20(18), 19822–19835 (2012).
    [Crossref] [PubMed]
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    [Crossref]
  11. N. Siegel, J. Rosen, and G. Brooker, “Faithful reconstruction of digital holograms captured by FINCH using a Hamming window function in the Fresnel propagation,” Opt. Lett. 38(19), 3922–3925 (2013).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  13. G. Brooker, S. McDonald, G. Adams, and J. Brooker, “Microscope attachment for high precision and efficient imaging,” US Patent 6,147,798A (2000).
  14. M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge University, 2009).
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    [Crossref] [PubMed]
  16. W. S. Rasband, “ImageJ,” NIH, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/ (1997–2014).
  17. G. Brooker and B. Storrie, “3D Holographic and 2-photon super resolution microscopy,” http://www.nist.gov/public_affairs/releases/2010_johnshopkins.cfm .
  18. R. Kelner, B. Katz, and J. Rosen, “Optical sectioning by confocal Fresnel incoherent correlation holography,” in Digital Holography and Three-Dimensional Imaging (Seattle, WA, July 13-17, 2014).
    [Crossref]
  19. R. Kelner, B. Katz, and J. Rosen, “Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system,” Optica 1(2), 70–74 (2014).
    [Crossref]

2014 (1)

2013 (3)

2012 (4)

2011 (3)

2008 (1)

J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photon. 2(3), 190–195 (2008).
[Crossref]

2007 (1)

1997 (1)

Bouchal, P.

P. Bouchal and Z. Bouchal, “Wide-field common-path incoherent correlation microscopy with a perfect overlapping of interfering beams,” J. Eur. Opt. Soc. Rapid Publ. 8, 13011 (2013).
[Crossref]

P. Bouchal, J. Kapitán, R. Chmelík, and Z. Bouchal, “Point spread function and two-point resolution in fresnel incoherent correlation holography,” Opt. Express 19(16), 15603–15620 (2011).
[Crossref] [PubMed]

Bouchal, Z.

P. Bouchal and Z. Bouchal, “Wide-field common-path incoherent correlation microscopy with a perfect overlapping of interfering beams,” J. Eur. Opt. Soc. Rapid Publ. 8, 13011 (2013).
[Crossref]

P. Bouchal, J. Kapitán, R. Chmelík, and Z. Bouchal, “Point spread function and two-point resolution in fresnel incoherent correlation holography,” Opt. Express 19(16), 15603–15620 (2011).
[Crossref] [PubMed]

Brooker, G.

N. Siegel, J. Rosen, and G. Brooker, “Faithful reconstruction of digital holograms captured by FINCH using a Hamming window function in the Fresnel propagation,” Opt. Lett. 38(19), 3922–3925 (2013).
[Crossref] [PubMed]

G. Brooker, N. Siegel, J. Rosen, N. Hashimoto, M. Kurihara, and A. Tanabe, “In-line FINCH super resolution digital holographic fluorescence microscopy using a high efficiency transmission liquid crystal GRIN lens,” Opt. Lett. 38(24), 5264–5267 (2013).
[Crossref] [PubMed]

N. Siegel, J. Rosen, and G. Brooker, “Reconstruction of objects above and below the objective focal plane with dimensional fidelity by FINCH fluorescence microscopy,” Opt. Express 20(18), 19822–19835 (2012).
[Crossref] [PubMed]

B. Katz, J. Rosen, R. Kelner, and G. Brooker, “Enhanced resolution and throughput of Fresnel incoherent correlation holography (FINCH) using dual diffractive lenses on a spatial light modulator (SLM),” Opt. Express 20(8), 9109–9121 (2012).
[Crossref] [PubMed]

J. Rosen, N. Siegel, and G. Brooker, “Theoretical and experimental demonstration of resolution beyond the Rayleigh limit by FINCH fluorescence microscopic imaging,” Opt. Express 19(27), 26249–26268 (2011).
[Crossref] [PubMed]

G. Brooker, N. Siegel, V. Wang, and J. Rosen, “Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy,” Opt. Express 19(6), 5047–5062 (2011).
[Crossref] [PubMed]

J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photon. 2(3), 190–195 (2008).
[Crossref]

J. Rosen and G. Brooker, “Digital spatially incoherent Fresnel holography,” Opt. Lett. 32(8), 912–914 (2007).
[Crossref] [PubMed]

Chmelík, R.

Hashimoto, N.

Kapitán, J.

Katz, B.

Kelner, R.

Kim, M. K.

Kurihara, M.

Lai, X.

Lv, X.

Rosen, J.

R. Kelner, B. Katz, and J. Rosen, “Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system,” Optica 1(2), 70–74 (2014).
[Crossref]

G. Brooker, N. Siegel, J. Rosen, N. Hashimoto, M. Kurihara, and A. Tanabe, “In-line FINCH super resolution digital holographic fluorescence microscopy using a high efficiency transmission liquid crystal GRIN lens,” Opt. Lett. 38(24), 5264–5267 (2013).
[Crossref] [PubMed]

N. Siegel, J. Rosen, and G. Brooker, “Faithful reconstruction of digital holograms captured by FINCH using a Hamming window function in the Fresnel propagation,” Opt. Lett. 38(19), 3922–3925 (2013).
[Crossref] [PubMed]

N. Siegel, J. Rosen, and G. Brooker, “Reconstruction of objects above and below the objective focal plane with dimensional fidelity by FINCH fluorescence microscopy,” Opt. Express 20(18), 19822–19835 (2012).
[Crossref] [PubMed]

B. Katz, J. Rosen, R. Kelner, and G. Brooker, “Enhanced resolution and throughput of Fresnel incoherent correlation holography (FINCH) using dual diffractive lenses on a spatial light modulator (SLM),” Opt. Express 20(8), 9109–9121 (2012).
[Crossref] [PubMed]

G. Brooker, N. Siegel, V. Wang, and J. Rosen, “Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy,” Opt. Express 19(6), 5047–5062 (2011).
[Crossref] [PubMed]

J. Rosen, N. Siegel, and G. Brooker, “Theoretical and experimental demonstration of resolution beyond the Rayleigh limit by FINCH fluorescence microscopic imaging,” Opt. Express 19(27), 26249–26268 (2011).
[Crossref] [PubMed]

J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photon. 2(3), 190–195 (2008).
[Crossref]

J. Rosen and G. Brooker, “Digital spatially incoherent Fresnel holography,” Opt. Lett. 32(8), 912–914 (2007).
[Crossref] [PubMed]

Siegel, N.

Tanabe, A.

Wang, V.

Yamaguchi, I.

Zeng, S.

Zhang, T.

Zhao, Y.

Zhou, Z.

J. Eur. Opt. Soc. Rapid Publ. (1)

P. Bouchal and Z. Bouchal, “Wide-field common-path incoherent correlation microscopy with a perfect overlapping of interfering beams,” J. Eur. Opt. Soc. Rapid Publ. 8, 13011 (2013).
[Crossref]

Nat. Photon. (1)

J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photon. 2(3), 190–195 (2008).
[Crossref]

Opt. Express (5)

Opt. Lett. (6)

Optica (1)

Other (5)

G. Brooker, S. McDonald, G. Adams, and J. Brooker, “Microscope attachment for high precision and efficient imaging,” US Patent 6,147,798A (2000).

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge University, 2009).

W. S. Rasband, “ImageJ,” NIH, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/ (1997–2014).

G. Brooker and B. Storrie, “3D Holographic and 2-photon super resolution microscopy,” http://www.nist.gov/public_affairs/releases/2010_johnshopkins.cfm .

R. Kelner, B. Katz, and J. Rosen, “Optical sectioning by confocal Fresnel incoherent correlation holography,” in Digital Holography and Three-Dimensional Imaging (Seattle, WA, July 13-17, 2014).
[Crossref]

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Figures (6)

Fig. 1
Fig. 1 Schematic diagram of a microscope offering a combination of simultaneous classical and holographic imaging in widefield or confocal modes. Widefield mode includes classical widefield fluorescence and holographic FINCH, and confocal mode includes classical spinning disk confocal and holographic confocal CINCH. This configuration allows for simultaneous holographic and classical imaging of the same exact field of a specimen. The output of an infinity objective passes through a microscope tube lens (L1) which acts as one lens of a 4f relay terminated by lens L2. At the intermediate image plane of the 4f relay system a spinning Nipkow disk can be inserted for CINCH imaging to present the holography optical train with a single plane image. The area marked with dashed lines represents the commercial confocal head (CARV) used in this work. The fluorescence excitation light from an arc source (Photofluor II) passes through an excitation filter and is directed by a dichroic mirror through the disk and then excites the sample. The emission light passes back through the dichroic mirror and the emission filter through the L2 lens of the 4f relay. A polarizing beam splitter (PBS) directs the s polarized light through another tube lens (L3) to respectively record either a confocal or widefield image on camera 2 depending upon whether the spinning disk is in the beam path. The widefield FINCH or confocal CINCH holographic image is likewise imaged by camera 1 depending upon the spinning disk placement after the image beam passes through lens L4, active GRIN lens G1, inactive GRIN lens G2, liquid crystal phase shifter ϕ and output polarizing filter (Pol.).
Fig. 2
Fig. 2 Widefield fluorescence and FINCH imaging of a sample with two layers of 1 μm beads separated by ca. 50 μm, taken with a 20x Nikon objective. (a-c) The FINCH holograms are shown as log(intensity) to emphasize the recording at the camera plane of holograms from both bead planes. (d-f) Hologram phase maps, (g-i) FINCH images and (j-l) widefield images are displayed in a linear scale with intensity bars indicating the relative intensity of each image. Each column of images results from image capture with the designated sample plane at the focal plane of the objective. Sample plane 1 contains the top layer of beads, sample plane 3 contains the bottom layer and sample plane 2 is equidistant between the two layers. The FINCH phase images contain the depth dependent phase information derived from the FINCH holograms and the FINCH images show the complex FINCH holograms propagated to the best focal plane.
Fig. 3
Fig. 3 CINCH and confocal imaging of a sample with two layers of 1 μm beads separated by ca. 50 μm, taken with a 20x Nikon objective. (a-c) The CINCH holograms are shown as log(intensity). (d-f) Hologram phase maps, (g-i) CINCH images and (j-l) confocal images are displayed in a linear scale with intensity bars indicating the relative intensity of each image. Each column of images results from image capture with the designated sample plane at the focal plane of the objective. Sample plane 1 contains the top layer of beads, sample plane 3 contains the bottom layer and sample plane 2 is equidistant between the two layers. The CINCH phase images contain the depth dependent phase information derived from the CINCH holograms and the reconstructed CINCH images show the complex holograms propagated to the best focal plane.
Fig. 4
Fig. 4 Transverse and axial comparative performance of four microscopy methods: widefield fluorescence, FINCH, confocal fluorescence and CINCH with a high-power objective (60x 1.49 NA Nikon TIRF) on an extended object (1 μm fluosphere bead). (a), (b) The focused images of the bead taken with all four methods are the best focused images selected from a z-series of images taken while stepping the sample through the objective focal plane in 200 nm steps. Profile plots through the peaks of the images shown in (a) and (b) show transverse profiles through the peak of the best focus image of the respective z-series. (c), (d) The xz surfaces for all four methods are created from the transverse intensity profiles through the overall peak taken from each z-series, as described in the text. Profile plots through the peaks of the images shown in (c) and (d) show axial profiles through the overall peak pixel in each z-series. Note that the FINCH axial-profile in (c) was shifted by 1.5 μm to more clearly show the increased depth of field in the FINCH image as opposed to the widefield fluorescence image.
Fig. 5
Fig. 5 Two different object planes, separated by 25 μm, of a fluorescent pollen grain sample studied by four microscope methods with a 60x Nikon CFI objective. The lobed pollen grain is in focus in plane 1 but out of focus in plane 2, while the spiked pollen grain is in focus in plane 2 but not in plane 1.
Fig. 6
Fig. 6 Confocal and CINCH images of a H&E Human Fundic Stomach section (top left and right respectively) taken using a 60x TIRF objective. Below each image is an intensity profile of the identical area from each image depicted by the red line. The CINCH image is the best plane of focus calculated from the Fresnel propagation. The confocal image is the image captured on the second camera during the capture of the CINCH holograms. The images without modification were opened in ImageJ [16] and the intensity profiles recorded. Images are 141 μm × 141 μm (confocal) and 145 μm × 145 μm (CINCH).

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