Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Non-scanning motionless fluorescence three-dimensional holographic microscopy

Nature Photonics volume 2, pages 190–195 (2008)Cite this article

Abstract

Holography is an attractive imaging technique as it offers the ability to view a complete three-dimensional volume from one image. However, holography is not widely applied to the field of three-dimensional fluorescence microscopic imaging, because fluorescence is incoherent and creating holograms requires a coherent interferometer system. Although scanning one beam of an interferometer pattern across the rear aperture of an objective to excite fluorescence in a specimen overcomes the coherence limitation, the mechanical scanning is complicated, which makes the image capturing slow, and the process is limited to low-numerical-aperture objectives. Here we present the first demonstration of a motionless microscopy system (FINCHSCOPE) based on Fresnel incoherent correlation holography, and its use in recording high-resolution three-dimensional fluorescent images of biological specimens. By using high-numerical-aperture objectives, a spatial light modulator, a CCD camera and some simple filters, FINCHSCOPE enables the acquisition of three-dimensional microscopic images without the need for scanning.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Comparison of the FINCH principle and conventional imaging.
Figure 2: FINCHSCOPE schematic in upright and inverted fluorescence microscopes.
Figure 3: FINCHSCOPE holography of polychromatic beads.
Figure 4: FINCHSCOPE fluorescence sections of pollen grains and Convallaria rhizom.
Figure 5: FINCHSCOPE and widefield fluorescence images of nerve fibres in a 70-μm-thick skin section.
Figure 6: Spinning disk confocal sections of nerve fibres in skin.

References

  1. Gabor, D. A new microscopic principle. Nature 161, 777–778 (1948).

    Article  ADS  Google Scholar 

  2. Garcia-Sucerquia, J. et al. Digital in-line holographic microscopy. Appl. Opt. 45, 836–850 (2006).

    Article  ADS  Google Scholar 

  3. Poon, T.-C. Scanning holography and two-dimensional image processing by acousto-optic two-pupil syntheses. J. Opt. Soc. Am. A 2, 521–527 (1985).

    Article  ADS  Google Scholar 

  4. Schilling, B. W. et al. Three-dimensional holographic fluorescence microscopy. Opt. Lett. 22, 1506–1508 (1997).

    Article  ADS  Google Scholar 

  5. Rosen, J. & Brooker, G. Digital spatially incoherent Fresnel holography. Opt. Lett. 32, 912–914 (2007).

    Article  ADS  Google Scholar 

  6. Rosen, J. & Brooker, G. Fluorescence incoherent color holography. Opt. Express. 15, 2244–2250 (2007).

    Article  ADS  Google Scholar 

  7. Indebetouw, G., Tada, Y., Rosen, J. & Brooker, G. Scanning holographic microscopy with resolution exceeding the Rayleigh limit of the objective by superposition of off-axis holograms. Appl. Opt. 46, 993–1000 (2007).

    Article  ADS  Google Scholar 

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

  9. McNally, J. G., Karpova, T., Cooper, J. & Conchello, J. A. Three-dimensional imaging by deconvolution microscopy. Methods 19, 373–385 (1999).

    Article  Google Scholar 

  10. Cowley, J. M. Off-axis STEM or TEM holography combined with four-dimensional diffraction imaging. Microsc. Microanal. 10, 9–15 (2004).

    Article  ADS  Google Scholar 

  11. Goodman, J. W. Introduction to Fourier Optics 2nd edn, 63–95 (McGraw-Hill, New York, 1996).

Download references

Acknowledgements

This work was supported by National Science Foundation grant no. 0420382 and CellOptic. We thank M. DeBernardi, B. Storrie and S. Krueger for valuable comments and A. Hermerschmidt, who provided the special firmware for the SLM. We also thank W.R. Kennedy and G. Wendelschager-Crabb for the immunolabelled slide of skin and K. Ryan for deconvolving the stack of images created from the hologram.

Author information

Author notes

  1. Joseph Rosen and Gary Brooker: CellOptic, 9605 Medical Center Drive Suite 224, Rockville, Maryland 20850, USAJoseph Rosen is on leave from the Department of Electrical and Computer Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.

Authors and Affiliations

  1. Johns Hopkins University Microscopy Center, Montgomery County Campus 9605 Medical Center Drive Suite 240, Rockville, 20850, Maryland, USA

    Joseph Rosen & Gary Brooker

  2. Johns Hopkins University Department of Chemistry, Zanvyl Krieger School of Arts and Sciences, and Advanced Technology Laboratory, Whiting School of Engineering, Baltimore, Maryland 21211, USA,

    Joseph Rosen & Gary Brooker

Authors
  1. Joseph Rosen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gary Brooker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed equally to this work.

Corresponding authors

Correspondence to Joseph Rosen or Gary Brooker.

Ethics declarations

Competing interests

Joseph Rosen and Gary Brooker are the founders of CellOptic and have an equity interest in the company.

Supplementary information

Supplementary Information

Supplementary Information S1 (PDF 77 kb)

Supplementary Information

Supplementary video S1 brookersupfig1.avi (AVI 1508 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rosen, J., Brooker, G. Non-scanning motionless fluorescence three-dimensional holographic microscopy. Nature Photon 2, 190–195 (2008). https://doi.org/10.1038/nphoton.2007.300

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2007.300

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing