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Non-scanning motionless fluorescence three-dimensional holographic microscopy

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.

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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.

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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.

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All authors contributed equally to this work.

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Correspondence to Joseph Rosen or Gary Brooker.

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

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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

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