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High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers.

Nisan Siegel1, Vladimir Lupashin2, Brian Storrie2

  • 1Department of Biomedical Engineering, Johns Hopkins University, 9605 Medical Center Drive Suite 240, Rockville, Maryland 20850, USA.; Microscopy Center, Johns Hopkins University Montgomery County Campus, Rockville, Maryland 20850, USA.; CellOptic, Inc., 9605 Medical Center Drive Suite 224, Rockville, Maryland 20850, USA.

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Fresnel incoherent correlation holography (FINCH) microscopy now achieves high-resolution biological imaging using birefringent crystals. This breakthrough overcomes previous limitations, enabling detailed visualization of cellular structures like the Golgi apparatus.

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Area of Science:

  • Biomedical Optics
  • Microscopy
  • Cell Biology

Background:

  • Fresnel incoherent correlation holography (FINCH) microscopy offers high-resolution imaging potential.
  • Previous FINCH implementations were limited to low-magnification and low-numerical-aperture configurations, hindering broader applications.
  • Aberrations and distortions affected image quality in earlier FINCH systems.

Purpose of the Study:

  • To develop and validate a novel FINCH microscopy system capable of high-numerical-aperture imaging.
  • To overcome the limitations of previous FINCH microscopy setups by employing birefringent elements.
  • To demonstrate enhanced resolution and image quality for biological samples.

Main Methods:

  • Utilized in-line incoherent interferometers constructed from uniaxial birefringent crystals (α-barium borate or calcite).
  • Integrated these birefringent elements with high-numerical-aperture oil immersion objectives.
  • Validated resolution using sub-resolution fluorescent beads and imaging of labeled proteins in HeLa cells.

Main Results:

  • Achieved a lateral point spread function of 149 nm at a 590 nm wavelength, surpassing standard wide-field fluorescence microscopy.
  • Successfully resolved three different GFP-labeled proteins and two other fluorescent dyes within the Golgi apparatus of HeLa cells.
  • Demonstrated image quality comparable to structured illumination microscopy for complex biological samples.

Conclusions:

  • Novel birefringent elements significantly enhance FINCH microscopy performance, overcoming previous aberration issues.
  • The improved FINCH system enables high-resolution imaging of subcellular structures with excellent detail.
  • This advancement positions FINCH microscopy as a powerful tool for detailed biological investigation.