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Quantitative phase and digital holographic microscopy

Most cells are almost transparent. In a conventional microscope they need stains or fluorescent labels to be seen, and those labels can change or harm the very thing being studied. Light passing through a cell is not dimmed much, but it is delayed, because the cell has a slightly higher refractive index than the medium around it. Quantitative phase imaging measures that delay at every pixel.

In off-axis digital holography, the light that went through the sample interferes with a tilted reference beam. The camera records straight fringes that the sample bends; a Fourier filter and an inverse transform turn them into a phase map. The phase is proportional to the optical path difference, so the image is a measurement, not just a picture: it reports thickness and dry mass, and how they change over time.

Sharper than the optics allow

Because holography records the full complex field rather than intensity alone, the field can be deconvolved with the microscope’s transfer function. We showed that this resolves structures below the classical Rayleigh limit, work that later led to three-dimensional phase nanoscopy (see holotomography).

Cells in microgravity

Digital holographic microscopy is fast and needs no scanning, which makes it robust enough for demanding environments. We built systems that monitored the shape of living cells under simulated microgravity and during parabolic flights for space-biology research.

Phase imaging for every lab

Holography usually needs a dedicated interferometer. A wedge prism placed in the imaging path of a standard microscope turns part of the beam into a tilted reference, making an ordinary instrument a quantitative phase microscope. We have also taken the method to the macroscale for label-free imaging of whole tissue sections, and to total internal reflection holography for imaging how cells meet a surface.

Try it: intensity versus complex field

An ordinary camera sees only what absorbs light. Holography records amplitude and phase; the phase converts into the thickness of transparent cells, the shape of micro-optics, or the height of a reflective surface.

Specimen
  1. 1

    Intensity image

    What an ordinary camera records with incoherent light: intensity only. Transparent structures barely show.

  2. 2

    Amplitude

    The first half of the complex field. It falls where the specimen absorbs or reflects less light.

  3. 3

    Phase

    The second half: how much the specimen delays the light. Click to move the profile line.

  1. 4

    Thickness or height in 3D

    Drag to rotate. Transmission: t = φ λ / (2π Δn). Reflection: h = φ λ / 4π.

  2. 5

    Line profile

    Solid: measured from the phase. Dashed: the true specimen.

Loading the simulation…

How the microscopes are built

Three typical holographic microscopes, animated: a Mach–Zehnder interferometer in transmission, a Michelson in reflection, and a lens-free in-line set-up.

Set-up
  1. 1

    Follow the light

    LaserBeam splitterMirrorSampleObjectiveTube lensReference arm, beam expanderObject armTilted mirrorCamera

    Schematic, not to scale. Light flows along the beams; the object arm is shown in the accent colour.

  2. 2

    On the camera

    The hologram this set-up records. The next tabs show how to reconstruct it.

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Try it: off-axis holography

The demo below runs the same reconstruction steps we use in the lab on a simulated hologram, including numerical refocusing and aberration compensation.

Specimen
  1. 1

    Record the hologram

    The object wave interferes with a tilted reference beam. The sample’s phase bends the straight fringes.

  2. 2

    Filter in Fourier space

    The tilt separates the spectrum into a zero order and two side orders. Drag the circle, or focus it and use the arrow keys.

  3. 3

    Read out the field

Numerical focusing and aberration compensation

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Try it: in-line holography

Without a separate reference beam the set-up becomes lens-free and compact, but the camera loses the phase and a twin image appears. Iterative phase retrieval with physical constraints removes it.

  1. 1

    Record an in-line hologram

    No separate reference beam: light that passes beside the object interferes with the light it scatters, so the camera sees diffraction rings.

  2. 2

    Propagate back to the object

    The camera records intensity only, so the reconstruction also contains the twin image: the conjugate wave, out of focus by twice the distance.

Loading the simulation…

Key publications

  1. Enhanced robustness digital holographic microscopy for demanding environment of space biology

    M. Fatih Toy, Stephane Richard, Jonas Kuhn, Alfredo Franco-Obregon, Marcel Egli, Christian Depeursinge

    Biomedical Optics Express, 3 (2), 2012Journal article

    DOIPDF
    Cite
    @article{toy2012enhanced,
  title={Enhanced robustness digital holographic microscopy for demanding environment of space biology},
  author={Toy, M Fatih and Richard, St{\'e}phane and K{\"u}hn, Jonas and Franco-Obreg{\'o}n, Alfredo and Egli, Marcel and Depeursinge, Christian},
  journal={Biomedical optics express},
  volume={3},
  number={2},
  pages={313--326},
  year={2012},
  publisher={Optica Publishing Group}
}

  2. Wedge prism assisted quantitative phase imaging on standard microscopes

    M. Fatih Toy

    Optics Communications, 451, 2019Journal article

    DOIPDF
    Cite
    @article{toy2019wedge,
  title={Wedge prism assisted quantitative phase imaging on standard microscopes},
  author={Toy, Muhammed Fatih},
  journal={Optics Communications},
  volume={451},
  pages={361--366},
  year={2019},
  publisher={Elsevier}
}

  3. Sub-Rayleigh resolution by phase imaging

    Yann Cotte, M. Fatih Toy, Etienne Shaffer, Nicolas Pavillon, Christian Depeursinge

    Optics Letters, 35 (13), 2010Journal article

    DOIPDF
    Cite
    @article{cotte2010sub,
  title={Sub-Rayleigh resolution by phase imaging},
  author={Cotte, Yann and Toy, M Fatih and Shaffer, Etienne and Pavillon, Nicolas and Depeursinge, Christian},
  journal={Optics letters},
  volume={35},
  number={13},
  pages={2176--2178},
  year={2010},
  publisher={Optica Publishing Group}
}

  4. Digital holographic microscopy real-time monitoring of cytoarchitectural alterations during simulated microgravity

    Christophe Pache, Jonas Kuhn, Kriss Westphal, M. Fatih Toy, Jerome Parent, Oralea Buchi, Alfredo Franco-Obregon, Christian Depeursinge, Marcel Egli

    Journal of Biomedical Optics, 15 (2), 2010Journal article

    DOIPDF
    Cite
    @article{pache2010digital,
  title={Digital holographic microscopy real-time monitoring of cytoarchitectural alterations during simulated microgravity},
  author={Pache, Christophe and K{\"u}hn, Jonas and Westphal, Kriss and Toy, M Fatih and Parent, J{\'e}r{\^o}me and B{\"u}chi, Oralea and Franco-Obregon, Alfredo and Depeursinge, Christian and Egli, Marcel},
  journal={Journal of biomedical optics},
  volume={15},
  number={2},
  pages={026021--026021},
  year={2010},
  publisher={Society of Photo-Optical Instrumentation Engineers}
}

  5. Accelerated autofocusing of off-axis holograms using critical sampling

    M. Fatih Toy, Jonas Kuhn, Stephane Richard, Jerome Parent, Marcel Egli, Christian Depeursinge

    Optics Letters, 37 (24), 2012Journal article

    DOIPDF
    Cite
    @article{toy2012accelerated,
  title={Accelerated autofocusing of off-axis holograms using critical sampling},
  author={Toy, M Fatih and K{\"u}hn, Jonas and Richard, St{\'e}phane and Parent, J{\'e}r{\^o}me and Egli, Marcel and Depeursinge, Christian},
  journal={Optics letters},
  volume={37},
  number={24},
  pages={5094--5096},
  year={2012},
  publisher={Optica Publishing Group}
}

  6. Quantitative phase macroscopic system for label-free imaging of tissue sections

    M. Fatih Toy

    Electrica, 21 (1), 2021Journal article

    DOIPDF
    Cite
    @article{toy2021quantitative,
  title={Quantitative phase macroscopic system for label-free imaging of tissue sections},
  author={Toy, Muhammed Fatih},
  journal={Electrica},
  year={2021},
  publisher={{\.I}stanbul University}
}

  7. Total internal reflection holographic microscopy for cellular imaging

    Tolga Gurcan, M. Fatih Toy

    In SPIE Photonics Europe 2022 Unconventional Optical Imaging III, Proc. SPIE 12136, 1213604, Strasbourg, 2022Conference paper

    DOIPDFLink
    Cite
    @inproceedings{gurcan2022total,
  title={Total internal reflection holographic microscopy for cellular imaging},
  author={G{\"u}rcan, Tolga and Toy, Muhammed Faith},
  booktitle={Unconventional Optical Imaging III},
  volume={12136},
  pages={18--22},
  year={2022},
  organization={SPIE}
}