Holographic Microscopy

Holographic microscopy is the most common form of quanti­tative phase imagingThe HoloMonitor® live cell imaging microscope employs digital holographic micro­scopy to allow non-invasive visualization and quanti­fication of living cells without compro­mising cell integrity.

Conventional Holography

A traditional hologram is recorded on a photo­graphic plate. The holographic image is created by illuminating the developed holo­gram with the laser that was used to create the hologram. This recreates the same light wavefield that originally came from the imaged object. The object appears to be 3-dimensional as each eye image a slightly different part of the wavefield, just like they would have done if the object was still there.

A quantitative phase image of a cell and the hologram it was created from

A cell phase image (left) and the hologram it was created from (right).

Digital Holography

The advent of high-resolution image sensors has made it possible to instead record a hologram digitally, allowing the holo­graphic image to be created by a computer, rather than re-illuminating the developed holo­gram. The computer actually creates two images: an amplitude (intensity) and a phase image. As unstained cells are trans­parent, most of the information resides in the phase image.

Wave interference

When light waves interact they create an interference pattern, just like water waves do.

The Holographic Microscopy Principle

Holographic microscopy creates (quantitative) phase images by letting a sample beam and a refer­ence beam interfere to create an interference pattern or hologram, as shown in the principle image below. The hologram is recorded by an image sensor and computer-processed to produce the final phase image.

Holographic microscopy principle

The principle design of HoloMonitor

It is useful to think of a phase image as a picture of the optical imprint created by the cells. When the illumi­nating sample beam passes through the sample, the sections of the beam that passes through the more optically dense cells are delayed in relation to the background. This shifts the phase of the parallel sample beam and imprints the morphology and 3-dimensional optical properties of the cells on the sample beam, similar to how beach waves are delayed and phase-shifted when they reach shallow water.

Delayed beach waves

Shallow water imprinted beach waves

Holographic Microscopy References

  • Label-free High Temporal Resolution Assessment of Cell Proliferation Using Digital Holographic Microscopy
    Birgit Janicke, Andreas Kårsnäs, Peter Egelberg and Kersti Alm
    Cytometry Part A (2017)

    The authors have developed a robust and label-free kinetic cell proliferation assay with high temporal resolution for adherent cells using HoloMonitor M4. Only two image processing settings were adjusted between cell lines, making the assay practical, user friendly, and free of user bias. In the recorded time-lapse image sequences, individual cells were automatically identified to provide detailed growth curves and growth rate data of cell number, confluence, and average cell volume. The results demonstrate how these parameters facilitate a deeper understanding of cell processes than what is achievable with current single-parameter and end-point methods.

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  • Digital Holographic Microscopy for Non-invasive Monitoring of Cell Cycle Arrest in L929 Cells
    Maria Falck Miniotis, Anthonny Mukwaya and Anette Gjörloff Wingren
    PLOS ONE (2014)

    We show that average cell phase volume results from DHM readings are comparable to the flow cytometry findings. DHM thus provides a non-disruptive alternative to flow cytometry. The technique has the potential to develop into a fast and cost-efficient method for pre-clinical monitoring of cancer treatment efficacy.

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  • Cells and Holograms — Holograms and Digital Holographic Microscopy as a Tool to Study the Morphology of Living Cells
    K. Alm, Z. El-Schich, M. Falck Miniotis, A. Gjörloff Wingren, B. Janicke and S. Oredsson
    Holography — Basic Principles and Contemporary Applications  (2013)
  • Refractometry of Microscopic Objects With Digital Holography
    M. Gustafsson, M. Sebesta
    Applied Optics (2004)

    Digital holography has some desirable properties for refractometry of microscopic objects since it gives phase and amplitude information of an object in all depths of focus from one set of exposures. We show that the amplitude part of the image can be used to observe how the Becke lines move between different depths of focus and hence determine whether an object has a higher or a lower index of refraction than its surrounding medium, i.e., the sign of the relief. It is also shown that one single-phase image provides an independent technique to determine the sign of relief between an object and the surrounding medium.

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