Label-free Live Cell Imaging & Analysis

The HoloMonitor® label-free live cell imaging system is based on the principle of quanti­tative phase imaging, enabling non-invasive visualization and live cell analysis of cultured cells without compro­mising cell integrity.

How Does It Work?

Just like water waves, light waves of a specific wavelength have two principal charac­ter­istics: amplitude and phase. Amplitude corre­sponds to light intensity and is the height of the wave, measured from crest to trough. Phase describes whether a wave is currently at its crest, in its trough, or some­where in between.

Invisible Cells

For a living cell to be visible to the naked eye or in a light micro­scope, the light arriving from the cell must differ in intensity from the back­ground, i.e. differ in amplitude. Unfortunately, living cells are as translucent as ice cubes in water. They neither absorb, emit nor scatter light to any significant extent and there­fore only slightly change the amplitude of the illuminating light, if at all.

Water glass with ice cubes

Phase-shifted beach waves.

The Live Cell Imaging Problem

To be visible in a light microscope, cells must be stained to absorb, emit or scatter light. Unstained living cells do, however, slow down and distort the light passing through them, just like beach waves are distorted by shallower water (above).

Quantification of phase-shifts make live cell imaging & analysis possible.

By using a phase contrast microscope these distortions or phase-shifts can be observed, making unstained cells clearly visible. However, conven­­tional live cell imaging using phase contrast micro­scopy cannot quantify phase-shifts, only visualize them.

The Live Cell Analysis Solution

With computer technology, it is possible both to quan­tify and visualize phase-shifts. This tech­nique is called quantitative phase imaging (QPI) or quanti­tative phase contrast microscopy to distinguish it from con­ventional phase contrast microscopy. QPI provides both quantitative and beautiful images of living cells, transforming phase micro­scopy and live cell imaging into a quantitative tool for detailed live cell analysis.

Live cell imaging: 3D quantitative phase image of Jimt-1 cells

An example of a quantitative phase image of living cells in 3D, created by HoloMonitor. The height of the cell and its color tone corre­spond to the optical thick­ness of the cell.

Live Cell Imaging References

  • Evaluation of Holographic Imaging Cytometer HoloMonitor M4 Motility Applications
    Y. Zhang and R. L. Judson
    Cytometry Part A (2018)

    The HoloMonitor software modules for cell tracking and wound healing analysis were evaluated and compared to the more conventional methods transwell migration and transwell invasion. Both HoloMonitor modules were found to be well-correlated with established standards, yielded reproducible results, and at the same time offered distinct advantages. The wound healing assay was the most tractable and automated method with good reproducibility, while the cell tracking module enabled identification of hypermobile subpopulations.

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  • 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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  • HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells
    Mikael Sebesta; Peter J. Egelberg; Anders Langberg; Jens-Henrik Lindskov; Kersti Alm; Birgit Janicke
    Proceedings, Quantitative Phase Imaging II (2016)

    Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitor M4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures.

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More Cell Imaging

3D Live cell image of Jimt-1 cells

Quantitative Phase Imaging

Quantitative phase imaging (QPI) provides both quantitative and beautiful images of living cells, transforming phase micro­scopy into a quantitative tool for detailed live cell analysis.
Learn more
A quantitative phase image of a cell and the hologram it was created from

Holographic Microscopy

Holographic microscopy is the most common form of quanti­tative phase imaging. The HoloMonitor® live cell time-lapse cytometers employ holographic micro­scopy to allow non-invasive visualization and quanti­fication of living cells without compro­mising cell integrity.
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HoloMonitor App Suite software

Time-lapse Imaging Cytometry

Time-lapse imaging cytometry allow non-invasive visualization and analysis of live cell populations by tracking and quantifying individual cells.
Learn more