Label-free Live Cell Imaging
Label-free live cell imaging using quantitative time-lapse imaging facilitates gentle cell imaging, enabling non-invasive visualization and singe-cell analysis of living cell cultures without compromising cell behavior.
When watching a time-lapse movie of a thriving cell population, it becomes evident that cellular interactions and individual cell behavior are much more elaborate and heterogeneous than we currently appreciate.
Live cell imaging promises to help us understand the contribution of individual cell behavior to the overall response of a cell population.
The Live Cell Imaging Predicament
Living mammalian cells are as translucent as ice cubes in water. To be visible in a standard or fluorescent light microscope, cells must, therefore, be stained or genetically modified to absorb, emit or scatter light. Unfortunately, the invasive preparations necessary to make cells visible are likely to affect cellular behavior, compromising the in vivo relevance of in vitro live cell observations.
Gentle Cell Imaging
Unstained cells do, however, slow down and distort the light passing through them, just like beach waves are distorted by shallower water (below). By using a phase contrast microscope these phase-shift distortions created by living cells can be observed, making unstained cells clearly visible.
Phase-shift distorted beach waves, created as a result of the lower wave speed in shallower water.
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.
When light passes through a cell submerged in cell media, the light amplitude is unaffected. But, the more optically dense cell slows down and delays the light slightly relative to the surrounding ambient light, creating the phase-shift which makes cells visible in a phase contrast microscope.
However, conventional phase contrast microscopy cannot quantify phase-shifts, only visualize them.
Quantitative Phase Imaging
Using a digital image sensor, low power diode illumination and sophisticated computer algorithms, the HoloMonitor live cell imager has the ability to both quantify and visualize phase-shifts. HoloMonitor employs a technique called quantitative phase imaging (QPI) or quantitative phase contrast microscopy, to distinguish it from its soon 100-year-old non-quantitative predecessor — the phase contrast microscope.
Gentle Time-Lapse Imaging
As the cell does not absorb any light energy, the cells are completely unaffected when observed using HoloMonitor — no energy exchange, no change. This allows HoloMonitor to gently acquire time-lapse image sequences over extended periods of time without compromising cellular behavior.
HoloMonitor provides both quantitative and beautiful time-lapse images of living cells, transforming phase microscopy and label-free live cell imaging into a quantitative tool for detailed analysis of living cells on a population and single-cell level.
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.
Label-Free Live Cell Imaging References
Evaluation of Holographic Imaging Cytometer HoloMonitor M4 Motility ApplicationsCytometry Part A (2018)Read more
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.
Label-free High Temporal Resolution Assessment of Cell Proliferation Using Digital Holographic MicroscopyCytometry Part A (2017)Read more
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.
HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cellsProceedings, Quantitative Phase Imaging II (2016)Read more
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.