Advancing Live Cell Analysis
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Phasefocus have recently launched Livecyte™ - a unique system for live cell analysis – or kinetic cytometry - which will enable a deeper understanding of the biology in many application areas including immunological, neurobiological, cancer and basic cell biology research.
We spoke to Tracey Zimmermann, VP of Global Sales at Phasefocus to learn more about the application areas for Livecyte™ and what unique new insights the technology will enable researchers and drug developers to reveal about the behaviour of cells’
TZ: Livecyte is a unique imaging system for live cell analysis which uses an optimized version of Quantitative Phase Imaging called Ptychography (tie-cog-rafee), to enable Kinetic Cytometry: the automatic tracking and analysis of phenotypic and kinetic behaviour of individual cells and cell populations over hours or days. The system produces high contrast, high fidelity images which are artefact free, quantitative and can be tracked with ease across the whole time-course, without the need for cell labelling or high intensity light imaging. Since the power of the illumination laser is thousands of times less than that used for traditional fluorescence light microscopy, cells suffer far less phototoxic shock-a particular advantage for fragile cells such as stem cells or primary cells.
JR: What challenges will this new system help researchers to overcome?
Another challenge facing time-course assays can be focal drift which can affect image quality, which in turn makes it very difficult to track cells. Livecyte’s ptychographic imaging technique ensures that cells are always in focus and since the system delivers a continuous field-of-view which is arbitrarily large (and can be up to a few millimetres in a single image), the need to stitch images is removed. Additionally, imaging continuously over multiple regions (at different magnifications if desired), within a single well of a multi-well plate or multiple regions within each well of a multi-well plate, ensures maximum productivity from each experiment.
JR: Phasefocus recently had a paper published in Nature Scientific Reports validating this new technology. Could you tell us about the significance of the results the paper described?
TZ: The paper was published in conjunction with researchers from University of York and highlights the power of ptychography for long-term imaging of complex primary neuronal cultures in conditions which are effectively “closer to real life”: http://www.nature.com/articles/srep22032. The paper reveals the key advantages to a researcher that a ptychographic imaging approach can bring. The non-invasive properties of the technique were demonstrated by observing the maturation of primary hippocampal cultures into neurons with extensive neurite connectivity, together with the proliferation of microglia and astrocytes. Quantitative phase time-lapse movies of cells within the three populations were generated, which can be viewed alongside the publication. Crucially, since quantitative data was available on every single cell, motility metrics such as speed, meandering index and Euclidean distance could be calculated and these could be linked with changes in morphology, thus deepening understanding of the behaviour of individual cells over time. In this paper, (which used VL21, the Livecyte predecessor), cell tracking was carried out manually using Image J, but using Livecyte, powerful tracking software (patent pending) allows tracking of cells automatically rather than the manual approach employed here.
JR: Livecyte allows researchers to interleave fluorescence measurements during different drug treatments or across cell types within a single experiment. Why is this an important step forward?
We spoke to Tracey Zimmermann, VP of Global Sales at Phasefocus to learn more about the application areas for Livecyte™ and what unique new insights the technology will enable researchers and drug developers to reveal about the behaviour of cells’
JR: Could you tell us about the key features of the Livecyte system?
TZ: Livecyte is a unique imaging system for live cell analysis which uses an optimized version of Quantitative Phase Imaging called Ptychography (tie-cog-rafee), to enable Kinetic Cytometry: the automatic tracking and analysis of phenotypic and kinetic behaviour of individual cells and cell populations over hours or days. The system produces high contrast, high fidelity images which are artefact free, quantitative and can be tracked with ease across the whole time-course, without the need for cell labelling or high intensity light imaging. Since the power of the illumination laser is thousands of times less than that used for traditional fluorescence light microscopy, cells suffer far less phototoxic shock-a particular advantage for fragile cells such as stem cells or primary cells.
JR: What challenges will this new system help researchers to overcome?
TZ: Time-lapse imaging is a fundamental tool for studying cellular behaviours. Live cell studies often involve fluorescent cell labelling techniques which produce high contrast images, but are compromised since fluorescent dyes require high intensity illumination to visualise their location and this can disturb normal cell functions, particularly over extended time-courses. The label free approach which Livecyte affords, allows longer observation of “happy” cells which have been minimally perturbed.
Another challenge facing time-course assays can be focal drift which can affect image quality, which in turn makes it very difficult to track cells. Livecyte’s ptychographic imaging technique ensures that cells are always in focus and since the system delivers a continuous field-of-view which is arbitrarily large (and can be up to a few millimetres in a single image), the need to stitch images is removed. Additionally, imaging continuously over multiple regions (at different magnifications if desired), within a single well of a multi-well plate or multiple regions within each well of a multi-well plate, ensures maximum productivity from each experiment.
Cells in culture and particularly in co-culture, are heterogeneous in nature and averaging these populations in assays may mask interesting characteristics of those cells which do not represent the majority, but may be significant in determining the ultimate behaviour of a population. Since Livecyte allows single cell analysis within cell populations, data on the typical, the median and the average cell can be obtained, whilst also allowing identification of outliers within one time-course experiment. This is exciting since in drug discovery, the outliers-clinically and drug resistance wise-could be the most interesting.
JR: Phasefocus recently had a paper published in Nature Scientific Reports validating this new technology. Could you tell us about the significance of the results the paper described?
TZ: The paper was published in conjunction with researchers from University of York and highlights the power of ptychography for long-term imaging of complex primary neuronal cultures in conditions which are effectively “closer to real life”: http://www.nature.com/articles/srep22032. The paper reveals the key advantages to a researcher that a ptychographic imaging approach can bring. The non-invasive properties of the technique were demonstrated by observing the maturation of primary hippocampal cultures into neurons with extensive neurite connectivity, together with the proliferation of microglia and astrocytes. Quantitative phase time-lapse movies of cells within the three populations were generated, which can be viewed alongside the publication. Crucially, since quantitative data was available on every single cell, motility metrics such as speed, meandering index and Euclidean distance could be calculated and these could be linked with changes in morphology, thus deepening understanding of the behaviour of individual cells over time. In this paper, (which used VL21, the Livecyte predecessor), cell tracking was carried out manually using Image J, but using Livecyte, powerful tracking software (patent pending) allows tracking of cells automatically rather than the manual approach employed here.
JR: Livecyte allows researchers to interleave fluorescence measurements during different drug treatments or across cell types within a single experiment. Why is this an important step forward?
TZ: Although there are clear advantages to a totally label free approach, the Livecyte system does have
the ability to combine label-free and fluorescence protocols automatically and seamlessly, offering new experimental approaches. Protocols can be used which are predominantly label-free in their sampling frequency, but allow periodic tracking of fluorescently labelled components. This still reduces phototoxic effects, but also enables additional verification, if it is required. Label-free and labelled results can then be accurately correlated, avoiding inconsistencies that may be experienced if using different instrumentation for each. An example of where this could be useful is in reporter gene assays, or where functional identification of intracellular features is required. The flexibility afforded by this approach could be particularly useful to extend the time-courses of more fragile cells, while still minimizing their perturbation.
Tracy was speaking to Jack Rudd, Editor for Technology Networks.
