Cell tracking casts doubt over fluorescent microscopy validity

ATA Scientific Pty Ltd
Wednesday, 14 April, 2021



Livecyte should be seen as a solution to a problem you didn’t know you had. It has the capacity to create a paradigm shift in your research.

This novel technology facilitates high-resolution images without the need for fluorescence. The software manages all the data and analysis on board. Analysis dashboards frequently clarify phenotypical behaviours that were previously unknown. All data is open for export.

Livecyte is quietly proving that fluorescent probes perturb your cells, at times killing them, fundamentally questioning the validity of research that is so heavily invested in fluorescent labelling. Fluorescent labels have helped with the expansion of live cell imaging but at what sacrifice?

Deciding whether a probe is harmful or not is at times ad hoc; “I transfected with the label, they lived”. So on the strength of this are you good to go? Labels have the potential to alter normal cell function and induce toxicity1,2 whilst phototoxicity presents an additional barrier to long-term imaging since the high intensity light required to excite fluorophores can also alter cell behaviour and induce cell death3.

How often can your cells be used after a week of imaging, especially if you have successfully isolated a sub population? Arguably never if you have perturbed them! Livecyte inherently enables the reuse of cells, providing confidence in cellular viability, while preserving natural behaviour and facilitating an extension to the experiment. Livecyte carefully borrows your precious cells and returns them to you at the end of your experiment.

Livecyte allows you to watch your cells grow over a week or so, segment them and then at a click of a button, track their every move, follow them through their cycle, watch their mitosis, follow the daughter cells, and analyse the added metrics provided by such functionality.

What you want to investigate, and your predictions about the cell behaviours are sometimes turned on their heads once you drill down into the data or move from a population behaviour to single cell analysis. Unleashing this power has transformed cellular microscopy, removing the dependence on fluorescence, enabling the study of natural behaviours resulting in confidence with the treatments.

How do we segment and track cells better?

Cell tracking is the process of following individual cells over time through mitosis or other cell cycle events and variations due to treatments. This can be an arduous task for just a few cells over a week long time-lapse. Now run along and do this over a bunch of fields across 96 wells… see you next year!

Automating cell tracking unlocks kinetic aspects of your experiment, logging fundamental changes, coupling dynamic phenotypes, directionality, track speed, mitotic time, cell lineage and meandering index to dry mass, morphology and proliferation. Remembering this can be on a population basis or down to single cell interrogation.

It is commonplace to attempt to assess cellular migration with a scratch wound technique. This is logical if you wish to understand the movement of fibroblasts but is not so ideal to injure other cells particularly if this is not the reason for the experiment. What kind of cellular cascade will this produce, will it be contrary to the experimental purpose?

Livecyte automatically collates image, single cell segmentation and cell tracking data into an interactive Cell Motility Dashboard enabling a label free, scratch free alternative.

Livecyte also calculates Cell Displacement and Cell Confinement Ratio for random migration studies. These metrics represent the distance a cell migrates relative to their point of origin and also considers the degree in which a cell meanders from its starting and end point. The random motility assay has been shown to be an effective, quantitative measure of cell migration without the contradictory activation of cellular processes, known or unknown or arbitrary measures such as gap closure in isolation. You can visualise this at https://vimeo.com/345641762.

Contact Peter Davis at ATA Scientific for more information or a virtual demo.

References
  1. lford R, Simpson HM, Duberman J, Hill GC, Ogawa M, Regino C, Kobayashi H, Choyke PL (2009). Toxicity of organic fluorophores used in molecular imaging: literature review. Mol Imaging 8(6):341-54.
  2. Coutu DL, Schroeder T (2013). Probing cellular processes by long-term live imaging-historic problems and current solutions. J Cell Sci. 126(Pt 17):3805-15.
  3. Magidson V, Khodjakov A (2013). Circumventing photodamage in live-cell microscopy. Methods Cell Biol. 114:545-60. https://www.phasefocus.com/resources/app-notes/sir-dna-phototoxicity
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