Page 5 - Live-cellanalysis handbook

Introducing Real-Time

Live-Cell Analysis

The biomedical world has come a long way since Anton van these systems gather cell images (phase contrast, bright-field and/
Leeuwenhoek first observed living cells with a basic microscope or fluorescence) from assay micro-plates automatically, repeatedly
in 1674. Using fluorescent probes and modern high resolution and around the clock. Image acquisition is completely non-
imaging techniques it is now possible to view labeled sub-cellular invasive and non-perturbing to cells, opening up the opportunity
structures at the 10-50 nanometer scale. For researchers working to capture the full, and as needed, long-term time course of the
with fixed (dead) cells, organelles can be studied at even higher biology. Acquisition scheduling, analysis and data viewing can be
resolution using electron microscopy. These methods provide conducted easily and remotely, without in-depth knowledge of
tremendous insight into the structure and function of cells down image processing. Data is analyzed on the fly, image by image,
to the molecular and atomic level. to provide real-time insight into cell behavior. We refer to this
paradigm, which is differentiated from straight live-cell imaging
The further development of cell imaging techniques has largely by the provision of analysed data at scale as opposed to simply
focused on resolving greater spatial detail within cells. Examples images, as ‘real-time live-cell analysis’.
include higher magnification, three dimensional viewing and
enhanced penetration into deep structures. Significant attention In an ideal world, the images acquired from a live-cell imaging
has also been paid to temporal resolution – time-lapse imaging device would be collected only from photons produced by the
has evolved for high-frame rate image capture from living cells to sample of interest, and in perfect focus. However, this is not
address “fast” biology such as synaptic transmission and muscle the usual case. There are multiple sources of confounding
contractility. Any consideration for technology advances at lower signal present in an image, each needing correction, removal, or
spatial or temporal detail may initially seem mundane, or even cleaning in order to reveal information which has been generated
unnecessary. However, this would fail to recognize some key unmet by the sample elements of interest. Corrections are needed
user needs. due to systematic aberrations in an imaging system stemming
from multiple sources. For example, detector anomalies (e.g.
First, there is an increasing realization that many important detector bias, dark current variability, field flatness and thermal
biological changes occur over far longer time periods than or gamma-ray noise), optical issues (non-flat optical components
current imaging solutions enable. For example, maturation and and illumination imperfections) or undesired signal introduced by
differentiation of stem cells can take hours, days and sometime the sample are common issues. Autofluorescence from cellular
weeks, which is hard to track using existing methods. Second, components or media, or non-biological signal sources such as
imaging techniques are not readily accessible to all researchers shading, or patterns arising from sample matrices or non-uniform
nor on an everyday basis. This lack of accessibility is either due to illumination due to meniscus effects in microwells must be
virtue of instrumentation that is expensive and use-saturated or removed before usable, replicable information can be extracted.
by complex software that renders image acquisition and analysis
the sole domain of the expert user. Third, and particularly with In order to perform these corrections, one must be aware of the
regard to time-lapse measurement, the throughput of current effects of each process, and manipulations on the raw images must
solutions is typically too low for frontline use in industrial be repeatable, to ensure faithful capture of the measured biological
applications. Finally and most importantly, researchers are signal across images, experiments, and devices. There are many
increasingly aware that any perturbance of the cells in the tutorials and software toolkits available to process images, however
process of imaging (e.g. fixing, loss of environmental control) systems that perform these corrections as a matter of course
can introduce unwanted and misleading experimental artefacts. provide consistency and ease of use, particularly when coupled with
Together, these factors frame up the requirement for solutions standardized assays, reagents and consumables which normalize
that enable longer-term, non-perturbing analyses of cells at a the experimental process (e.g. the IncuCyte Live-Cell Analysis
throughput and ease of use commensurate with non-specialist System, and the assays and reagents available from Sartorius). The
users, and at industrial scale. consistency with which images are acquired and processed strongly
influences the ability to analyze the collected data. This can be a
A new generation of specialized compact microscopes and live-cell time-consuming task, and purpose-built software that presents only
imaging devices, are now emerging to meet this need. Designed to the tools necessary for a specific scientific question can remove
reside within the controlled, stable environment of a cell incubator, what can be a significant hurdle in the image analysis workflow.

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