systems biology Systems biology is the computational modeling, computational and mathematical analysis and modeling of complex biological systems. It is a biology-based interdisciplinary field of study that focuses on complex interactions within biological syst ...

, live single-cell imaging is a

live cell imaging Live-cell imaging is the study of living cells using time-lapse microscopy. It is used by scientists to obtain a better understanding of biological function through the study of cellular dynamics. Live-cell imaging was pioneered in the first de ...

technique that combines traditional live cell imaging and

time-lapse microscopy Time-lapse microscopy is time-lapse photography applied to microscopy. Microscope image sequences are recorded and then viewed at a greater speed to give an accelerated view of the microscopic process. Before the introduction of the video tape ...

techniques with automated cell tracking and feature extraction, drawing many techniques from

high-content screening High-content screening (HCS), also known as high-content analysis (HCA) or cellomics, is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a ...

. It is used to study signalling dynamics and behaviour in populations of individual living cells. Live single cell studies can reveal key behaviours that would otherwise be masked in population averaging experiments such as

western blot The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...

s. In a live single cell imaging experiment a fluorescent reporter is introduced into a cell line to measure the levels, localisation or activity of a signalling molecule. Subsequently, a population of cells is imaged over time with careful atmospheric control to maintain viability, and reduce stress upon the cells. Automated cell tracking is then performed upon these time series images, following which filtering and quality control may be performed. Analysis of features describing the fluorescent reporter over time, can then lead to modelling and generation of biological conclusions from which further experimentation can be guided.

History

The field of live single-cell imaging began with work demonstrating that

green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...

(GFP), found in the jellyfish ''Aequorea victoria'', could be expressed in living organisms. This discovery allowed researches to study the localisation and levels of proteins in living single cells, for example the activity of

kinase In biochemistry, a kinase () is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule don ...

s, and

calcium Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar to ...

levels, through the use of

FRET A fret is any of the thin strips of material, usually metal wire, inserted laterally at specific positions along the neck or fretboard of a stringed instrument. Frets usually extend across the full width of the neck. On some historical instrume ...

reporters, as well as numerous other phenotypes. Generally, these early studies focused on the localisation and behaviour of these fluorescently labelled proteins at the subcellular level over short periods of time. However, this changed with pioneering studies looking at the tumour suppressor p53 and the stress and inflammation related protein NF-κB, revealing there levels and localisation respectively to oscillate over periods of several hours. Live single cell approaches were also applied around this time to understand signalling in single cell organisms including bacteria, where live studies allowed the dynamics of competence to be modelled, and yeast revealing the mechanism underpinning coherent cell cycle entry.

Experimental work flow

Fluorescent reporters

In any live single cell study, the first step is to introduce a reporter for our protein/molecule of interest into a suitable cell line. Much of the growth in the field has come from improved gene editing tools such as

CRISPR CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacte ...

, this leading to development of a wide variety of fluorescent reporters. Fluorescent tagging uses a gene encoding a fluorescent protein that is inserted into the coding frame of the protein to be tagged. Texture and intensity features can be extracted from images of the tagged protein. Molecules can also be tagged

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...

and introduced into the cell with

electrophoresis Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...

. This enable the use of smaller and more photostable

fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...

s but requires additional washing steps. By engineering expression of FRET reporter such that donor and emitter fluorophores are only in close proximity when an upstream signalling molecule is either active or inactive, the donor to emitter fluorescence intensity ratio can be used as a measure of signalling activity. For example, in key early work using FRET reporters for live single studies FRET reporters of

Rho GTPase The Rho family of GTPases is a family of small (~21 kDa) signaling G proteins, and is a subfamily of the Ras superfamily. The members of the Rho GTPase family have been shown to regulate many aspects of intracellular actin dynamics, and are found ...

activity were engineered. Nuclear translocation reporters use engineered

nuclear import A nuclear localization signal ''or'' sequence (NLS) is an amino acid sequence that 'tags' a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines o ...

and

nuclear export A nuclear export signal (NES) is a short target peptide containing 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. It has the opposite ...

signals, which can be inhibited by signalling molecules, to record signalling activity via the ration of nuclear reporter to cytoplasmic reporter.

Live imaging

Live cell imaging of fluorescently labelled cells must then be performed. This requires simultaneous incubation of cells in stress free conditions whilst imaging is being performed. There are several factors that must be taken into account when choosing imaging conditions such as phototoxicity,

photobleaching In optics, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between t ...

, tracking ease, rate of change of signalling activity, and Signal to noise. These all relate to imaging frequency and illumination intensity. Phototoxicity can result from being exposed to large amounts of light over long periods of time. Cells will become stressed, which can lead to apoptosis. High frequency and intensity imaging can cause the fluorophore signal to decrease through photobleaching. Higher frequency imaging generally makes automated cell tracking easier. Imaging frequencies should be able to capture necessary changes to signalling activity. Low intensity imaging or poor reporters may prevent low levels of signalling activity within the cell from being detected.

Live cell tracking

Following live cell imaging, automated tracking software is then employed to extract time series data from videos of cells. Live cell tracking is generally split into two steps, image segmentation of cells or their nuclei and cell/nuclei tracking based on these segments. Many challenges still exist in this stage of a live single cell imaging study. However recent progress has been highlighted in the field first objective comparison of single cell tracking techniques.

Quantitative phase imaging __FORCETOC__ Quantitative phase contrast microscopy or quantitative phase imaging are the collective names for a group of microscopy methods that quantify the phase shift that occurs when light waves pass through a more optically dense object. Tr ...

(QPI) is particularly useful for live cell tracking. As QPI is label-free, it does not induce phototoxicity, nor does it suffer from the photobleaching associated with fluorescence imaging. QPI offers a significantly higher contrast than conventional phase imaging techniques, such as phase-contrast microscopy. The higher contrast facilitates more robust cell segmentation and tracking than achievable with conventional phase imaging. New techniques that use a combination of traditional image segmentation techniques and deep learning to segment cells are also becoming more widely used as well.

Data analysis

In the final stage of a live single-cell imaging study modelling and analysis of time series data extracted from tracked cells is performed. Pedigree tree profiles can be constructed to reveal heterogeneity in individual cell response and downstream signalling. A large overlap between analysis of single cell live data, and modelling of biological systems using

ordinary differential equations In mathematics, an ordinary differential equation (ODE) is a differential equation whose unknown(s) consists of one (or more) function(s) of one variable and involves the derivatives of those functions. The term ''ordinary'' is used in contrast w ...

exists. Results from this key data analysis step will drive further experimentation, for example by perturbing aspects of the system being studied and then comparing signalling dynamics with those of the control population.

Applications

By analysing the signalling dynamics of single cells across entire populations, live single cell studies are now letting us understand how these dynamics affect key cellular decision making processes. For example, live single cell studies of the growth factor ERK revealed it to possess digital all-or-nothing activation. Moreover, this all-or-nothing activation was pulsatile, and the frequency of pulses in turn determined whether mammalian cells would commit to cell cycle entry or not. In another key example, live single-cell studies of CDK2 activity in mammalian cells demonstrated that bifurcation in CDK2 activity following mitosis, determined whether cells would continue to proliferate or enter a state of quiescence; now shown, using live single-cell methods, to be caused by stochastic DNA damage inducing upregulation of

p21 p21Cip1 (alternatively p21Waf1), also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1, is a cyclin-dependent kinase inhibitor (CKI) that is capable of inhibiting all cyclin/CDK complexes, though is primarily associated ...

, which inhibits CDK2 activity. Moving forward, live single cell studies will now likely incooperate multiple reporters into single cell lines to allow complex decision making processes to be understood, however challenges still remain in scaling up live single cell studies.

References

{{reflist Cell imaging