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Fluorescence Live-Cell Imaging [Synopsis]

A crucial analytical tool in the study of cellular biology, pharmacology, developmental biology, neurobiology and other biomedical research is live-cell imaging. With live-cell imaging, researchers are now able to study the very essence of cellular and subcellular functioning and structure (1).

With advancements in instrumentation, technologies, and the overcoming of a number of technical challenges, live-cell imaging has enabled researchers to study cellular dynamics with enhanced spatial and temporal resolution (2).

History of fluorescence microscopy

A cinematographic change took place in the life sciences in the late nineteenth and early twentieth century. The Scottish medical doctor, John McIntyre filmed the movement of a frog’s leg in 1897 (3). In 1907, the Swiss researcher Julius Ries, while working at the Marey Institute in Paris, was the first to take a time-lapse biological film. In this case, he filmed the fertilization and embryonic development of the sea urchin. By this, he wanted his students to visualize the biological process of living cells originating only from other living cells (4).

With the availability of microcinematography apparatuses for biology in Europe by 1914, and, phase contrast microscopy by the 1930s, researchers could now delve into the cell, and also have a peep of intracellular organelles (5).

These discoveries stimulated the life sciences field. Many researchers started using time lapse microcinematography to study cells in greater detail. However, not all scientists were enamoured of live cell imaging. The Nobel laureate Peter Medawar in the 1940s argued that while biologists who made films of living cells had been ‘delighted, distracted, and beguiled by the sheer beauty’ of these cells, these approaches who not ‘solve biological problems’. Thankfully, Medawer’s opinion did not stop other researchers (6).

A brief timeline of fluorescence live cell imaging history
A brief timeline of fluorescence live cell imaging history

Labelling is important for live-cell imaging in many instances. These include three-dimensional imaging of subcellular structures, tissues, and living cells (7). In the late 1800s, synthetic fluorescent dyes began to be developed. In the early 1900s, these dyes were used with fluorescence microscopy to examine bacteria, other pathogens, and tissue samples. With the availability of cell permeable acetomethoxy dyes, researchers could now study living cells in greater detail (8).

The discovery, purification, and characterization of the Green Fluorescent Protein (GFP) from the jellyfish Aequorea Victoria from the 1960s onwards showed that this marker was highly suitable for in vivo fluorescence in different types of cells and organisms. This seminal work resulted in the 2008 Nobel Prize in Chemistry being jointly awarded to Osamu Shimomura of Boston University, and, Martin Chalfie and Roger Y. Tsien of the University of California (9).

The Essentialities of live cell imaging

A huge challenge faced by researchers is the need to maintain a healthy cellular environment during the process of live cell imaging (10).

Cell health

The environment must be such that the cells are healthy and alive. This becomes crucial and more challenging when imaging is continued over hours and days. On these occasions, the various reasons for altered metabolic functioning must be addressed. These include pH, osmolarity, temperature, and oxygenation. It is necessary that the CO2 concentration is properly maintained for a physiological pH. A CO2 concentration of 5 percent is possible with special incubation chambers (11).

Temperature regulation is another crucial factor since even a slight fluctuation in temperature can alter the physiology of the cells. Stage-top incubation chambers are available that heat the sample. Since the objective acts as a heat sink, it also needs to be heated. Various options are available for this purpose, including closed loop heaters and copper tubing water jackets. With sufficient media volume in the cell chamber, a thermal mass is created that reduces fluctuations in the temperature (10).

When a stable temperature is needed for the long term, special custom designed boxes are used. They will enclose the cell culture, objective, stage, and either part of or the whole microscope body. With this, thermal equilibration is obtained and the complete system is maintained at a common temperature (11).

However, these custom designed boxes come with their own limitations. They may require customised modifications when microscopic components are upgraded. This can be expensive. There can be leakage of temperature, and/or gases. In addition, only one sample can be examined at any given time (11).

It is due to these limitations that the best solutions for long term live-cell imaging are imaging devices.

Imaging Devices

The imaging device in which the cells are maintained must keep them healthy and viable. Imaging devices include imaging chambers, incubator devices, imaging boxes, and stage top incubators (12).

When the imaging experiment is of a short duration (30 minutes or less), the cells are grown on glass coverslips (standard thickness of 0.17 mm), placed on microscopic slides and sealed to avoid contamination. Various sealant options are available, including vacuum grease, agarose, and VALAP (a 1:1:1 ratio of VAseline, LAnonin, and Paraffin) (13).

When the imaging experiment is of longer duration, more sophisticated cell chambers are essential. They are of two types, open cell chambers and closed cell chambers. The former, like Petri dishes, allow access. Thus, researchers are able to manipulate the cells by performing microinjections, adding drugs, or changing the media.

In the case of closed cell chambers, the cells are insulated from the external environment. A port may be provided for addition of drugs (11).

Label-free and labelled imaging microscopy

When researchers conduct experiments with live-cell imaging, they have two options: Label-free imaging (Brightfield, holotomography), and labelled imaging (fluorescence). When the optical microscopy system is chosen for live cell imaging, three variables need to be looked into: the speed needed for acquirement of images; viability of the specimens; and, sensitivity of the detector (signal-to-noise-SNR) (14, 15, 16).

For live-cell imaging experiments to be successful, considerable attention must be paid to maintaining the cells in a healthy, living state. It is only with living cells that critical information with regard to the basic nature of cellular and tissue functioning is obtained. The same is not possible with fixation of cells (17).

Currently available widefield epifluorescence, total internal reflection fluorescence (TIRF) microscopy, and spinning disc confocal microscopes have comparable optical and mechanical components.

There must be utmost exploitation of light and the least number of optical elements in the path of light. For optimal SNR, the filters used for purpose of imaging and the spectral profiles of the fluorophores should match as much as possible.

Fast mechanical shutters are used to control the excitation light. Nowadays, shutters are not required for certain excitation sources like LEDs and lasers. Motorized stages are used to acquire multiple regions of the specimen in parallel.

Focus correction strategies in the form of autofocus or hardware-based strategies are needed to control mechanical drift. This drift may arise due to gradients in the temperature and instability of the stage.

Sensitive detection is essential to obtain images when samples are weakly fluorescent. The camera must be able to detect the dynamic processes and capture minute details. Additionally, the detection system should be able to accurately measure differences in intensity (1).

Depending on the type of experimentation, researchers can choose different types of detection systems. Electron multiplying charge-coupled device (EMCCD) cameras are used for living cells with weak signals. sCMOS (scientific Complementary metal–oxide–semiconductor) cameras are preferred for fast live imaging (18).

Fluorescence imaging

As mentioned earlier in this essay, the discovery of GFP revolutionized live-cell imaging. Variations of this protein include Blue Fluorescent Protein (BFP), Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), and Discosoma Red (DsRed) Fluorescent Protein. These derivatives have altered absorption and emission properties, thus allowing use of different fluorescent protein probes in the same experiment (19).

The popularity of these fluorescent proteins (FPs) is that external labelling is not required. The introduction of the FP encoding sequence in combination to the sequence coding for the protein of interest is sufficient. A mutated version of the desired protein is expressed with fluorescence properties and intact functional properties. This allows for precise labelling and analysis.

Researchers need to take into account the following caveats for FPs: Aggregation and dimerization that is dependent on temperature and cell lines; and, maturation times that can vary between different FPs (20).

Photoactivatable GFP (PA-GFP), Dronpa, and the photoconvertible green/red fluorescent protein (mEos) are some of the currently available FP alternatives that are photoconvertible, photoactivatable, or photoswitchable (21).

The organic fluorescent molecules, the sulfonated rhodamine derivatives, are now commonly used in routine live-cell imaging experiments. They have several advantages over FPs such as smaller size, better spectral range, and greater photostability (22).

Reactive intermediates of the sulfonated rhodamine derivatives (hydrazides, succinimidyl esters, and maleimides) pre-conjugated to various antibodies, dextrin, and other molecules have increased water solubility, photostability, and better emission (22).

Fluorophores such as the cell trackers (green/red) and the Annexin XII based probe, polarity Sensitive Indicator of Viability and Apoptosis (pSIVA) are very alluring for use in live-cell imaging because they allow the sample to be viewed immediately after addition.

Inorganic fluorescent probes

The inorganic fluorescent probes include silicon nanoparticles, lanthanide-doped oxide nanoparticles, fluorescent nanodiamonds, and quantum dots (QDs-semiconductor nanoparticles (23).

These are used in place of organic fluorophores to give better fluorescence yield and reduced photobleaching.

Fluorescence applications

Fluorescence live-cell imaging has numerous applications (10). Some of these are:

  • Three-dimensional imaging of living cells
  • Three-dimensional imaging of tissues and intracellular structures
  • Quantification of calcium, magnesium, and other ions
  • Kinetics of cell migration
  • Dynamics of protein structure, organization, interactions
  • Signal transduction studies
  • Monitoring of molecules in living animals

Importance during the COVID-19 pandemic

With physical distancing and occupancy restrictions in many research laboratories and institutions, every researcher has to adapt to the new world brought about by COVID-19.

When live-cell imaging facilities are available, researchers are able to monitor their experiments off-site. The limited time available in the laboratory is now utilized for crucial work. As a result, the efficiency within the facility is improved, and, physical distancing and occupancy restrictions are maintained (24).

The problems, solutions, and the need for fluorescence live-cell imaging

Fluorescence live-cell imaging has transformed the way in which researchers study molecular interactions, subcellular and cellular structures, and also cells within living animals. These observations occur in real time, and across time.

Fluorescent live-cell imaging troubleshooting tips
Fluorescent live-cell imaging troubleshooting tips

When compared to snapshots that are available by imaging studies of fixed cells, live-cell imaging gives the researcher crucial, reliable, and relevant information.

Chemical fixation of cells for microscopy has been vital for researchers in life sciences. Fixatives have been used for preserving cellular organization. However, chemical fixatives introduce artifacts that make the identification of true structures baffling. Hence, the cellular arrangement of chemically fixed cells is not the same as that of living cells (25).

The field of drug discovery and development is one such area. In chimeric antigen receptor (CAR) T-cell therapy, candidate antigens are screened and identified. In addition, live-cell imaging has helped check the effectiveness of candidate CAR T-cells (26).

At times researchers may encounter one or more problems when they use live-cell imaging. These could be background fluorescence, photobleaching, bleed-through, or phototoxicity.

Background fluorescence (background noise) is unwanted fluorescent signals that are of two main types. It could result from autofluorescence from the imaging media, the sample, the vessel, or even from unbound fluorophores. The second type results from ambient light, camera noise, or light from the excitation source. Depending on the source of the background noise in the experiment, researchers can try one or more of the following troubleshooting techniques: Wash the sample several times; optimize the concentration of the fluorescent dye; check the medium and the vessel (27).

In photobleaching, photochemical destruction of the fluorophore results in signal fading during the experimental process. This problem is particularly troublesome during image quantification, since it distorts the data. Troubleshooting steps include the use of a different dye; use of neutral density filters; establish a photobleach curve; use of commercially prepared mounting media with antifade protection; and, try and minimize exposure (28).

Bleed-through occurs when more than one fluorophore is used for labelling, and, fluorescence from a neighbouring channel contaminates your channel of interest. In such a scenario, the researcher should choose spectral non-overlapping fluorophores; best suited filters; or, fluorophores with a narrow spectrum (29).

Phototoxicity results when the experimental cells become stressed and unhealthy. The light used for exciting the fluorophores may damage the cells; furthermore, cells may detach from the vessel, display enlarged mitochondria, plasma membrane blebbing or large vacuoles. What needs to be done in this situation is to optimize the light path as efficiently as possible; and, use the lowest intensity and shortest exposure time if possible (30).


It is now clear that over the years, fluorescence live-cell imaging has become an integral part of research. It has shown its benefits, its pros and cons, but, it is clear that this technique cannot be ignored anymore in the field of life sciences.

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