What is live-cell imaging
Advancements in microscopy over the last two decades have created a revolution in biology, allowing complex, dynamic processes to be recorded with high spatial and temporal resolution. From 3D imaging of developmental events in whole organisms to single molecule tracking in individual cells, live-cell imaging has become a cornerstone of modern biology.
“Live-cell imaging” is an umbrella term that refers to a wide variety of methods that use time-lapse microscopy to observe, track, and quantify dynamic processes in whole tissues, cells, and/or subcellular structures in living systems.
Live-cell imaging is essential for many fields, including cell biology, developmental biology, neuroscience, biophysics, molecular biology, and cancer biology, just to name a few.
The benefits of live-cell imaging
While fixed cells offers useful snap-shots of complex cellular behaviors, live-cell imaging removes the guess-work about events that occur between data points and provides information about dynamic processes that may otherwise be difficult or arduous to acquire.
Further, the information held in living cells may be more physiologically relevant. For example, live imaging eliminates artifacts that can be introduced during the fixation process, and can therefore provide more reliable information about a sample. Indeed, different fixation methods can influence the apparent structure of the cytoskeleton1,2 or drastically alter cell morphology3.
Challenges of live-cell imaging
A central challenge in live-cell imaging is keeping cells healthy and alive, and maintaining physiological cell behavior. Specimens removed from their native source may exhibit morphological or behavioral changes. For example, fibroblasts exhibit distinct modes of migration on 2D surfaces versus 3D environments/in vivo4.
Cell health and behavior can be controlled during imaging by keeping cells in a culture system that closely resembles their physiological growth environment5,6. This is especially important when performing extended imaging experiments, on the scale of hours to days, where cells can quickly undergo stress response signaling and die5-7.
The most straightforward way to ensure cells are kept in a controlled environment is by performing imaging procedures directly within cell culture incubators. This can be achieved using a small microscope placed within the incubator, which eliminates the need for specialized equipment and provides a straightforward method for imaging that minimizes environmental shock to the cells.
Further, basic cell subculturing methods can strongly influence cell health and live imaging outcomes. For example, the seeding density of cells, cell confluency at the time of splitting, and the total number of passages can all influence cell behavior8.
As concerns about experimental reproducibility continue to grow, it has become increasingly essential to standardize experimental procedures and cell culture techniques, especially in preclinical fields where irreproducibility can impact both economics and human health9,10. Microscopes that can monitor cell health by performing live imaging and time-lapse recording within a cell culture incubator can help support this effort.
Recognizing cell stress and death
To ensure cells or tissue samples are behaving in a physiological manner during the duration of an experiment, the imaging environment must adequately preserve cell health. As a result, it is crucial that the sample is closely monitored for signs of cell stress and death in a given experimental setup.
For tissue culture cells, common indicators of cell stress and apoptosis include membrane blebbing, vacuole formation, detachment from the culture dish/rounding up, and multi-nucleation (Figure 1)5,11,12. These phenotypes can be easily visualized using phase contrast or DIC microscopy. In addition, fluorescent protein aggregation and abnormal mitochondrial morphology can be used as indicators of cell stress5,11,12. Cell viability indicator dyes, such as trypan blue and alamarBlue (ThermoFischer), can also be used5,11,12.
Controlling the imaging environment
Different cell and tissue types will require unique culturing and imaging conditions. In all cases, however, the temperature, osmolarity, humidity, pH, and oxygen content of the imaging environment must be strictly maintained (Figure 2) 5-7. This can be achieved by imaging directly within the cell culture incubator, using imaging chambers, or using additional microscope hardware.
Imaging chambers, stage top incubators, and environmental control chambers
For some experiments, a biological sample may be sealed inside of an imaging chamber for the duration of the imaging process7,13. For example, yeast are typically sealed inside a slide chamber using VALAP (1:1:1 vaseline:lanolin:paraffin)13, while the lids of glass-bottomed dishes can be sealed using vacuum grease.
If a sample must be accessed during imaging, such as for drug treatment, dishes with removable lids and filled with excess culture medium may be used. More complex imaging chambers are commercially available or can be lab-built, including perfusion systems and microfluidic devices14-16.
In cases where a more complex imaging modality is required, imaging chambers are typically used in conjunction with heated stage inserts, stage top incubators, or large chambers that enclose the microscope5-7.
Temperature control is crucial, as even minor fluctuations in temperature can alter cell health and behavior, such as changing the length of the cell cycle17. Different cell types will require different environmental temperatures: while 37°C is optimal for most mammalian cell types, many insect cells, such as Sf9 cells, must be maintained at 27°C18, while fission yeast must be kept at 30°C19.
Stable temperature control is easier to maintain with environmental control chambers that enclose the microscope, but are more expensive and bulky, restricting access to the stage. However, if an immersion objective is used in combination with a heated stage or stage top incubator, an objective lens heater should also be used to ensure the sample is actually maintained at the desired temperature. This is because the objective itself can act as a heat sink, reducing the temperature of the sample5-7.
Osmolarity and humidity
The liquid in an uncovered tissue culture vessel will quickly evaporate at 37°C, which will increase the osmolarity of the culture medium and lead to changes in cell behavior. A stage top incubator can be humidified to 97-100% humidity to maintain osmolarity within the correct range (260mOsm - 320mOsm) by bubbling CO2 through a container of deionized water. In addition, the initial volume of media used in the imaging chamber can be increased, and wet paper towels can be placed in the incubation chamber7.
The majority of culturing media are buffered to physiological pH (7.0-7.4) by sodium bicarbonate and CO25,7,11. Without a buffering agent in the medium or CO2 exposure, however, the media’s pH will quickly increase, leading to changes in cell physiology within minutes7,11. Therefore, for even very short imaging experiments, physiological pH should be maintained by using 10-25mM HEPES to buffer the culturing medium7.
For longer imaging experiments, a stage top incubator connected to a pressure-regulated tank of CO2 should be used. Depending on the unique needs of the sample, more expensive gas mixers with stricter control of the CO2 content can be purchased. For example, while many established tissue culture lines, like NIH/3T3 fibroblasts and CHO-K1 cells, require a 95% air/5% CO2 environment for optimal growth5-7, primary mouse keratinocytes require a 7% CO2 atmosphere20.
Oxygenation: from hypoxia to normoxia to hyperoxia
Oxygen is a crucial component of a cell’s microenvironment, and serves a variety of functions in metabolic homeostasis and cell signaling processes. In vivo, oxygen tension varies widely across different tissues and cell types (Figure 3)21-23.
Figure 3. In vivo oxygen tension in different tissues21-23.
However, the typical mammalian cell culture incubator, which exposes cells to the ambient atmosphere, is not designed to maintain physiological, or normoxic, oxygen levels (Figure 3)21-23.
Keep in mind that efficient oxygenation of the culture media is dependent on many factors, including the size and depth of the culture chamber, the oxygen consumption rate of a particular cell type, and the seeding density of the cells10. This means that intracellular oxygen content may differ from extracellular oxygen levels (Figure 4)21-23.
Figure 4. The influence of extracellular oxygen content on intracellular oxygen levels in common cell culture models21-23.
As a result, cultured cells are often grown in hyperoxic environments, where oxygen levels are higher than physiological levels. This can decrease cell proliferation rates, plating efficiency, and lead to a reduction in metabolic activity10,24. On the other hand, hypoxic conditions can trigger the unfolded protein response (UPR), mTOR signaling, and drive hypoxia-inducible factor (HIF)-mediated gene expression, ultimately resulting in reduced metabolic rates, cell cycle arrest, and pro-survival signaling25,26.
In addition, there is a growing appreciation that specific experimental conditions require strict maintenance of oxygen levels. Hypoxic conditions, where oxygen levels are low, are actually advantageous in the generation of induced pluripotent stem cells and mesenchymal stem cells, the differentiation of organoids, and the identification of effective therapeutics in cancer cell research21,27-29.
For example, embryonic stem cells exhibit greater pluripotency, less spontaneous differentiation, and more robust responses to differentiation stimuli when grown under low oxygen conditions21.
Further, a central therapeutic challenge in cancer treatment is targeting the cells at the hypoxic cores of solid tumors28. It was recently shown that hypoxic culture conditions could increase the toxicity of a novel chemotherapeutic, the thioredoxin inhibitor AW464 (NSC706704), in colorectal cancer cells and reduce expression of the proangiogenic factor VEGF in endothelial cells28.
Therefore, the oxygen content of the culture environment should be maintained using a dedicated oxygen source. In addition, the initial volume of media used in an imaging chamber can be increased during an imaging experiment7.
Microscopy techniques compatible with live-cell imaging
Live-cell imaging can be used to study a vast array of biological processes and is compatible with both labeled and label-free samples. Biological samples may be labeled using a variety of fluorescent probes, including fluorescently tagged proteins introduced through molecular biology methods (ex. GFP, RFP, etc.), organelle-specific dyes (ex. MitoTracker, LysoTracker, BODIPY, Hoechst), protein tags (ex. SNAP, CLIP, Halo), inorganic fluorescent probes (ex. Quantum Dots), and bioluminescent tags (ex. Luciferase)30.
However, the use of fluorescent dyes and exogenously introduced fluorescent probes can induce changes in cell behavior and cell health5,31,32. For example, the genetically encoded actin probe Lifeact can drastically alter F-actin organization and cellular behavior33.
Further, the imaging process itself can lead to light-induced damage. Upon exposure to high illumination light, fluorescent molecules can react with molecular oxygen and produce deleterious free radicals, resulting in phototoxic damage and cell death5. As such, experiments using fluorescent probes can only be performed for a limited time before the cells die.
Alternatively, label-free microscopy techniques eliminate the need for exogenous probes, which can induce toxicity, and prevent photodamage5,11,30. Transmission light microscopy, including bright field, phase contrast, and differential interference contrast (DIC) microscopy are all compatible with long-term live-cell imaging34.
Many cellular behaviors can be examined over time using label-free live-cell imaging. In addition to assays that focus on the morphology, migratory behaviors, and subcellular structures of single cells in a field, more high-throughput experiments can also be performed.
Whole cell populations can be assayed for a variety of characteristics during time-lapse imaging, including proliferation rate, apoptosis rate, cell attachment and detachment rates, colony formation, and drug responses35,36. Complex cell migratory behaviors, such as migration speed and persistence, can also be examined in wound healing assays, where a scratch is made in a confluent monolayer of cells.
At the organismal level, individual tissues or even whole organisms may be imaged over the course of development or at adult stages. For example, live imaging of zebrafish embryonic and larval morphology can be used to assess the effect of potential toxins on embryonic development37.
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