How Live Cell Research is Changing the Face of Biological Research
If you want to see in real time what’s going on inside living cells, then you should be performing live cell imaging. Live cell imaging techniques allow real time examination of almost every aspect of cellular function under a variety of normal and experimental conditions, including:
Stem cells
Stem cells are so sensitive, conventional fluorescence microscopy (the imaging of live cells at high resolution over long periods) is lethal. That’s why researchers often image dead or dying cells and miss dynamic processes that are essential to our understanding of cell biology.
With the live cell imaging techniques of today, cell biology is in the midst of a microscopy boom. Stem cell researchers are coming up with inventive techniques for capturing live images in their native habitats, and researchers can catch glimpses of stem cell activity by focusing their scopes on cells cultured in dishes or in tissue extracted from animals. Observing living stem cells under such conditions is resulting in medical advancements for cancer, degenerative diseases such as Alzheimer and heart disease.
Mitosis
Perhaps the most amazing thing about mitosis is its precision, a feature that has really come to light with advances in light microscopy. Researchers now know that mitosis is a highly regulated process involving hundreds of different cellular proteins and the dynamic nature of mitosis is best appreciated when this process is viewed in living cells. Advances in fluorescence live cell imaging have allowed scientists to study this process in great detail, providing important insights into the biological control of this process and how it might go wrong in diseases such as cancer.
Why live cell research?
The study of cells is the study of basic biology, and living cells offer one of the most accessible models of biological processes scientists have. Continual advances in imaging techniques and design of fluorescent probes improve the power of this approach, ensuring that live cell imaging is an important tool in biology.
Live cell imaging is the study of living cells using time-lapse microscopy. It was pioneered in the first decade of the 20th century – one of the first time-lapse microcinematrographic films of cells ever made was by Julius Reis, showing the fertilisation and development of the sea urchin egg. Since then, several microscopy methods have been developed, which allow researchers to study living cells in greater detail and with less effort.
Live cell research techniques
The growing number of live cell research techniques means you can obtain greater amounts of information without stressing out your cells (or yourself). The three most common techniques are:
Widefield Fluorescence Microscopy
The most basic technique for live cell imaging, widefield fluorescent microscopy yields valuable results if you are imaging adherent cells, large regions of interest (such as organelles) or very thin tissue sections (less than 5 metres). A CCD camera is used to capture images, and then the epi-fluorescence illumination source can be a mercury lamp, xenon lamp, LED’s, etc. Each of the light sources require carefully matched interference filters for the specific excitation and emission wavelengths of your fluorophore of interest.
Widefield and Contrast
Widefield fluorescence microscopy can be used in combination with other common contrast techniques such as phase contrast and differential interference contrast (DIC) microscopy. This combination is useful when performing live-cell imaging to examine general cell morphology or viability while also imaging regions of interest within cells. The combination of contrast and fluorescence microscopy is usually carried out in two separate image captures, using the transmitted light for contrast followed by epi-fluorescence imaging for detection of fluorophores. The two images are then combined in post-image-analysis.
Optical Sectioning
Optical sectioning can alleviate blurring, since only information from the region that corresponds to the objective depth of field is extracted. This technique also enables volume rendering of stacks to generate three-dimensional images. Aside from using confocal techniques, optical sections can also be obtained in widefield fluorescence microscopy using structured illumination.
With all live-cell imaging experiments, the main challenges are to keep your cells alive and healthy over a period of time while they are on the stage of the microscope. Your cells must be kept in a temperature- and pH-stable environment, which is usually achieved by using a chamber where the cells are placed, or larger environmental chambers around the microscope itself.
Benefits of live cell research
With live cell imaging, kinetic processes such as enzyme activity, signal transduction, protein and receptor trafficking, and membrane recycling (endocytosis and exocytosis) can all be interrogated and:
- Cellular enzymes and other cytosolic molecules remain in the cell
- Scientists can observe dynamic cellular processes as they happen
- Cellular structures can be studied in their native environment, meaning less experimental artifact
- Cellular biomolecules and structures can be tracked over time
- Interactions between cells can be observed
Factors to consider, however, include:
- Cells must be kept in their natural physiological ranges for pH, temperature, and osmolarity
- You must have a specific way to label your target, whether it is a molecule, a cellular function, or a cellular state (and illuminate it with minimum toxicity)
- Living cells are not generally permeable to large molecules (i.e., antibodies)
- Moving objects can be more difficult to keep in focus
- Interrogation techniques can be harmful to living cells
To best manage this, you need to make a plan.
Making a plan
When considering a live cell imaging experiment, it is critical to devise an experimental plan. Successful live cell imaging experiments can be a major technical challenge and the conditions under which cells are maintained on the microscope stage, although widely variable, often dictate the success or failure of an experiment.
An important caution is to ensure that cells are in good condition and function normally while on the microscope stage with illumination in the presence of synthetic fluorophores or fluorescent proteins. The goal is to design your experiment to be as non-invasive as possible, since fluorescent imaging can have unwanted side effects due to illumination, which isn’t something your cells are exposed to in the incubator.
The good news is that normal atmospheric oxygen tension levels are suitable for most cultures. With regard to osmolarity, most of the cell lines have a large tolerance for osmotic pressure, with good growth at osmolarities between 260 and 320 milliosmolar. When cells are grown in opennplate cultures or Petri dishes, hypotonic medium can be used to cope with evaporation.
Choosing Livecyte from Phasefocus
Live-cell imaging requires you to keep the cells functioning during the experiment, while being able to assess whether the experimental method is causing problems that will complicate the interpretation of your results. With Livecyte from Phasefocus (a unique system for live cell analysis that enables the study of phenotypic and kinetic behaviour of individual cells and cell populations over hours or days), this process is made simple.
Livecyte uses an optimised version of Quantitative Phase Imaging (QPI) called Ptychography to generate quantitative data without the need for cell labelling. Livecyte exploits the inherent contrast mechanisms that cells possess; refractive index and thickness variations to produce high contrast, high fidelity images without halos or speckling. Cells can be observed with minimal perturbation which is especially useful for primary cells and stem cells.
With Livecyte, you can:
- Directly measure cell motility and separate cell motility from cell proliferation
- Characterise morphological and behavioural cell phenotypes during wound healing
- Perform non-invasive time-lapse imaging to quantify cell death without labels, dyes or phototoxic damage
- Automatically identify cells undergoing mitosis and extract the mitotic index
- Calculate cell death dose response curves without the use of fluorescent labels
- Analyse and extract parameters to identify heterogeneity within mixed cancer cell populations
- Enable investigation of combination therapies on primary cancer cultures
- Understand the underlying mechanisms of angiogenesis.
Livecyte comes complete with automated cell tracking software (patent pending), which can follow all cells for a complete time-course, even if those cells pass over each other. Cells are always in focus irrespective of focal drift or uneven sample holders.
To learn more about Livecyte from Phasefocus download the product brochure or contact ATA Scientific to make a product enquiry. We’re the specialist analytical instrument provider for Australia and New Zealand, and we offer expert support with every decision you make.
Contact us to discuss your requirements.