Live-cell imaging has gained momentum in the ever-changing biological research landscape as an integral tool that allows insight into the subtle dynamics of living in the cell. This mighty approach helps scientists to observe and analyze cellular processes right in real-time to let them get a glimpse of the dynamic nature of living systems. Being one of those 'edge' sciences standing between biology and technology, live-cell imaging proceeds with enlarging the known borders of what is understood about cellular function and cellular behaviour.
Fundamentals of Live-Cell Imaging
Live-cell imaging has its origins in the first pioneering work at the start of the 20th century by a scientist named Julius Ries, who produced one of the first time-lapse microcinematographic films, showing fertilization and subsequent development of sea urchin eggs. Since then, the technology has experienced a radical transformation due to continued improvement in microscopy techniques and fluorescent labeling methods, together with advances in digital imaging technologies.
By its very definition, live-cell imaging attempts to maintain cells in a near physiological state while observing them for extended periods. It is this delicate balance between observation and preservation that distinguishes live-cell imaging from the fixed-cell microscopy of yesteryear.
Key Imaging Modalities for Live Cells
Widefield Fluorescence Microscopy
Despite all these newer methods, widefield fluorescence microscopy remains one of the cornerstones of live-cell imaging because of its simplicity and low phototoxicity. Modern widefield systems coupled with high-performance cameras, using advanced filter technologies, can capture fast cellular events at very good spatial and temporal resolutions. With deconvolution algorithms applied, widefield microscopy rivals confocal microscopy in image quality while generating considerably lower levels of photodamage to living specimens.
Confocal Laser Scanning Microscopy (CLSM)
CLSM has long been a workhorse in the cell biology laboratory due to its unparalleled optical sectioning capability, which makes three-dimensional imaging of thick specimens possible. However, the high light intensity required for confocal imaging can be detrimental to live cells. Advances in detector technology, such as the introduction of Gallium Arsenide Phosphide photomultiplier tubes, have improved quantum efficiency for confocal systems allowing reduced laser power and improving cell viability during imaging.
Two-Photon/Multi-Photon Microscopy (MPM)
MPM has now become a gold instrument for live-cell imaging, particularly for thick specimens and in vivo studies. By employing longer-wavelength excitation light, MPM reduces photo-damage and increases depth penetration. The localized excitation occurring in MPM also minimizes photobleaching outside the focal plane. For these reasons, it is an excellent choice for long-term imaging of sensitive samples.
Light Sheet Fluorescence Microscopy (LSM)
It has revolutionized the field of developmental biology and is increasingly used for cellular imaging. The principle of LSM is based on illuminating the sample with a thin sheet of light and detecting fluorescence perpendicular to the plane of illumination, a principle that drastically reduces phototoxicity and enables high-speed 3D imaging. This is an excellent method for the long-term imaging of developing embryos and large tissue specimens.
Advanced Cell Imaging Techniques
Super-Resolution Microscopy
The development of super-resolution techniques, such as Structured Illumination Microscopy (SIM), Stimulated Emission Depletion (STED), and Single-Molecule Localization Microscopy (STORM/PALM), has made this possible. In addition, these approaches have pushed the optical resolution for live-cell imaging far below the diffraction limit of light, revealing previously unseen views of cellular architecture and molecular interaction details.
Holotomography (HT)
Holotomography is an emerging label-free imaging technique used for the quantitative measurement of a 3D refractive index distribution within living cells. By combining principles of holographic and tomographic imaging, HT provides quantitative, high-resolution 3D images of cellular structures without fluorescent labeling. This non-invasive technique is of especial value for the long-term observation of sensitive samples.
Fluorescent Labeling for Live-Cell Imaging
One of the prime advantages of live-cell imaging became possible with the advent of fluorescent proteins, pioneered by the discovery of Green Fluorescent Protein. These genetically encoded fluorescent tags can be used for specific labeling of proteins and cellular structures in living cells and enable a researcher to trace molecular dynamics and interactions with unsurpassed precision.
Recent advances in fluorescent protein engineering have produced brighter, more photostable variants for all colors of the visible spectrum. Moreover, the development of photoactivatable and photoswitchable fluorescent proteins has enabled new super-resolution imaging modalities and pulse-chase experiments in live cells.
Challenges and Considerations in Live-Cell Imaging
Live-cell imaging is very promising, but it poses special problems. One of the most critical issues is the health of cells being imaged, which requires a controlled environment: temperature, humidity, CO2. Recent advances in the field have mostly overcome phototoxicity and photobleaching, but the aim is still to reduce light exposure to a minimum while keeping a good signal-to-noise ratio.
Balancing the spatial and temporal resolution against cell viability will always be a challenge in live-cell imaging. Therefore, all experimental designs must be very carefully considered for the trade-offs among image quality, acquisition speed, and potential cellular stress.
Future Directions in Cell Imaging
The field of live-cell imaging keeps evolving at an enormous velocity, with several trends being exciting to watch in the future:
1. Artificial Intelligence and Machine Learning: integrating both for image analysis and adaptive microscopy.
2. The development of new biosensors enabling real-time monitoring of cellular physiology.
3. CLEM approaches that bridge the gap between dynamic live-cell imaging and high-resolution ultrastructural analysis.
4. Enhanced capability for in vivo imaging in order to explore cellular processes in intact organisms.
As these technologies continue to mature, they hold the bright promise of affording deeper insights into the complex and dynamic world of cellular life, furthering our understanding of fundamental biological processes and enabling new discoveries in fields reaching from developmental biology to neuroscience and beyond.
Live-cell imaging has transformed our ability to study cellular processes in their native context. It puts life into a dynamic view at the microscopic scale. As we go on pushing the frontiers of what is possible in biological imaging, then so shall we continue with increasingly refined and complex understandings of the elaborate molecular dance constituting life in all its forms.