Benefits of Confocal Microscopy for Live Cell Imaging
Confocal microscopy is a powerful tool that can be used to create 3D images of the structures within living cells and to examine the dynamics of cellular processes1.
For high-speed imaging of fluorescent molecules and structures within living cells, microscopes must provide rapid field-of-view scanning that eliminates any out-of-focus light planes that can obscure fluorescence2.
Several confocal microscopy methods ensure such light planes are removed2 and these techniques have become increasingly popular over recent years, owing to their exceptional image quality, relative ease-of-use and multiple applications in various different research areas.
Two of the main types of commercially available confocal microscopes are the confocal laser scanning microscope and the spinning-disk confocal microscope.
Confocal microscopy technologies were introduced more than 40 years ago, but advances in optical design and camera technology have fuelled the expansion and versatility of the techniques3 and particularly the spinning-disk technology. Recently, Andor, a leading manufacturer of high-performance cameras and microscopy systems, has optimized spinning-disk technology to develop a powerful spinning-disk confocal imaging platform called Dragonfly.
Using confocal laser scanning microscopes, researchers can generate 3D images of organelles within living cells and examine changes that occur in cells over time. A single beam is focused on the specimen plane to sequentially scans points in the region of interest and emission light is filtered through a pinhole to eliminate light from out of focus regions3.
However, although these microscopes are effective at eliminating unwanted light from these regions, they only allow for a relatively slow image acquisition speed3. These single-beam lasers generally scan at rate of 1 microsecond per pixel, which is too slow to capture the dynamic, millisecond events that would reveal the complex molecular processes within living cells3.
When the confocal laser scanning microscope is used with high numerical aperture lenses, the fluorescence speed is limited because only about one cubic micron of light can be obtained from the fluorophore within the scanned beam’s focus4.
When a fluorophore is excited at a moderate level, the light emitted is proportional to the strength of the incident excitation, but the excited states lasts a significant amount of time. As the excitation level is increased, most of the fluorophores are therefore eventually pumped up to an excited state leaving the ground state depleted. Increasing the excitation source at this point does not generate anymore signal because the fluorophore is saturated and the image is degraded4.
Reducing the power of the excitation source does improves the images, but it lowers the speed at which an image with a given signal-to-noise ratio can be obtained4.
These speed limitations can be overcome using parallelism – the application of a line or an array of pinholes. This approach was first introduced by a German physicist called Nipkow4.
A “Nipkow” disc or spinning disk is opaque, with the exception of the presence of thousands of pinholes that are often covered with tiny focusing lenses arranged in spiral patterns2.
When light passes through the series of pinholes, each is imaged by the objective to a spot on the specimen which emits a fluorescence that can be recorded once it passes back through the pinholes. The disc effectively serves as several thousand confocal microscopes all operating in parallel meaning several thousand points on the specimen are illuminated at the same time. This parallelism means that saturation is avoided and higher excitation levels can be used4.
As the disc spins at high-speeds of 6000 rpm, spaces between the pinholes are filled in, generating a real-time confocal image that can be seen with the naked eye2.
As somewhat of a game-changer in the field, Andor’s Dragonfly builds on and further optimizes the Nipkow or spinning disk technology to offer an unprecedented combination of speed, sensitivity and resolution5.
The Dragonfly features a dual spinning disc which is equipped with an array of micro-lenses for each pinhole to maximize the transmission of light. This ensures a highly efficient coupling of the laser to the pinholes, meaning less laser power is needed to achieve high quality confocal images4.
The benefits of delivering higher-efficiency imaging at lower laser powers are that the Dragonfly has more accurate cell physiology, less photobleaching, phototoxicity, and lower fluorophore concentration. It also is less expensive than CLSM and has superior speed and image quality than conventional spinning disc technology, the Dragonfly also operates at least ten times more quickly, further improving throughput time. The benefits of a higher speed include the ability to view live cells, to view larger regions of specimen in 3D stacks in a fraction of the time required by other methods, and the ability to view dynamic cell processes.
In the life science research setting, which increasingly demands more expedient delivery of results and accelerated throughput for more involved study, this powerful new platform provides a fast, sensitive and high-resolution confocal imaging platform for the high-throughput, real-time visualization of the molecules, structures and dynamic events within living cells. Furthermore, Imaris software allows further visualisation and analysis of your image data.
- Confocal Microscopy – A Visual Slice of the Cellular World. The Science Creative Quarterly 2004. http://www.scq.ubc.ca/confocal-microscopy-a-visual-slice-of-the-cellular-world/
- Dailey, M et al. Education in Microscopy and Digital Imaging. Zeiss. http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/techniques.html
- Education in Microscopy and digital Imaging. http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html
- Oxford Instruments. Confocal Dual Spinning Disk. Andor. https://andor.oxinst.com/learning/view/article/confocal-dual-spinning-disk
- Oxford Instruments. Dragonfly. https://andor.oxinst.com/products/dragonfly
- Confocal Laser Scanning Microscopy (CLSM). Wageningen. https://www.wur.nl/en/show/Confocal-Laser-Scanning-Microscopy.htm.