Revolution ’ in the high speed imaging of cells

Live cell imaging has a problem – how do you image fast cellular events without loosing spatial information? The answer, it turns out, is to get your optics in a bit of a spin say Carl Zeiss

WHEN Carl Zeiss made the first phase contrast microscope, it transformed biological science; kick-starting the science of cell biology, making live cell imaging possible, and providing the foundation for its rapid advancement in the latter half of the 20th century.  Its direct descendants are today’s powerful research microscopes and, especially, the Laser Scanning Confocal Microscope.

In fact, live cell imaging (LCI) and the laser scanning microscope (LSM) are quite synonymous for many scientists as they seek to unravel the complex processes that control every aspect of cellular function and the molecular mechanisms underlying disease.  With the ability to capture effortlessly all the spatial information of an individual cell and pass it directly to the PC for display, analysis or archiving, it is no wonder that LCI is one of life sciences fastest growing techniques, probing the interactions of the cell's protein partners, mapping the movement of individual intracellular molecules or measuring the dynamics of the cytoskeleton during such processes as cell adhesion, cell motility and cell signalling.
 
However, despite all the advances in laser technology, piezo movement systems, computer processing power, storage technology, digital cameras and sophisticated image analysis software, fluorescence microscopy of the dynamics of living cells still presents special challenges to a microscope imaging system.

The first test that every system confronts is the need to simultaneously offer both high spatial resolution and high temporal resolution whilst working with illumination levels low enough to prevent fluorophore damage and cytotoxicity. How quickly you can move the laser spot while still capturing as much of the emitted signal as possible determines three critical factors for live cell imaging:
• How long must you expose the cell to high intensity laser light and risk cellular damage?
• How many individual sites or cells can be imaged in a given timeframe?
• Can you capture highly transient events, such as the movement of individual molecules?

Every LSM makes a trade-off between scanning speed and resolution but the

Figure 1: The Cell Observer SD

different technologies employed mean that some trade-off less.  And, the faster you can image with any given amount of light is an advantage in all three critical areas outlined above and impact significantly on the results achieved.

However, some live cell experiments may last for many days and the challenge of keeping the cells alive over this period is beyond many LCI workstations.

Most laser scanning microscopes in use today concentrate a single laser beam into a focused, diffraction-limited spot which is moved over the specimen. The use of a single spot means that they are limited in the speed with which they can scan and acquire images over a given area.  If three-dimensional imaging is required, then this slowness is exacerbated as the region under investigation is repeatedly scanned at different depths to build a stack of images along the Z-axis. In the several minutes that the sample is illuminated while the image series is acquired, bleaching of the fluorochromes or phototoxic damage may occur. Moreover, when it comes to fast events, such as calcium sparks and nerve impulses, researchers then have to choose between speed and resolution.  Image fast enough to capture the highly transient events under investigation and spatial information is sacrificed.  Maintain resolution and you miss the event altogether.  Even worse, try to go faster by increasing the light intensity and you risk damage and destruction of the sample itself.

It seems obvious that if we could replace the single point-by-point approach with two or more points simultaneously, an increase in speed with a concomitant reduction in the risk of photodamage should result. It is this approach that Zeiss is taking with the Cell Observer SD – a dedicated live cell imaging workstation that radically alters the balance that can be struck between speed, resolution, sensitivity and cell longevity (Figure 1).

The confocal scanning unit of the Cell Observer SD integrates a technology proposed by a German engineering student, Paul Nipkow, when he patented the world's first electromechanical television system in 1884.  He took a disc and cut into it a spiral series of holes positioned so that they could scan every part of an image in turn as the disk spun around.  This raster scanning concept has been translated into the Nipkow disk, real-time scanning confocal microscope in which thousands of pinholes are arranged along a series of spirals.  Illumination and detection occur through the pinholes and it is this parallel processing that explains the high frame rates that can be achieved.

The Cell Observer SD integrates the Yokogawa CSU-X1 spinning disc and, for the first time, optimises the unit's features for the exacting requirements of live cell imaging.  As the disc rotates the entire sample is swept with the microbeams of light, with every rotation of 30° capturing the cameras entire field of view.  Yielding frame rates that clearly outstrip those of traditional single-point scanners, when combined with fluorescence detection from a range of high quantum efficiency AxioCam CCD cameras, illumination intensity can be minimised and, thus, phototoxic effects.

In conventional Nipkow disc systems, a large proportion of the incident light

Figure 2: Schematic diagram of the beam path in the CSU-X1 confocal scanning unit.

would be blocked by the opaque areas between the pinholes. The SD’s scanning unit features a special tandem-disc system that incorporates a second, mechanically coupled disc.  This contains a matched array of microlenses that focus the incident excitation light precisely through the pinholes for efficient use of the available light.  A schematic diagram of the beam path in the CSU-X1 unit is shown in Figure 2.

The Nipkow disc’s lower illumination intensity is advantageous in live cell imaging in reducing photobleaching and allowing observation over several days.  Moreover, the high quantum efficiency of the latest generation of CCD cameras allows the Nipkow disc system to be more optically efficient than the point scanner alternative.

We have seen how the lower illumination intensity can contribute to long periods of experimental observation through the minimising of photodamage.  However, a complete live cell imaging workstation must offer more.  It should have an incubation system that will maintain the cells in optimum physiological condition throughout the time span of the experiment.  And, it should also be controlled by a software environment that controls every aspect of all the equipment above whilst collecting and interpreting multiple 3D data sets for multiple cells, multiple times in experiments that might span several days.

At core of the Cell Observer SD is the inverted Axio Observer research microscope, which exhibits outstanding optics and brilliant fluorescence performance with flexibility, stability and ergonomics.  These attributes make it the perfect platform to mount a range of peripherals designed to work together to provide optimal biophysical conditions under the lens. The incubation concept ranges from compact stage-top incubators to the XL incubator, with its particularly impressive temperature stability.  Environmental controls are provided by stacking modules that can be added to either new or existing microscopes and the AxioVision software will take full control of temperature, CO2, O2 and humidity. From simple temperature control for relatively short-tem experiments up to complete environmental chambers and cell cultivation systems, each component is compatible with the microscope and each other.

Cell Observer SD combines two high performance technologies – the research grade Cell Observer microscopy platform and the CSU-X1 Nipkow disc technology. Together, they represent the epitome of flexible, precise, high-speed confocal microscopy.