SubscribeWhen Carl Zeiss Jena built the first phase contrast microscope, it transformed the world of biology.
It kick-started the science of cell biology, made live cell imaging possible and provided the foundation for its rapid advancement in the latter half of the 20th century.
Figure 1: The first Phase Contrast Microscope Prototype, Carl Zeiss Jena 1936
Live Cell Imaging is now one of life sciences' fastest growing techniques, utilised in biomedical facilities around the globe to unravel the complex processes that control every aspect of cellular function. Its power is allowing us to probe the interactions of the cell's protein partners and map the movement of individual intracellular molecules or measure the dynamics of the cytoskeleton during such processes as cell adhesion, cell motility and cell signalling.
It can be argued that laser scanning microscopes, cheap and readily-available computing power, sophisticated image analysis software, megapixel digital camera systems and a plethora of highly specific fluorescent tags mean that cellular studies are routine, ordinary even. However, live cell imaging still poses a number of challenges and, like most technological challenges, they are most clearly visible where scientists and researchers are pushing hardest at the boundaries.
Slow, slow, quick, quick, slow.
Even where the challenge appears to have been met, subtle differences in the technology employed in different microscopes can have a substantial impact on the results that can be achieved. For instance, today's laser scanning microscopes allow users to capture all the spatial information of an individual cell simply and quickly. Layer upon layer of two-dimensional images created by scanning the focal plane with a laser spot are electronically combined to build a 3D image of the cell. Add a motorised stage and populations of cells can be automatically scanned, rendered in 3D and compared.
But how quickly you can move that spot of light while still capturing the entire emitted signal determines three critical factors:
- How long do you have to expose the cell to this high intensity light and risk cellular damage?
- How many cells can be compared in any period of time?
- Can you capture highly transient events, such as the movement of individual molecules?
Every laser scanning microscope makes a trade-off between scanning speed and resolution but the different technologies employed mean that some trade-off less. And, the faster you can scan AND resolve gives you an advantage in all three critical areas outlined above. Fast imaging technology, then, is an area where the technology boundary is being pushed.
At the other end of the cell's time continuum, some live cell experiments, such as documenting the slow growth and reproduction of cells, may last for many days. In this case, just keeping the cells alive is a challenge that many live cell imaging workstations cannot meet. Maintaining them in optimum physiological condition throughout the long observation phase is another place where the technology boundary is actually being pushed.
A life support system for cells
A complete live cell imaging workstation should offer:
- an advanced motorised microscope or laser scanning microscope.
- a motorised stage.
- a highly sensitive digital camera or photomultipliers.
- optical technology that minimises damage to the cell whilst collecting a high-resolution image.
- an incubation system that will maintain the cells in optimum physiological condition throughout the time span of the experiment.
- software 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.
A decade ago, laboratories had no option but to build their own workstations around their existing microscopes. Recently, various microscope manufacturers have attempted to ‘patchwork’ together multi-dimensional analysis systems around their microscopes using motorised stages, cameras, image analysis software, incubation systems and automation software from different suppliers.
Carl Zeiss has gone one step further and developed a complete solution for the observation and digital documentation of living processes using perfectly matched components. Cell Observer [Figure 2] is a modular, ready-to-use system that can be exactly tailored to the exacting requirements made by live cell imaging's diverse applications [Table 1].
Figure 2: Cell Observer - the complete system
Table 1.Live Cell Imaging Applications
- Dynamic Fluorescence - Fast Imaging of Calcium Signals, Gene expression and localisation with GFP and its derivatives
- Cell Motility - Cellular movement and differentiation, morphological response to stress and environment
- Neurobiology - Interaction of neurons and neuroglial cells, the growth of axons/dendrites
- Dynamic Gene expression with Luminescent reporters
- Elucidation of cellular signalling pathways
- Protein & vesicle tracking
- Cell cycle and development studies
- FRET/FLIM analysis of molecular interaction
It is based around the Axiovert 200, our research grade inverted microscope, which exhibits outstanding optics and brilliant fluorescence performance with superior flexibility, stability and ergonomics. Available as a manual or fully motorised version, the Axiovert 200 is the perfect platform to mount a range of peripherals designed to work together to provide optimal biophysical conditions under the lens.
Figure 3: Live Cell Imaging is possible only if the cells are Alive!
A flexible range of imaging options is possible with four Carl Zeiss AxioCam digital cameras available. At the top end, the AxioCam HR offers 12-Megapixel resolution (4164 x 3120 pixels), professional 14-bit signal digitisation and a dynamic range of 1:2000. These raw numbers assume huge significance in fluorescence imaging where faint signals often need to be recorded in the presence of signals hundreds of times brighter. The AxioCam HR can resolve 2000 levels of brightness and is ideally suited to distinguishing subtle colour changes at the full resolution capacity of the microscope.
The key to the Cell Observer's flexibility is a vast range of life support options that enables individual users to choose exactly the right combination of components for their specific application. These include a wide selection of heatable mounting frames, inserts, stages and plates together with control units for 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 fully compatible with the microscope and each other.
Supporting the system
The glue that binds together every aspect of the complete Cell Observer system is software. The AxioVision software collects every single piece of data generated after co-ordinating every aspect of the system's operation.
AxioVision is an intelligent, modular software suite that begins by controlling the basic functions of the microscope and camera, including the stage, light source, shutters and filters. As any individual Cell Observer option is added to the system, AxioVision recognises its presence and integrates its control into the user interface
Other options then allow the full capability of the system to be utilised in complex, long-term experiments. For instance,Mark & Find eliminates searching and simplifies the collection of multiple datasets by taking control of the XY stage so that pre-determined positions on a sample are automatically relocated. Large samples are dealt with byMosaiX, which combines images of individual fields of view to form a single, large image of an entire surface. Whilst retaining the pixel accuracy of the original individual images, MosaiX combines the overlapping images so precisely that all the detail of the specimen is maintained. Users can then simply and quickly navigate around the object like a map and then drill down for a high power view of the detail.
Autofocus delivers 100% sharpness on all focal planes, Z-stack controls the motorised focusing as it processes up or down through the image, and OrthoView analyses the resulting Z-stacks. Multichannel records up to eight simultaneous wavelengths whilst Time-lapse is the master correlator, co-ordinating and controlling all the other modules to document the cells living processes as they happen.
In perfectly marrying together the microscope, camera, and sample movement with a life support system for cells, Cell Observer does more than just keep the cells alive, valuable though this is. It gives researchers a means to keep cells in an optimum environment during the entire length of the experimental process and places brand new investigative opportunities before them.
There are, though, still those issues of speed and light intensity.
Finding safety in the blue light district
One way of reducing the phototoxic effect in live cell imaging is to expose the cell to less and less light through faster and faster scanning techniques. I'm going to cover this point shortly but an alternative may be to use 'safer' light.
Despite what seems an abundance of GFP-like fluorphores that fluoresce at different wavelengths, many need to be excited by a UV light source. This has limited their use in laser scanning confocal imaging as the UV light itself easily damages the samples under observation. These near-UV fluorophores include DAPI, Hoechst, CFP and Elexa Fluor® 405.
What is needed is a light source that will excite these fluorophores without damaging the cells. With the introduction of the 405nm Blue Diode Laser, Carl Zeiss has done just that.
Available as an option for the LSM 5 PASCAL, LSM 510 and LSM 510 META microscopes, the new laser is simply coupled into the scan head using an additional fibre. Full control of the laser intensity is offered in 0.1% increments and the shorter wavelength also increases resolution for reflectance imaging. The wider range of fluorophores opens up new research opportunities and decreases the chance of spectral overlap at the same time. As well as extending the boundaries for near-UV fluorophores, the blue diode laser will excite neuronal tracer-fluorogold, until now the sole preserve of UV systems.
It's a speed thing
Safer 405nm light will play a vital role in future live cell imaging systems. However, when it comes to dynamic cell events, the way that microscope manufacturers cope with the trade-off between resolution and scanning speed is key. In this respect, the award-winning LSM 510 family of microscopes sets the pace.
A standard technique in many cell experiments is to bleach a small area of the cell by exposing it to high intensity light for few seconds. The photobleaching process wipes out the signal from fluorescence-tagged molecules within that area. However, if the fluorescence signal begins to recover, it is an indication that other fluorescence-tagged molecules from surrounding areas within the cell are mobile. This technique is referred to as fluorescence recovery after photobleaching or FRAP and has been used to show that distinct populations of cellular proteins are constantly moving within the cell. A complementary technique, FLIP, continuously photobleaches a small area of the cell. If the signal throughout the cell gradually declines it indicates intracellular mobility. If any areas remain fluorescent, then we can infer that internal compartments are keeping those populations of tagged proteins separate.
Graham Dunn and his colleagues in London have developed a new method for tracking molecules within living cells following photobleaching. In their FLAP (fluorescence localisation after photobleaching) method, the molecule to be tracked carries two fluorophores. One is photobleached, just as in FRAP and FLIP, and the other acts as a reference label enabling the distribution of the molecules to be easily located merely by image differencing.
The group has used FLAP to study actin dynamics during protrusion in cancer cells and has shown that actin is delivered to the leading edge of cells at a speed much faster than can be accounted for by diffusion. This is a very significant finding, suggesting that cancer cells can rapidly propel materials to the places where they need them and may point the way to preventing cancer cells from growing and spreading if a way can be found of stopping the active transport mechanism. Using the technique, they can track both the fast relocation of monomeric (globular) actin and the much slower dynamics of filamentous F-actin simultaneously in living cells.
Figure 4: FLAP ratio image of the leading lamella of a T15 cell immediately after a strip (red band) was photobleached for 4s. Three regions of strongly labelled transported actin can be seen at the top right leading edge. Zicha et. Al., 2003
However, the key to their research was the ability to be able to scan relatively large areas of the cell very rapidly immediately following cessation of photobleaching. This they owe to their purchase of a Carl Zeiss LSM 510 laser scanning confocal microscope, which is capable of scanning 2,600 lines per second at full resolution. Put another way, that's five frames per second or just 0.35 nanoseconds per laser spot.
Fast into the Future
The LSM 510 showed that fast frame rates and reduced phototoxicity are possible. Now, with the LSM5 LIVE confocal laser scanning microscope, Zeiss has moved the technology on further and is set to make a significant contribution to the future of this exciting branch of science. My next article will cover this breakthrough instrument in detail. For now, just consider the raw facts:
- LSM 5 LIVE collects up to 120 confocal images per second at a resolution of 512 x 512 pixels, approximately 20 times faster than any other confocal system, and the ultrafast Z-drive solution permits 3D image stacks to be acquired every second.
Fast frame rate technology, high illumination efficiency and high resolution - never mind the Foxtrot, the LSM5 LIVE confocal laser scanning microscope will trip the light fantastic.
Figure 5: The LSM5 LIVE confocal laser scanning microscope