by Laura Lane
Nature’s breathtaking sites and scenes already offer more than a lifetime’s worth of exploration. Now, with the latest confocal imaging technology, catching a glimpse of all the vistas and views could take an eternity. Not only do the latest instruments reveal an endless intricacy of structure and organization, but they also allow researchers to observe and record activities and dynamic changes. For years, that capability also came with hefty price tags. But today, researchers have access to a large selection of more affordable designs.
Even with lower-end models as expensive as $150,000, the discovery power of confocal imaging technology is worth the cost. With its ability to measure live events and monitor the movements of individual cells and molecules, confocal imaging systems can provide precise, real-time information. This is important because “many physiological and pathological events affect individual molecules or organelles,” according to a paper published in Nature Chemical Biology1. Other imaging methods that assess static specimens provide merely an average measurement, which represents “a state that does not exist at the microscopic level.” With the ability to visualize such fine detail, confocal imaging also allows you to understand the sequence of events and their exact location within a single cell.
Confocal history
While the earliest confocal instruments didn’t offer live cell imaging capabilities, the technology certainly opened up windows to the molecular world. In 1953, Princeton University researcher Marvin Minsky was among the first to achieve confocal imaging. By exclusively viewing light focused through a pinhole, which blocked unfocused light, the resulting images were crisper and more detailed than ever. Confocal microscopes have traditionally relied upon lamps, such as the tungsten-halogen varieties and those found in conventional widefield microscopes. In addition to light-emitting diodes, halogen lamps still remain popular modes of illumination. In the 1980’s, confocal microscopes developers took advantage of the bright illumination of lasers. Able to modulate the size of its focus and to dictate the specific wavelength of the light, many companies have harnessed lasers to push the envelope in imaging.
Choosing a confocal system
The type of confocal system you choose will largely depend on your specimens and project goals, but you have to consider many other factors. You can typically find that high end instruments are shared among several labs, such as in core facilities, says Bernhard Zimmerman, senior product manager of advanced imaging microscopy for Carl Zeiss Microimaging GmbH. “On the other hand, if you have very specialized applications and complex experimental setups, then you should purchase your own unit.”
The tradeoffs: light source, speed and resolution
If your budget is severely restricted, your choices may be limited to confocal systems that employ lamps as the light source. If you have more to spend, you may want to consider laser-equipped systems. More expensive because of the high cost of lasers, these systems illuminate the sample one point at a time, which produces images of higher quality. Also, the lasers allow you to see structures deep within the specimen and to produce three-dimensional images. However, the intensity of lasers can harm the specimen and cause photobleaching such that fluorescent probes can no longer be visualized.
Over the years, companies have taken optimal advantage of the benefits of the various types of light sources, while also making modifications to moderate the downsides. However, picking the correct light source is only one of many factors to weigh when choosing the right confocal system. You have to consider your throughput, resolution and efficiency needs. Deciding on what’s most important to you will help you get started. If you need speed, then you may have to sacrifice resolution. If protecting your specimen is priority, then you may want to perform a test run of a laser system before purchasing it.
“There are so many tradeoffs in microscopy,” says Nicolas B. George, group manager of research microscopes for Olympus America Inc.
With systems that perform high content screening, “you’re not worried about the details. You just want to see which cells are positive or not,” George says. “With regular confocal microscopes, the answer is in the details and, for example, you’re looking at a Drosophila larva and quantifiying motorneurons in the ventral nerve cord.”
Lasers and fluorescent probes
The fluorescent probes that you use will also affect your purchasing decision as they determine which specific set of lasers you need to incorporate in the instrument, says Zimmermann. Using probes that provide powerful signals allow you to use a system with dimmer light. With a laser scanning system, this means turning down the laser intensity to a minimum, which also preserves the integrity of your sample and minimizes probe bleaching.
“The key to all this is to maximize sensitivity, which is not a trivial task,” he says. “Whatever you do, increasing sensitivity also improves your ability to achieve higher resolution, greater acquisition speed and increased depth penetration.”
Spectral confocal imaging
Attempts to address all of these needs with one system have resulted in a number of novel technologies. Spectral confocal imaging is one example. Introduced by Zeiss in 2001, these devices can collect and process all the fluorescent light coming from the sample.
“Standard confocal instruments are color blind,” Zimmerman says. “The detector is just an intensity detector,” requiring the use of filters to perceive color.
Lon Nelson, marketing manager for Leica Microsystems, explains that spectral confocal systems can “take in a broad range of fluorescence input or output and very specifically delineate it down to a certain range of wavelengths”.
“Basically, it allows you to very clearly differentiate between two to five different probes at the same time, in the same specimen,” he says. “In addition, these systems offer the versatility of high-resolution scanning for fixed cells, high-speed scanning for live cell experiments and matrix screening protocols for high content analysis.”
A prism-like technology, the acousto-optical beam splitter, acts like a “switching valve for light,” according to Leica Microsystems’ description2, which adjusts according to the probes that need to be excited. A prism-based spectral detection system then receives the emitted light in the reverse direction. This signal is split into a spectrum of wavelengths and each specific emission profile is sent to its own detection device.
“By doing wavelength separation in a very exacting manner, you can make sure you’re only getting fluorescence signal from the protein that you’re looking at, as opposed to autofluorescence, other fluorophores or background noise,” says Nelson.
Spinning disk systems
Another option to consider is the Nipkow spinning disk. Light from lamps or lasers shine through the disk’s many pinholes or slits. With multiple beams simultaneously scanning the same sample, the spinning disk can provide increased efficiency and speed of image aquisition. In addition, spinning disk systems are usually equipped with charged coupled devices (CCD) as detectors, which “readily capture images with an array detector,” according to the Olympus FluoView Resource Center3. Instead of the traditional photomultipler detector tubes found in many confocal systems, the cooled CCD system is much more efficient in the capture and detection of photons.
Software and database management
Maximizing assay efficiency also means increasing the speed of image analysis, according to Jan Hughes, vice president of BioResearch for MDS Analytical Technologies. “We already have the most cutting-edge in hardware,” Hughes says. “What we now need is to increase our efforts in software development.”
One area that requires serious consideration is database management. “It’s the Achilles’ heel of many of our customers’ infrastructures,” he says, explaining the importance of being able to easily access vast amounts of specific information and images that are stored in imaging databases.
Efficiency can also be optimized by using more flexible image acquisition software that can be readily programmed to obtain a set of specific parameters during acquisition. Instead of allowing the system to acquire images of the entire microplate well, you can instruct the software to record a subset of data. For example, you can program the software such that the system acquires data for only 5,000 cells – rather than the potential tens of thousands of total cells – within each well of a multiwell plate.
“As soon as the system has acquired the images for the set number of cells, the system proceeds to the next well,” Hughes says. “Then you don’t over- or under-image for more efficient assays.”
References:
1Jaiswal JK and Simon SM, “Imaging Single Events at the Cell Membrane”, Nature Chemical Biology, 3(2):92-98, February 2007.
2AOBS® Leica TCS SP5: The High Sensitivity Broadband Confocal for High-Quality Results
3Olympus FluoView Resource Center
