Confluence of Electronics, Sensing, and Optics
Camera sensor technology employed in microscopy is changing rapidly, says Paul Jantzen, marketing manager for core microscopy at Olympus America (Center Valley, PA). Fifteen years ago, customers used still or video cameras to capture images; CCD sensor-based cameras emerged ten years ago and dominated until very recently. “Now, scientific-grade CMOS cameras are taking over that market. CMOS-based cameras are faster and less expensive, and for high-end research require special computer configurations to capture all the data.”
Disclaimer of sorts: Olympus, which manufactures consumer cameras, does not produce CMOS cameras for scientific markets. It relies on third-party companies with interest in security, microscopy, defense, and machine vision imaging markets.
“But CMOS happens to be great for scientific imaging as well,” Jantzen says.
The higher speed of CMOS enables microscopies to monitor biological events that occur very quickly, such as structural or chemical dynamics within cells, movement of ions or vesicles, and protein trafficking. “Rapid biological events demand rapid imaging, and CMOS fits the bill,” Jantzen notes. “This has significantly reduced the cost of entry into high-end imaging for many laboratories.”
Formerly, scientific-grade CMOS cameras cost upward of $80,000. Thanks to the same technical breakthroughs that have revolutionized consumer imaging products, today’s top-end CMOS cameras have come down dramatically in cost, to the $10,000- $15,000 range. “This technologic shift has been driven by companies that specialize in camera sensors,” Jantzen says.
Because the sensors are large and capture data extremely rapidly, sophisticated software and hardware are required to acquire data and store it quickly for later evaluation.
Microscopy itself often combines two or more technologies, and the same applies for applications as more labs employ “combinatorial microscopy,” which combines two or more advanced microscopy techniques in one experiment. “Newly introduced instruments allow scientists to merge these modalities easily,” says Lynne Chang, PhD, senior applications scientist at Nikon (Melville, NY).
One example merges superresolution microscopy with confocal imaging. The latter provides standard resolution limited by diffraction limits, which gives a detailed overview of the sample through such techniques as optical sectioning or stitching. “You get a nice 3D image of the entire tissue,” Chang tells Lab Manager. Then, operators zoom in with super-resolution imaging for a very high-resolution image. A type of light microscopy, super-resolution imaging acquires images beyond the diffraction limit. “The combinatorial technique puts super-resolution information within the context of the larger tissue,” Chang adds.
Another potential combination uses a device to illuminate one region of a cell to activate a signaling pathway, followed by monitoring of the effects through super-resolution imaging. Still another idea merges light microscopy with electron microscopy (EM) for what Dr. Chang terms “correlative imaging,” which bridges the resolution capabilities of two or more techniques.
All experts interviewed for this article agreed that, at the high end at least, microscopes are becoming feature-rich but simultaneously too rich for individual research groups. “It’s hard to find funds for sophisticated imaging systems for each lab that uses them,” says Lynne Chang. More universities are writing instrument grants for locating high-end microscopes into core facilities. “Many of these installations will be multi-modality systems.”
Lights, camera, automation!
Dennis Doherty, national sales manager at Prior Scientific (Rockland, MA), agrees that the trend in microscope sales is toward fewer instruments with greater capability. “Sales volume is down, but sophistication is up,” he says. “Today, groups pool funds, and instead of purchasing two or three microscopes that do different things, they purchase something that can do everything they need plus more.”
As much as any instrument, microscopes have evolved with the disciplines they serve. “At one time 10- or 20-micron resolution was fine,” Doherty observes. “Then it was 5 microns, then 1, and now we’re looking at nanometer resolution.” In the past, cameras were limiting factors. Today, laser and LED light sources switch in microseconds; motion control and speed issues are improving, both for data/image acquisition and for mechanically moving from one position within the field to another.
Conversion to solid-state lighting from mercury lamps has been advantageous on several levels. Mercury is toxic, and lamps employing that element are energy hogs. Because mercury lamps take time to warm up to operating status, they typically remain on all day. Solid-state lamps are on only when needed. “They last for tens of thousands of hours and are exponentially more efficient,” Doherty says.
Automation has positively affected microscopy, particularly as investigators move to microplate-based, multichannel experiments. As with HPLC and spectroscopy, vendors have introduced plate handlers specifically for microscopes, as well as automation for more traditional slides. “As your acquisition speed increases, you don’t want someone sitting in front of a microscope plunking a slide down every five minutes,” Doherty notes.
Pushing the limits
Phil Bryson, VP for nanotechnology systems at Hitachi High Technologies America (Gaithersburg, MD), says that extending scanning electron microscopy (SEM) resolution from a long-standing limit of 4 angstroms to 1 angstrom (download the Hitachi paper at http://bit. ly/1hZFdHs) enables significantly improved throughput for semiconductor manufacturers.
“The instrumentation allows a chip company to do a hundred samples per day, compared with ten or twelve using lower-resolution technology,” Bryson says.
Compared with nominally higher resolution transmission electron microscopy (TEM), SEM involves little or no sample preparation: Scanning typically occurs on bulk samples. With TEM, technicians must cut a sample to the proper thickness—30 to 40 nm—to allow electrons to pass through. The process is costly and time-consuming.
“TEM instrumentation alone is ten times more expensive than SEM, but to prepare samples you also need a focused ion beam, which costs about $1 million.”
Until recently, labs that required resolution better than 4 angstroms were limited to TEM. By extending the capabilities of less expensive SEM, analysts have gained resolution and throughput, at lower cost.
“This represents a significant extension of SEM imaging, which has been very well received,” Bryson says.
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