Microscopy is evolving toward greater functionality and capabilities. The Auriga FIB SEM platform from Carl Zeiss (Thornwood, NY) has been around for 11 years yet undergoes constant improvement. The system combines two distinct technologies: scanning electron microscopy (SEM) and focused ion beam (FIB). SEM’s capability of providing very high-resolution surface analysis is well known in the life sciences, materials, and semiconductor industries. SEM provides detail significantly beyond the diffraction limit of light microscopy, illuminating structures and events down to about 1nm.
The only drawback of SEM is its limitation to surfaces. This is where FIB enters the picture. FIB is a type of very-small-domain milling technology that uses ionized gallium to generate cross sections perpendicular to the plane of SEM observation, exposing them to SEM analysis. Like some advanced medical imaging technologies, the combination of FIB and SEM creates as many cross sections as the sample size will allow. Images generated in this way may be combined through appropriate analysis software into a complete 3-D image of the sample.
Markus Wiederspahn, communications manager at Zeiss, emphasizes that unlike using a sharp knife or scissors, where material is theoretically preserved, FIB removes extremely small volumes of sample material. “It’s more like milling than cutting,” he says. “It can even cut steel, but it is not meant to remove a huge block of material.” FIB channels are just a few microns wide.
Wiederspahn notes several interesting applications of FIB-SEM. In brain research, the technique can provide 3-D maps of lesions common in neurological structures, which may help in research on Parkinson’s or Alzheimer’s. Another example involves analyzing semiconductor chips for fabrication characteristics or even failure. “FIB-SEM can provide the most detailed view of isolation or insulation structures within microchips,” Wiederspahn notes. Successful deployment of—and the ability to analyze—these structures are in part responsible for the geometric increase in storage density (and price drops) for pen drives and other chipbased storage systems. In addition, some systems are capable of mapping elemental doping within chips—an important quality check that fabrication is proceeding as intended.
Fluorescence microscopy allows, among other capabilities, simultaneous detection of multiple events at the cellular level and live-cell imaging. In Archives of Pathology and Laboratory Medicine in early 2011, University of Massachusetts pathologist Andrew Fischer, M.D., wrote that live-cell imaging has transformed microscopy from relying on static events to interpreting dynamic cellular process. “New techniques have bypassed by about 100-fold what had long been believed to be a limit to the resolution of light microscopy” and allows visualization of “events in living or fixed cells that are immeasurable by other molecular techniques.”
Meanwhile, Prof. Gabriel Popescu and coworkers at the University of Illinois’ Beckman Institute have developed a novel fluorescence microscopy technique that for the first time examines critical cellular transport events at multiple spatial and temporal scales to study diffusive and directed motion transport in living cells.
The technique, dispersion-relation fluorescence spectroscopy (DFS), attaches fluorophores to molecules whose motion produces spontaneous fluorescence intensity fluctuations that when analyzed provide information on mass transport dynamics—what is moving, where, and when—within the cell. Says graduate student Ru Wang, “The beauty of this method is that you can use a commercial fluorescent microscope that is found everywhere to collect and analyze data in a very simple way. You don’t need complicated expertise. Everyone can use it.”
Renaissance in visible microscopy
“This is the most exciting time in microscopy since Abbe,” says Paul Goodwin, science director at Applied Precision (Issaqua, WA), a GE Healthcare company. He is referring to Ernst Karl Abbe (1840–1905), an optics pioneer who, with Karl Zeiss and Otto Schott, practically invented the microscope industry.
According to Goodwin, microscope optics have not changed much since Abbe’s day. “Improvements to optics have been evolutionary, not revolutionary.” What have spawned the new age in microscopy have been the confluence of optics, camera technology, and mechanical systems and the emergence of cheap, powerful computing.
Several decades ago the idea of attaching cameras and computers to microscopes was the stuff of science fiction. Digital photography was in its infancy, and data storage was prohibitively expensive. “Today this setup is standard for any organization doing serious biology,” Goodwin says.
GE has been committed to high-content cell analysis and screening for some time. Its IN Cell Analyzer HCA imaging systems allow simultaneous analysis of morphology and multiple markers within the same cell. GE’s acquisition of Applied Precision in 2011 expanded its portfolio with additional high-end optical, computational, and machine control technologies.
The ability to study cells in three dimensions with a time element has revolutionized biology and greatly expanded the utility of cells in pharmaceuticals, industrial toxicology, fermentation, and basic science.
Early optics pioneers recognized that the wavelength of light introduced a diffraction limit—a fundamental boundary of what was observable under a microscope. Since many cellular details of interest fell just below the magnification capability under visible illumination, diffraction limits were like a thorn in the side of microscopists. Mitochondria, for example, are approximately 0.25 micron in diameter.
Recently the combination of computation, optics, and computer control has pushed resolution well beyond this value. “Instead of being limited to 0.25 micron laterally and 0.6 micron between section planes, we’re now looking at 100nm laterally and 300nm axially,” Goodwin tells Lab Manager. The additional resolution enables not just visualizing mitochondria but also looking inside them.
“And in the past year we’ve demonstrated this capability not only in fixed cells but also in living cells.” Applied precision is working on other super-resolution methods that currently obtain resolution on the order of a few tens of nanometers, but only on fixed samples.