One of the challenges of Blink, as with other high-resolution imaging methods, is data analysis. “Lots of quality control is needed for image setup and data analysis. Many researchers must custom-build their own analytic techniques for each sample since what is supplied from manufacturers often does not meet individual needs.”
For Dr. Neumann, the Blink studies provided a new view of how DC-SIGN is organized. “We found that DC-SIGN localized to a small nanodomain of ~80 nm capable of binding viruses. Further, these nanodomains can be clustered into larger microdomains capable of forming a receptor platform for larger pzathogens such as yeast.”
Hyperspectral Confocal Imaging
Confocal microscopy is an indispensable biological tool that allows probing living or fixed cells with great sensitivity and spatial resolution in three dimensions. However, the technology suffers from key limitations, notes David M. Haaland, Ph.D., senior scientist, Sandia National Laboratories.
“Traditional confocal fluorescence microscopes generally utilize optical filters to define the spectral bands for photons reaching the detector. However, emission spectra can overlap, creating common problems of autofluorescence and spectral crosstalk.”
Dr. Haaland and colleagues developed a special type of technology called hyperspectral confocal fluorescence imaging.“This technology overcomes these challenges by recording the complete emission spectrum of the entire volume of the image voxels (i.e., 3D pixel). It allows imaging hundreds of spectral wavelengths per image using a custom prism spectrometer. By eliminating autofluorescence and crosstalk, the new technology can identify in the image all emitting fluorescent species and their concentrations without prior information.”
A major advantage of the technology is its exceedingly high spectral acquisition speed. “The hyperspectral microscope employs 488 nm laser excitation while collecting 512 spectral emission wavelengths at each voxel. This is all done over the spectral range of 500–800 nm at a spectral resolution of 1–3 nm and an imaging rate of 8,300 spectra per second. This high acquisition speed is accomplished by the incorporation of an electron multiplying charge coupled device with a new readout mode. Other similar microscopes do not have this extremely high speed.”
The sophisticated microscope generates highly complex data that requires an equally sophisticated mode of analysis. To handle the mounds of data collected, the researchers developed multivariate curve resolution (MCR) software.
“Our algorithmic approach provides for dramatically faster computation of the rigorous, constrained alternating least-squares MCR analysis. With the hyperspectral microscope and MCR analysis, we often discover features and components in living samples that no one has seen before.”
Atomic Force Microscopy
Sounding like something out of a science fiction novel, atomic force microscopy (AFM) and its cousin, mode synthesizing AFM (MSAFM), provide a powerful means to probe the subtleties of solid surfaces. “This is a new approach to material characterization over a wide frequency range. It can provide a wealth of information on the physical properties of the surface and subsurface of materials such as plant cell walls,” says Laurene Tetard, Ph.D., Wigner Fellow, Oak Ridge National Laboratory.
Dr. Tetard and colleagues from the BioEnergy Science Center are utilizing MSAFM on plant systems. “Multifrequency AFM technologies are still in their infancy, but they are emerging as important tools. MSAFM was imagined to offer a multimodal platform for nanoscale spatial resolution of complex materials such as plants.”
The first application of this technology to study plant cell walls was reported by Dr. Tetard and colleagues in Industrial Biotechnology and Nanotechnology. “We are employing this technology to learn how to improve the effectiveness of further chemical treatments that are involved in the conversion of polysaccharides to simple sugars for fermentation into ethanol and for biofuel.”
How does MSAFM work and what’s its resolution? “A standard episode of MSAFM consists of mechanically actuating the probe (a microcantilever) and the sample. The nonlinear nature of the tip-sample interaction results in synthesizing new operational modes in the system. The phenomena can be compared to difference and sum frequency generation in nonlinear optics. The resolution capability is similar to that of the atomic force microscopy, but offers the advantage of simultaneously mapping the properties of the sample with the same resolution.”
According to Dr. Tetard, the new technology has a number of advantages and applications. “Today, electron microscopy and scanning probe microscopy are the main techniques used to image and study materials at the nanoscale. MSAFM offers the advantage of simultaneous channels over a wide frequency range to study the properties of the sample at the nanoscale.
“In addition, MSAFM is nondestructive, can be operated in air or liquid, and can be configured for subsurface imaging. Possible applications for the new technique include cracks and defects detection for the semiconductor industry, studies of nanoparticles-cell interactions in nanotoxicity, and in plant cell biology.”
Since this is a young and emerging technology, there are some issues that need addressing. “Some of our current work involves quantitative calibration and development of the technique for liquid imaging.”
According to Dr. Tolar, fluorescence microscopy will continue to break the barriers of resolution limitations and interface with other technologies.
“As resolution continues to improve, it will eventually reach into the subnanometer range where it can see the architecture of cellular complexes, much like electron microscopy. It also is a nice complement to the atomic resolution of crystallography, nuclear magnetic resonance, and cryo-electron tomatography. One of the most compelling prospects is the further delineation of mechanistic processes of molecular signaling.”