November 1, 2012 (Vol. 32, No. 19)

Super-resolution microscopy is revolutionizing the ability to probe subcellular features as small as single molecules. Surpassing traditional limitations of conventional optical microscopes, these new technologies permit very precise visualization and measurement of features that are below the limits of diffraction.

The super-resolution toolbox includes localization microscopy that identifies the location of individual fluorophores a few at a time, structured illumination microscopy that allows a three-dimensional view, and hyperspectral confocal imaging, among others. Applications for the new technologies range from depicting the fine architecture of cellular immune processes to creation of improved biofuels.

Pavel Tolar, Ph.D., program leader in the division of immune cell biology at MRC National Institute for Medical Research, provides a perspective on several current imaging technologies.

“Traditional fluorescence microscopy is hampered by the resolution limit set by the diffraction of light that is ~200–300 nanometers. Recently, a number of novel instrument-based fluorescence approaches have been employed to circumvent these limitations. We are now seeing imaging down to a resolution that approaches molecular scale (10–15 nm).”

An example of such nanomolar-scale imaging is that of fluorescent resonance energy transfer (FRET), a single molecule technology in which energy is transferred from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor).

“FRET is well-suited to detect protein interactions as well as conformational changes. This provides a remarkable visualization of dynamic protein function to monitor protein-protein interactions (e.g., how signaling complexes assemble) where the distance of interaction is from 2–10 nm. For that reason it is useful for drug screening. But, it is also limited by its low throughput.”

According to Dr. Tolar, the most accessible of these new technologies are photo-activated localization microscopy (PALM) and the related stochastic optical reconstruction microscopy (STORM).

“PALM/STORM are based on the detection and very precise localization of individual molecules. Both employ photo-activatable fluorescent labels for localizing many molecules in a sequential manner using repetitive cycles of activation and imaging. The end result is generation of a high-resolution image that maps the positions of all the molecules monitored. The technology currently is only limited by the number of molecules that can be activated at one time, their brightness, and the rate of photobleaching.”

Deciphering Spatial Relationships

Although signaling complexes have been extensively studied along a number of fronts with numerous biochemical and imaging techniques, many questions remain. An example is signaling downstream of the T-cell antigen receptor (TCR).

“The spatial organization of individual molecules within T-cell signaling complexes or microclusters is largely unknown,” notes Valarie Barr, Ph.D., staff scientist, Laboratory of Cellular and Molecular Biology (LCMB), Center for Cancer Research at NCI. “Issues include heterogeneity in complexes, complete size distribution, arrangement in the plasma membrane, and mechanism for formation.”

Dr. Barr and Eilon Sherman, Ph.D., in collaboration with other colleagues in the LCMB, utilized PALM technology to characterize the organization of signaling molecules during T-cell activation. According to Dr. Barr, “To decipher receptor-regulated cellular signaling (a process involving transient, heterogeneous complexes of undefined structure), we decided to use single and two-color PALM. This is useful particularly to study complexes in the plasma membrane of intact T cells that are downstream of the TCR.”

They first examined the organization of the TCR adapter protein LAT by conventional fluorescence microscopy (diffraction, confocal, and total internal reflection microscopy). Dr. Sherman says they found a few surprises.

“We found that LAT microclusters accounted only for a small portion of the total LAT molecules on the cell. We next employed PALM imaging because it allowed us to observe individual LAT molecules that were tagged with photo-activatable fluorescent proteins. We measured the probability density of locating individual LAT-tagged molecules with a precision of ~20 nm. We also analyzed the scale of LAT clustering using pair-correlation functions. We determined that the majority of LAT molecules were localized to very small nanoclusters of 2–4 detected molecules.”

Dr. Sherman notes that PALM also permitted following of the interactions of the phosphorylating enzyme ZAP-70 and other associated TCR-signaling molecules. Dr. Sherman concludes, “Zap-70 preferentially phosphorylates LAT especially in sites where LAT co-resides with the TCR zeta chain. Overall, our observations provide an example of how PALM technology can help to better discern the complexities of the organization of such signaling complexes as well as the mechanisms involved in shaping how they are arranged in the plasma membrane.”

PALM image of an activated Jurkat T cell: Two signaling proteins are shown. Linker of activation in T cells (LAT) conjugated to the photoactivatable protein dronpa is shown in green, while SH2 domain-containing leukocyte protein (SLP) conjugated to the photoactivatable protein PAmcherry is shown in red. The dots show individual molecules and the insert shows a close-up of the middle portion of the cell. [NCI]

Blink Technology

A super-resolution imaging technique, Blink Microscopy (Blink), uses sequential, sparsely distributed single molecule imaging to create maps identifying the precise locations of individual molecules down to a level of ~30 nm. “Blink can exploit the fluctuating emission of fluorophores that are able to photoswitch from a bright to a dark state,” explains Aaron K. Neumann, Ph.D., professor, department of pathology, University of New Mexico School of Medicine.

“My colleagues, Ken Jacobson, Ph.D., principal investigator at the University of North Carolina at Chapel Hill, and Philip Tinnefeld, Ph.D., principal investigator at Technische Universitat Braunschweig, and I used Blink to investigate the lateral distribution of a dendritic cell membrane protein called DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin, also called CD209). This protein binds to a large range of pathogens such as HIV and Ebola virus. DC-SIGN presents as tetramers in microdomains on the cell surface.”

Because Blink is based on the sequential localizations of single dyes used for immunofluorescent labeling, the precise location of individual DC-SIGN probes could be determined. “Basically we generate movies taken while the dyes were induced to blink in the presence of reducing and oxidizing agents. Most dye is driven into a dark state under chemical conditions used, while a small number of total dyes activate at one time. Each point in a Blink image represents a precise location of a single molecule generated by fitting the spatial distribution of the signal to a Gaussian function.”

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.”

Mode synthesizing atomic force microscopy scheme for lignocellulosic biomass characterization at the nanoscale. The signal of the detector, S(t), is decomposed in a frequency spectrum using a spectrum analyzer. The new synthesized modes (modes 1 to n) correspond to frequency mixing between the tip and the sample oscillations, as a result of their nonlinear interaction. [Oak Ridge National Laboratory]

Shining New Light on Bacterial Division

Australian researchers are utilizing GE Healthcare’s super-resolution microscope, the Delta Vision OMX Blaze™, technology to probe the process of bacterial cell division, a project that may ultimately contribute to the development of novel antibiotics. A recent paper in PLoS Biology described research conducted at the ithree institute at University of Technology Sydney that used this technology.

Because of the tiny size of bacterial cells, it is a challenge to image the spatial organization of cellular proteins. In their study, the investigators used the GE Healthcare system, which is a new type of super-resolution microscopy called three-dimensional structured illumination microscopy, to localize cell division bacterial proteins in both the spherical Staphlococcus aureus and the rod-shaped Bacillus subtilis. Using this new technology, the researchers say they’ve completely revised the model of how bacteria divide and multiply.

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