Microscopy is an important analytical tool for evaluating the architecture of cells, and valuable information can be obtained using light and electron microscopy methods. Conventional microscopy techniques, however, are generally limited to the investigation of isolated cells and often require the fixing, staining, and dehydration of the cells before examination. The evaluation of living cells using microscopy techniques has the potential to provide additional information on the native structures, functions, and kinetics of cells and their components. Nongjian (NJ) Tao, director of the Center for Bioelectronics and Biosensors at Arizona State University’s Biodesign Institute, is addressing this issue through the development of new microscopy methods that combine multiple imaging technologies and can be applied to the evaluation of molecular-scale phenomena in living systems.
Specifically, Tao has developed a microscopy technique based on plasmonic resonance that can be used to evaluate cellular chemistry and biology in real time. “The trick to studying cells at this level is to use a system that has very high resolution but also can record images at very rapid rates,” he says. Previously Tao applied plasmonic imaging to the analysis of single cells, cell organelles, viruses, and nanoparticles. Currently, he is focused on the investigation of single protein-protein interactions and recently published results of a study of membrane proteins (1).
Overcoming physical limitations
The spacial resolution of conventional optical microscopy techniques, such as fluoresce and confocal imaging, is limited to 200–300 nm due to the diffraction limit of light, which prevents the examination of cellular components and biochemical compounds such as proteins. Although electron microscopy can achieve the necessary resolution, it is not suitable for living systems because electrons typically can’t penetrate through water and in addition can cause damage to biological material. Atomic force microscopy, meanwhile, can be used to evaluate biological samples but does not have the necessary imaging speed. Furthermore, as mentioned above, sample preparation for even the most advanced microscopy methods poses additional issues. Finally, label-based methods often require complex synthetic steps, are often only end-point assays that do not follow fast kinetics, and there is always concern that the label will affect cellular activity.
Surface plasmon resonance microscopy (SPRM) on the other hand can achieve high resolution imaging at very rapid rates. In plasmonic electrical impedence imaging (P-EIM), an electrical impedance signal from a living cell is detected optically via surface plasmon resonance. Cell components with different charge and ion distributions affect the signal differently, and the responses to these changes in an electric field are used to generate images. Similarly, plasmonic-based electrochemical current imaging (P-ECM) creates precise images of electrochemical reactions based on changes in an optical signal produced by surface plasmon resonance caused by variations in the electrochemical current density. With the new plasmon resonance microscope, it is possible to noninvasively investigate fast biochemical reactions and interactions that take place in living cells without labels.
In addition, with the new technique, surface plasmon resonance microscopy can be carried out simultaneously with optical and fluorescence imaging, so that if desired, label-based techniques can be employed as well.
Why membrane proteins
“We have elected to study membrane proteins because they are involved in many basic cellular processes. They are the target of many new drug candidates, and many have been recognized as biomarkers of a number of diseases,” notes Tao. “Understanding the binding behavior of membrane proteins to potential drug molecules and ligands for biomarker detection is very important, but to date has been very difficult. With our new label-free imaging method, we are able to measure the binding kinetics of membrane proteins in single living cells and thus investigate their natural behavior,” he continues.
Using the new microscopy method, Tao and his colleagues were able to monitor the interactions between lectin and glycoproteins, the binding activity and distribution of nicotinicacetylcholine receptors (nAChRs) on the membrane of a single cell, and cell-migration behavior on a millisecond time scale with a spatial resolution of submicrons to microns.
Glycoproteins, or proteins with attached sugar chains, were chosen because they are ubiquitous and involved in cellular communication and recognition via interactions with numerous biochemicals. Lectins are proteins that bind to specific sugar chains of glycoproteins on cell membranes. Thus, investigation of these interactions can provide information about the location and role of sugar structures in cellular activity, according to Tao. Specifically, lectin wheat-germ agglutinin (WGA) was studied by the researchers because it selectively binds to N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (sialic acid) sugar residues and can provide information about cell-surface glycosylation of SH-EP1 human epithelial cells (1).
Using SPRM, it was possible to observe the WGA interacting and associating with the glycoproteins on individual cells and then dissociating from the cell surface (1). In addition, a comparison of the behavior of fixed and unfixed cells demonstrated that while the association rate was similar for both, the dissociation rate in the live cells was much slower than that in the fixed cells. Further experiments confirmed a first-order binding kinetic process, and that the WGA did indeed bind to the GlcNAc of the membrane proteins on the top surface of the cells.
Observation of cellular migration
Cell migration and other cellular processes occur as the result of polarization or redistribution of glycoproteins on the cell membrane, thus Tao and his colleagues used SPRM to investigate glycoprotein polarization and redistribution during chemotaxis of a living cell (1). Images of live SH-EP1 cells were obtained before and after inducing cell migration using the chemoattractant fetal bovine serum (fbs). The WGA was bound at the leading edge of the cell prior to migration, and after exposure to the fbs, the average glycoprotein density in the leading edge of the cell increased by 28% and redistribution occurred even before the leading edge of the cell migrated.
"Because SPRM can be used to investigate chemical and electrochemical reactions and the interactions of proteins and other cellular biochemicals within living cells, we believe that this new microscopy method will make it possible to gain new insights into cellular activity, information that can be applied to the development of new drugs,” says Tao. This new technique may also have the potential for use in the analysis and monitoring of cell-based biopharmaceutical manufacturing processes.
1. W. Wang et al., Nat. Chem. 4 (10) 846-853 (2012).