47 | No. 1 | 2009. Features. Microscopy and. Image Analysis. A Closer Look ... he says. His team uses a focused beam of light to measure elastic (angular) ...
Features
Tech News Microscopy and Image Analysis
ultimately to visualize lipid domains— including the interaction of ligands with their receptors—in living cells.
Shining a Light
A Closer Look
A variety of techniques are allowing a closer look into the lives of individual cells and even the organelles and molecules they contain. Approaches include the use of intra-vital confocal microscopy and in vivo flow cytometry to visualize cell migration in intact animals, and near-infrared night vision technology to follow cells in the lymphatic system. These technologies rely on the use of fluorescent labeling, though non-fluorescent strategies are being developed as well.
Natural Fluorescence Ahmed A. Heikal is interested in understanding how molecules behave in different cellular environments and conditions. Currently Associate Professor of Bioengineering at Pennsylvania State University (University Park, PA), Heikal has accepted the position of Associate Professor of Chemistry and Biochemistry and Associate Professor of Pharmacy at the University of Minnesota, Duluth (Duluth, MN), where he will start in late August 2009. He studies nicotinamide adenine dinucleotide (NADH)—a coenzyme abundant in the inner membrane of mitochondria— and exploits this naturally fluorescent molecule as a biomarker in metabolic studies. His group is using a combination of spectroscopy and microscopy to measure NADH concentration in cells, and to determine how much is bound and how much is free. The goal is to monitor mitochondrial activity and to distinguish between cells in different disease or metabolic states. Heikal notes that his group’s approach is non-destructive to the cells being observed since it avoids the use of labels (e.g., f luorescent compounds), which are potentially toxic. These labels can also bind nonspecifically to structures of little interest (the group’s strategy is explained in Figure 1). Future targets of research include an animal model for skin cancer in which different stages of tumor progression can be studied. They are also interested in both artificial and natural biomembranes, how lipids form domains, and how proteins are “given permission” to cross membranes into and out of cells. Vol. 47 | No. 1 | 2009
Figure 1. Intracellular structures in living cells can be visualized using a non-destructive, auto-fluorescence technique. (A) The conformation and surrounding environment of intracellular NADH in living cells can be probed using two-photon (2P) fluorescence lifetime imaging microscopy (FLIM). (B) These 2PFLIM images, in a calibrated microscope, allow for converting fluorescence intensity into intracellular NADH concentration in living cells. This approach provides an alternative to biochemical techniques that require cell destruction. (C) Using time-resolved autofluorescence anisotropy of intracellular NADH, the relative population of free and enzymebound cofactors could be directly quantified at the single-cell level. Courtesy of Ahmed Heikal, Pennsylvania State University.
Heikal thinks that understanding the biophysical properties of membranes may lead to an understanding of biological properties, such as cell signaling. Giant unilamellar vesicles (GUVs) have been used as membrane models with fluorescent labeling, but these models contain only a few of the many types of lipids found in cell membranes. Blebs, which are isolated from living cells, contain more natural bilayers, and although they are isolated from the cytoskeleton, they may provide a more natural model. Heikal is hoping
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Andrew Berger, Associate Professor of Optics at the Institute of Optics at the University of Rochester (Rochester, NY), is using a light-only, non-label approach to analyze single cells. “The idea we are working on is using a single beam of light to tease two pieces of information out of the cell,” he says. His team uses a focused beam of light to measure elastic (angular) scattering by photon diffusion (which provides structural information) and inelastic scattering by Raman spectroscopy (which provides information about the relative abundance of chemicals such as proteins and DNA). They are currently looking at human CD8+ cytotoxic T lymphocytes that have been negatively selected by flow cytometry. Though Berger says that there were a number of technical hurdles to overcome, he says it was most exciting to be able to sort immune cells that had been activated from those that had not. He would like to look at an isolated cell and observe the time sequence of immune activation, the speed of response, and what the changes are both in organelle size and in chemical content. “People have speculated that they can see decondensation of chromatin,” Berger says, explaining that his team is correlating reduced amounts of DNA measured by spectroscopy with decondensation, and therefore a possible increase in transcription. He says that his group has also observed a drop in DNA signal strength relative to the protein signal in stimulated immune cells.
“People have speculated that they can see decondensation of chromatin.” For Berger, avoiding f luorescence labeling is a “self-imposed boundary. We are doing nothing to cells, so there is no prep time and no alteration of cellular function. FACS [fluorescence activated cell sorting] is the gold standard, but there is a niche for www.BioTechniques.com
Features
not labeling. If you label a cell, what it does over time will be different.”
A Place for Fluorescence
Benjamin K. Chen of the Mount Sinai School of Medicine (New York, NY) and colleagues at the University of California Davis Center for Biophotonics Science and Technology (Davis, CA) recently showed how human immunodeficiency virus (HIV) can be transferred directly from an infected T-cell to an uninfected T-cell through structures they call “virological synapses.” The creation of an infectious clone of HIV that expresses GFP made visualization of viral transfer possible. Previously, cell-free virus was the focus of studies on HIV infectivity. Strategies to block HIV infection at the cellular level will now have to take both mechanisms of infectivity into account. “It’s appropriate that the discovery and development of GFP was awarded the Nobel prize,” says Chen. “GFP is transforming cell biology in the same way that PCR has transformed molecular biology.” There are caveats to the use of GFP, he says, noting that it is a big, globular protein, often fused to a protein of interest that is
not much larger. Nevertheless, he says, it’s possible to track biologically meaningful events using GFP as long as one knows that one is studying a biologically intact system. In the future, smaller peptide tags may be developed to overcome this problem. He also suggests that better infrared (IR) dyes for tissues would be another forward-looking approach, though current IR technology is expensive and “not there yet.” He does believe that true IR fluorophores should, like GFP, exist in nature and be able to be introduced similarly into animal models. Chen’s group has utilized the knowledge of HIV biology to insert the GFP gene into a whole HIV provirus while maintaining infectivity. HIV’s core gag protein is modular, consisting of four major subdomains connected by linker regions that are cleaved by viral proteases. Chen assumed that it might be possible to insert GFP with similar linker regions and maintain viral infectivity; this assumption was correct. The packaged viral particles contain the four native gag subunits plus GFP in stoichiometric amounts, so that there are ~5000 fluorescent molecules per virus. “That makes it easy to use as a label to track where it’s going,” Chen explains.
His group examined the construct’s infectivity in T-cell lines and were “surprised at a couple of fronts.” The gag protein, although already on the surface of the infected cell, was not concentrated at any locus. Once the infected cell adheres to an uninfected cell, the gag protein moves in the membrane, which was a finding not seen in previous studies of fixed cells. “Also not appreciated,” says Chen, “was that the transfer process morphologically is an endocytic process. The recipient cell internalizes the virus into some kind of endosome; we don’t know yet what kind. It’s challenging to characterize endosomes in T-cells.” Chen says that until his work, T-cells had not been considered a “highly endocytic cell type.” He would like to be able to use more than one color fluorophore at a time, and observe T-cells in their native environment, in tissues, and ultimately in intact animals. “We will need to discover how the virus is moving around in vivo. It’s bound to be different than in vitro.” Written by Lynne Lederman, Ph.D. BioTechniques 47:579-581 ( July 2009) doi 102144/000113189
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