Tracking Cells with Light
by Tina Zhang and Nicole Pepperl
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| The highest ion velocity corresponds with low calcium levels, indicating that they are inversely related. |
Overview of Two-Photon Microscopy
Two-photon microscopy uses a high-intensity laser to excite certain dye compounds called fluorophores, which are bound to cellular structures of interest such as proteins. The fluorophores, in turn, emit characteristic photons that are detected to form an image as the microscope scans across a cell. Two photons are required since they each carry half of the energy needed to excite the fluorophore. The lower energy photons result in highly localized excitations that minimize both photobleaching of the fluorophores and photodamage to the cells, allowing powerful magnification of living cells.
The Role of Calcium
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| An image of mouse kidney cells taken from a two-photon microscope. These microscopes can provide detailed images of tissue. This image is 134 nanometers^2. |
Lewis' research began with a serendipitous discovery. His group found that partial depletion of calcium ions from the endoplasmic reticulum of mature T cells caused big calcium ion oscillations. The researchers then went on to ask whether oscillations might be useful for cell signaling, and it turned out that they were - they increased the efficiency and the specifi city of gene expression in T cells studied in vitro.
However, "there are certain things that cells do in vivo that just aren't well replicated in vitro. In the body, cells interact in a three-dimensional matrix, not on a two-dimensional surface," Lewis explains. On the other hand, measuring calcium signals on a 2-D surface such as a petri dish is much easier than measuring signals in a 3-D environment such as the body because in the latter, light from the microscope is scattered by surrounding cells, and the resulting image is blurred. In order to study thymocytes (developing T cells) in vivo and also produce a clear image, Lewis turned to two-photon microscopy.
Effect of Calcium on Cell Movement
As a result of two-photon microscopy's dexterity, Lewis' research group made a remarkable discovery. Thymocytes labeled with calcium indicator dyes were observed to have low calcium levels during movement, but high calcium levels when they were stationary. Lewis treated the thymocytes with varying levels of calcium, and observed that once thymocytes reach an antigen (a foreign substance), calcium signals they generate act as a positive feedback to keep the thymocytes stationary. Decreasing calcium signals sets them in motion again. Lewis concluded a causal relationship: "if you increase the calcium inside the cell it will stop moving, and if you let the calcium level go back down, the cell will start crawling away."
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| Slide preparation: Thymocyte motion and signaling is visualized by thymic slice preparation with two-photon microscopy. |
Future Applications of Two-Photon Microscopy
The Lewis group also uses two-photon microscopy to determine "what role differences in calcium signaling play in telling cells to live or die." Understanding more about calcium signaling will help researchers discover new ways to boost our immune systems or make them more effective. "Usually researchers are just looking at the cells themselves, seeing whether they touch each other," explains Lewis. "By looking at how cells behave you can formulate lots of hypotheses, but sooner or later you have to look at where the molecules are and what the signals being generated are."
Other research groups at Stanford are also benefiting from two-photon microscopy. Professor Mark Schnitzer's group in the Departments of Biological Sciences and Applied Physics recently created a portable two-photon fluorescence microendoscope, useful for two-photon imaging in biomedical applications. This microscope uses two-photon microscopy as well as tiny fiber-optic tubes that can explore and transmit images of deeper areas that two-photon microscopy can't reach. Who could have imagined the profound implications for biomedical research E.T.'s glowing fingers held?
To Learn More
Optics Letters - a scientific journal featuring papers discussing the latest advancements in imaging technologies: http://ol.osa.org
Nature Immunology - another journal featuring papers on wide range of immunological research, including more information on thymocytes: http://www.nature.com/ni/
Cell Sciences Imaging Facility (CSIF) website - the Stanford research facility that houses 2-photon and confocal microscopes: http://taltos.stanford.edu
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