Two Photon Microscopy

Two-Photon Microscopy: by Tina Zhang and Nicole Pepperl

engineering + technology

Tracking Cells with

Light Richard Lews

T

ry pressing a flashlight against your fingertip, and you’ll find that it produces an eerily pink glow reminiscent of E.T.’s glowing fingers. This phenomenon occurs because light can penetrate a small distance through living tissues. Transillumination is used in two-photon microscopy to create nanoscale images of living tissues. Stanford scientists have been using and perfecting this method of imaging live cells to gain a better understanding of cellular function in our bodies. 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 Richard Lewis, Professor of Molecular and Cellular Physiology at the School of Medicine, uses two-photon microscopy to investigate the effects of calcium signaling in lymphocytes, a type of white blood cell, on gene transcription and cellular development. During lymphocyte development in the thymus, each new T cell undergoes a process that determines whether the cell survives or dies. Although the mechanism is not yet completely understood, calcium signaling is involved in this life-or-death choice. Lewis’ research began with a serendipitous discovery. His group found that partial depletion of calcium ions from the endoplasmic http://www.grc.nasa.gov/WWW/RT/2003/6000/6712fischer.html reticulum of mature T An image of mouse kidney cells taken from a cells caused big calcium two-photon microscope. These microscopes ion oscillations. The can provide detailed images of tissue. This researchers then went image is 134 nanometers2. on to ask whether

layout design: design:Anita Anita Verma

oscillations might be useful for cell signaling, and it turned out that they were – they increased the efficiency and the specificity 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 Richard Lews the body, cells interact in a three-dimensional matrix, The highest ion velocity corresponds with not on a two-dimensional low calcium levels, indicating that they surface,” Lewis explains. On are inversely related. 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

The lower energy photons result in highly localized excitations that minimize both photobleaching of the fluorophores and photodamage to the cells. 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

volume iv 41

engineering + technology

stop moving, and if you let the calcium level go back down, the cell will start crawling away.” Oscillating calcium levels in thymocytes create a balance between their ability to move from cell to cell and their ability to stop and generate calcium signals when they encounter antigens. According to Lewis, a problem arises when thymocytes barely move at all and fail to reach any antigens. On the other hand, some thymocytes are so motile that they whiz by antigens, not stopping long enough (at least half an hour) to receive a signal for destruction of the antigen..

See-Saw Microscopy:

Comparing Confocal and Two-Photon Microscopy

http://www.ornl.gov/info/ornlreview/v37_3_04/article06.shtml

Courtesy Richard Lews

Future Applications of Two-Photon Image of thymocytes taken by a two-photon The light beam of a confocal fluorescent Microscopy microscope. Only thymocytes were dyed so microscope. Confocal microscopes use The Lewis group also uses two-photon all other cells appear black. Red dots are the only one photon of light to create images by microscopy to determine “what role deepest cross-section of cells and gray dots are exciting the target to emit light. differences in calcium signaling play in the most superficial. telling cells to live or die.” Understanding • Laser scanning with light in the visible spectrum, • Pulsed-laser scanning with infrared light, which more about calcium signaling will help where severe blasting of cells with lasers leads to has longer wavelengths and is able to penetrate researchers discover new ways to boost phototoxicity, damaging cells permanently. tissues farther by at least 100 microns. our immune systems or make them more • Emits a single beam that illuminates a cone of • Emits a single beam that illuminates only the effective. “Usually researchers are just light above and below the focal point. focal point. looking at the cells themselves, seeing whether they touch each other,” explains • Has a pinhole that rejects some light from • Less light is rejected so the signal is stronger, Lewis. “By looking at how cells behave everywhere but the focal point and light is focused at the focal point. you can formulate lots of hypotheses, but • Because light detection is low, prolonged • Only molecules at the focal point are excited to sooner or later you have to look at where illumination leads to progressive fading of fluoresce, resulting in less damage to cells and the molecules are and what the signals the emitted light, a phenomenon called prolonging the time during which the cells can being generated are.” photobleaching. be examined. Other research groups at Stanford are also benefiting from two-photon microscopy. Professor Mark Schnitzer’s group in the Departments of Biological Sciences Like many freshmen, Nicole Pepperl is undeclared and undecided. In and Applied Physics recently created a portable two-photon her spare time she enjoys reading, writing and providing a running fluorescence microendoscope, useful for two-photon imaging commentary during movies. in biomedical applications. This microscope uses two-photon microscopy as well as tiny fiber-optic tubes that can explore and Tina Zhang, on the other hand, is among the dedicated pre-meds. She transmit images of deeper areas that two-photon microscopy can’t does not have spare time, but if she did she would enjoy jogging and reach. Who could have imagined the profound implications for playing Go. biomedical research E.T.’s glowing fingers held? S

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 a wide range of immunological research, including more information on thymocytes. http://www.nature.com/ni/

Slide preparation: Thymocyte motion and signaling is visualized by thymic slice preparation with two-photon microscopy. High calcium levels in cells

42 stanford scientific

Cell Sciences Imaging Facility (CSIF) website – the Stanford research facility that houses 2-photon and confocal microscopes. http://taltos.stanford.edu