Introduced in 1981, video-enhanced differential interference contrast (DIC) provides the ability to visualize subresolution phase objects such as single 25-nm-diameter microtubules. These organelles serve as tracks in cells, along which motor proteins move to transport cargo. In the early years of the technique, researchers struggled with the question of how to find a stable and uniform light source that had a narrow bandwidth. A fiber optic light scrambler introduced in 1985 addressed this issue by transforming the nonuniform light emission from an arc lamp into a uniform light disk.Investigators use arc lamps with fiber optic light scramblers as light sources for video-enhanced DIC imaging. However, the heat emission from these devices can create trouble during experiments. “The arc lamp typically used [for DIC imaging] is normally the hottest object in the room,” said Volker Bormuth, an investigator with Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany. “Temperature gradients cause thermal drift, which is detrimental during single-molecule experiments with nanometer precision.”Researchers have reported the use of LED illumination for video-enhanced differential interference contrast imaging of single microtubules. The low heat emission from LEDs reduces thermal drift during experiments. Also, LEDs generally have a very narrow bandwidth, simplifying the experimental setup. Images reprinted with permission of the Journal of Microscopy.For this reason, Bormuth and his colleagues devised a study in which they used LED illumination for video-enhanced DIC imaging of single microtubules. As reported in the April issue of the Journal of Microscopy, they showed that the low heat emission from the LEDs reduced thermal drift during the experiments. They therefore recommend the source for use with other single-molecule techniques, such as optical trapping.Initially used only as indicator lamps in electronics equipment, LEDs have evolved over the past couple of decades to become bright, stable light sources that offer the possibility of microsecond switching. Investigators are replacing conventional lamps with LEDs for fluorescence applications, and commercial providers have begun to offer LED illumination for bright-field microscopy.In the Journal of Microscopy study, the researchers developed a simple, compact LED condenser using Luxeon Star devices made by Philips Lumileds of San Jose, Calif. Introduced about seven years ago, these “power LEDs” operate at much higher power than conventional indicator LEDs — 350 mA or more compared with 20 to 25 mA — and, therefore, offer much higher light output. According to the company’s Steve Landau, the performance of the LEDs is continually improving. Whereas seven years ago a white Luxeon LED operating at 350 mA might give off 8 to 13 lm, the same drive current today will give off 70, 80 or even 90 lm. Thus, the LEDs offer a viable alternative for use as light sources in microscopy.Bormuth noted several advantages to using the Luxeon Star devices, and LEDs in general. First, although the diodes still are not as bright as arc lamps, they are “absolutely in the range where no compromise is necessary.” Additionally, because LEDs have a very narrow bandwidth, they do not require filters to select colors. Thus, the need for many of the optical elements used with arc lamps is eliminated, simplifying the setup and facilitating adjustments during experiments.Also, LEDs have very long lifetimes when compared with arc lamps. “Our light is always on; when we come in the next day, everything is thermally equilibrated so we can directly perform our measurements without further delay,” Bormuth said. “Normally, we would not leave a light source on overnight or for several days because of limited lifetimes.” The investigators tested blue, red and green LEDs for video-enhanced DIC imaging by visualizing and tracking the ends of single microtubules. The experimental setup was based on an inverted Zeiss microscope, from which the scientists had removed the arm that typically holds the bright-field light source and replaced it with a stand-alone condenser with an LED mounted in it. Light from the LED was sent through several lenses to a 40×, 1.3-NA oil-immersion objective, also made by Zeiss. All components of the objective were mounted on rods fixed to a thick aluminum base plate, enabling free vertical positioning of the components. Furthermore, the LED and iris, as well as the complete condenser, were laterally adjustable to achieve Koehler illumination.For imaging, they used a Zeiss 100×, 1.3-NA oil-immersion objective in conjunction with a standard video camera, made by Watec of Tsuruoka, Japan, with a pixel size of 8.6 × 8.3 μm, a field of view of 735 × 572 pixels and a frame rate of 25 Hz. They obtained images at up to double the magnification with an adjustable zoom positioned before the camera. A video card made by National Instruments of Austin, Texas, acquired the frames.Using a blue LED and 200× illumination, researchers produced images of single microtubules. Shown here are a single frame acquired at video rate (a), the average of 25 consecutive frames of the same area (b) and the same image after a Fourier bandpass filter was applied.The investigators visualized microtubules with all three LEDs and found that the blue yielded the best images, qualitatively. The blue also yielded the highest signal-to-noise ratio, allowing them to image single microtubules with a 25-Hz video rate and a signal-to-noise ratio of 3.4 ±0.2. They also could see the microtubules with the red and green LEDs, with signal-to-noise ratios of 3.1 ±0.3 and 4.1 ±0.6, respectively. They expected to see a higher ratio for the red than for the blue because the former has a smaller emitter area and, therefore, a higher luminous density. They noted that the lower signal-to-noise ratio may have resulted from the large variability in the light output of the LEDs.The researchers also have reported that LED light sources minimize thermal drift during experiments with optical tweezers. They showed that they could reduce temperature gradients considerably, with respect to 100-W arc or halogen lamps.In the meantime, Philips Lumileds continues to develop the power LEDs. The company is focusing on improvements in three key areas, Landau said: lumens per watt, lumens per area and lumens per dollar. Lumens per watt is a measure of efficiency, and improvements in this area could help drive adoption of the LEDs as light sources for microscopy applications. For example, an incandescent light might emit 10 to 15 lm/W, with 95 percent of the energy coming out as heat and only 5 percent as light. Power LEDs offer about 70 lm/W and will be increasingly efficient with further development.Contact Volker Bormuth, Max Planck Institute of Molecular Cell Biology and Genetics; e-mail: bormuth@mpi-cbg.de; Steve Landau, Philips Lumileds Lighting Co.; e-mail: steve.landau@philips.com.