Cool Tunable Laser Suitable for Microscopy

Hank Hogan

A team of scientists at the State University of New York at Albany in Colonie and at the New York state Department of Health in Albany has demonstrated an inexpensive excitation method for applications in confocal microscopy. The scientists exploit temperature-dependent changes in a semiconductor laser's output to improve contrast and to excite different dyes with the same laser. A room-temperature 635-nm laser diode, for example, has an output wavelength of 609 nm when it is chilled to 2196 °C, the temperature of liquid nitrogen.

Researchers captured these confocal microscope images from the same sample of filter paper soaked in Cy5 using a laser diode excitation source at 6 °C and at 2196 °C. Because of a temperature-dependent blueshift in the wavelength of the laser, the low-temperature image has a higher signal-to-noise ratio and reveals more details. The technique could be used to create an inexpensive, tunable light source.

"The goal is to create inexpensive, tunable light sources for fluorescence confocal microscopy," said Susanne M. Lee, an assistant professor of physics and director of the university's Metastable Materials Manufacturing Laboratory. Another objective is to extend the number of fluorochromes that can be used simultaneously for imaging, while improving the signal-to-noise ratio as well.

The better signal-to-noise ratio arises because of the change in the laser's output. A 25-nm shift creates greater spectral separation between the incoming light and the resulting fluorescence. The fluorochrome Cy5, for instance, fluoresces from 610 to 800 nm. With a source close to 600 nm, less of the fluorescence is obscured by reflected light making its way past any filters.

The scientists demonstrated the method in a Bio-Rad MRC 600 laser scanning confocal microscope equipped with a 3-mW Hitachi laser diode. They operated the laser from just below room temperature to that of liquid nitrogen. They discovered that the response of the Cy5 was almost unchanged but that the signal-to-noise ratio improved fivefold at the lowest temperature.

The researchers' next step is to cool the laser so that it operates at various wavelengths and to use it to excite multiple fluorochromes. This will allow different parts of a cell to be imaged at the same time, because the individual fluorochromes will respond differently as the wavelength changes.

Making sense of the results will require extracting multiple and perhaps overlapping spectra -- something that is not possible right now, Lee explained.