Multispectral Microscopy Accelerates

Richard Gaughan

Multispectral microscopy provides information about the spatial variation in wavelength-dependent parameters of features on the scale of a few microns, but when elements of a sample rapidly change with time, it becomes difficult to capture the changes and identify the stimulus. A new microscopy system can capture multispectral images of samples with a time resolution of milliseconds.

A multispectral microscope developed at Marquette University offers a time resolution of milliseconds. Three- and two-dimensional plots of the response of cholesteric liquid crystal to continuous-wave illumination by an 805-nm diode laser reveal differences among the unit cells. Courtesy of Chieu D. Tran.

The microscope developed by Chieu D. Tran and his colleagues at Marquette University uses an acousto-optic tunable filter to direct a single wavelength from a broadband source into a 5-mm-diameter liquid lightguide from Lumatec GmbH of Munich, Germany, through a 20x microscope objective to the sample. The sample is imaged with a progressive-scan silicon area camera from Dalsa Inc. of Waterloo, Ontario, Canada, and the images are transferred to a digital frame grabber from Dipix Corp. of Ottawa.

The instrument can gather successive images at a single wavelength or step the wavelength with each successive frame. In single-wavelength operation, the system acquires images at a rate of 33 fps; for imaging as a function of wavelength, the maximum rate is 16 fps.

In the Feb. 15 issue of Analytical Chemistry, the researchers report the capabilities of the microscope, measuring commercially available cholesteric liquid crystal sheets. They heated the liquid crystal with 805-nm light from a diode laser, which induced color changes in its 3- to 5-µm unit cells.

When irradiated with 200-ms pulses from the laser, the crystal showed an early change in intensity of reflected light at 520 nm, with a narrower, later change at wavelengths up to 620 nm. When illuminated with a continuous-wave beam, the color change was not uniform across the entire crystal but varied from unit cell to unit cell.

Extending capabilities

The success of the prototype is encouraging Tran to extend the capabilities of the instrument into the ultraviolet and near- and middle-infrared regions of the spectrum. The team is also shortening its time resolution. The imaging system is being evaluated for macroscopic applications, including agricultural field monitoring, but its primary focus is the microscopic world, such as observing the cellular interaction of drugs without the use of fluorescence or radioactive labeling.