Microscopy Focus | June 2006
UV Thermal Lens Microscope Shows Promise for Lab-on-a-Chip Applications
by Anne L. Fischer
The miniaturization of various analytical systems greatly simplifies chemical and biochemical research. Because such lab-on-a-chip solutions involve microscale sample volumes and device dimensions, the sensitivity of the detection methods employed is of paramount importance.
The principle of thermal lens signal generation is shown for conventional continuous-wave (left) and quasi-continuous-wave excitation (right).
To that end, scientists at Kanagawa Academy of Science and Technology in Kawasaki, Japan, and at the University of Tokyo recently demonstrated an ultrasensitive, label-free method for the detection of nonfluorescent molecules. In the work, they employed a thermal lens microscope with a 266-nm pulsed laser as the excitation source.
In a thermal lens microscope, an excitation and a probe laser are focused onto a sample. When the excitation beam is absorbed, the temperature of the sample increases, forming a refractive index distribution that ultimately produces a concave excitation beam. The strength of this “lens” is proportional to the sample concentration and is detected by a change in transmittance in the probe beam.
The researchers chose a UV excitation source because a wide range of molecules absorb in that spectral region. Although other studies have used a continuous-wave UV laser for thermal lens detection, Kazuma Mawatari of Kanagawa Academy said that this was the first demonstration of a thermal lens microscope incorporating a pulsed UV laser.
The UV thermal lens microscope displayed its potential for label-free detection with both a microchip and a high-performance liquid chromatography system.
A problem with using a pulsed laser involves achieving lock-in amplifier detection to realize precise measurement, as with conventional continuous-wave excitation. They employed quasi-continuous-wave excitation by modulating the pulse trains at ∼1 kHz and detected the synchronous signal with a lock-in amplifier. They obtained a pulse repetition frequency of 80 kHz, which Mawatari indicated is important for achieving a high signal-to-noise ratio.
Another challenge was finding the right flow velocity to avoid a loss of sensitivity from photochemical reactions. The scientists found that a permissible flow velocity is in the range of 6.6 to 19.8 mm/s and could sensitively detect adenine aqueous solutions on a microchip without labeling. The results indicate that the technique offers a sensitivity 350 times higher with a volume approximately three orders smaller than they could have achieved using a spectrophotometric method.
They also used the UV thermal lens microscope for liquid chromatography detection. In a microcolumn, they separated fluorine and pyrene, which were detected with 150 times greater sensitivity than could be achieved with a spectrophotometric approach.
The researchers have launched the Institute of Microchemical Technology, a private venture in Kawasaki, to provide tools for microchip research. They plan to offer the UV thermal lens microscope in the near future.
Analytical Chemistry, April 15, 2006, pp. 2859-2863.
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