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Laser sheets analyze live bacteria

17 Jun 2002

In an oceanography first, thin laser-sheet microscopy explores live bacteria in their natural habitat.

Researchers at the US-based University of California, San Diego, have made a breakthrough for oceanographers by using thin laser-sheet microscopy (TLSM) to study mobile bacteria in water. Eran Fuchs and colleagues say they can image these fast-moving microbes very simply and with greater speed and accuracy than existing techniques.

The researchers designed the new TLSM by first passing blue light from an argon laser through a beam expander to create a 1 mm by 1 mm by 23 µm sheet of light. The light sheet was then projected into the field of view of a microscope objective so that, when in use, only the focused area of the sample is illuminated.

"This technique concentrates the light only where it is needed, which maximizes the efficiency of the excitation source," said Fuchs. "And most importantly, the technique minimizes the background noise while maximizing image sharpness. Since particles that are not in focus are not illuminated, they do not fluoresce."

The researchers have tested their new system on microbes suspended in sea water, held in a standard cuvette. Over a 15 ms exposure time and within an area of 0.3 mm2, they clearly discriminated individual organisms that ranged in size from 1 to 50 µm. High resolutions also meant that they could measure bacteria densities 2.2 times higher than tests that simulated more conventional techniques such as standard epi-fluorescence.

Fuchs is confident that the technique is better than existing techniques such as two-photon microscopy. "Since the entire scene is evenly illuminated there is no need to build the image point-by-point," explained Fuchs. "This is the only existing system that allows us to look at mobile bacteria in their natural habitat and investigate in real time how they interact with each other in their surroundings."

The researchers hope to swap the argon laser for a 457 nm laser diode to make their system more compact. "Our ultimate goal is to develop an in-situ instrument that can be deployed in the field," added Fuchs. "This technology has the potential to make fundamental contributions to microbial oceanography by making an unexplored realm of marine ecosystems now accessible."

Rebecca Pool is news editor on Optics.org and Opto & Laser Europe magazine.

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