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13 Aug 2004
European scientists demonstrate a fluorescent microscope that captures images up to 0.5 mm inside a living fish embryo.
Researchers can now take a much deeper look inside living organisms thanks to a state-of-the-art fluorescence microscope developed by scientists at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. (Science 305 1007)
Using a technique known as selective plane illumination microscopy (SPIM) the team has managed to image living fish and insect embryos at depths of up to 0.5 mm with a resolution better than 6 microns. The setup is compatible with conventional microscope objective lenses.
Thick biological specimens present a challenge to existing optical imaging techniques because of their absorptive and scattering properties. "Over the years we've seen current microscopes falling short of what scientists need," EMBL scientist Ernst Stelzer commented. "This new microscope is easy to build, is about one-third the cost of current technologies, and gives scientists improved resolution by a factor of about five."
Importantly, and unlike rival techniques such as confocal laser scanning microscopy, only the focal plane under observation is illuminated and therefore exposed to photo damage. As a result SPIM is able to generate a sharp image by eliminating out-of-focus fluorescence.
A thin light sheet from either an Argon-ion or HeNe laser illuminates the sample which is embedded in a cylinder of agarose gel immersed in an aqueous chamber. The microscope's objective lens, with its optical axis at right angles to the light sheet, dips into the chamber and collects fluorescence from the sample.
"We use a high-precision four axes (xyz & theta) motor to move the sample through the fixed optical system," Stelzer told Optics.org. "[Power] is below 4 mW per [light] sheet, that is about 4 µW per line or pixel,". The team report no detrimental effects on the living organisms when imaging over three day periods.
Scientists at EMBL have used SPIM to investigate the heart of a Medaka fish embryo, a structure that is hard to image due to the surrounding yolk sphere. Using a fast frame technique they recorded moving images of the heartbeat. The team claim that previously this could have only been demonstrated at stages when the heart is exposed and by cooling the embryo to reduce the heart rate.
Stelzer hinted to Optics.org that there was interest in commercializing the setup, but did not wish to comment at this stage.
Author
James Tyrrell is reporter on Optics.org and Opto & Laser Europe magazine.
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