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Opt Lett. 2004 Mar 15;29(6):569-71.

Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy.

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  • 1Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.


We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al (5 x 10(-18) l) at a water-glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.

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