We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al (5×10-18>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.
© 2004 Optical Society of America
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(190.4350) Nonlinear optics : Nonlinear optics at surfaces
(260.6970) Physical optics : Total internal reflection
Thomas Ruckstuhl and Stefan Seeger, "Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy," Opt. Lett. 29, 569-571 (2004)