Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy.
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.
Ruckstuhl
Thomas
T
Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. t.ruckstuhl@pci.unizh.ch
Seeger
Stefan
S
D001671:Biochemistry / Q000379:methods; D004867:Equipment Design; D018613:Microscopy, Confocal / Q000295:instrumentation; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D012542:Scattering, Radiation