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 magnit...

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Bibliographic Details
Published in:Optics letters 2004-03, Vol.29 (6), p.569-571
Main Authors: Ruckstuhl, Thomas, Seeger, Stefan
Format: Article
Language:English
Subjects:
Biochemistry - methods
Equipment Design
Microscopy, Confocal - instrumentation
Microscopy, Fluorescence - instrumentation
Scattering, Radiation
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Summary: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.
ISSN:0146-9592
1539-4794
DOI:10.1364/OL.29.000569