Abstract

We explore a new confocal microscope for the detection of surface-generated fluorescence. The instrument is designed for high resolution imaging as well as for the readout of large biochips. Special feature is the separated collection of two different fluorescence emission modes. One optical path covers the emission into the glass at low surface angles, the other captures high angles, exceeding the critical angle of the water/glass interface. Due to the collection of the supercritical angle fluorescence (SAF) the confocal detection volume is strictly confined to the interface, whereas the low angles collect much deeper from the aqueous analyte solution. Hence the system can deliver information about surface-bound and unbound fraction of fluorescent analyte simultaneously.

© 2004 Optical Society of America

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References

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    [CrossRef] [PubMed]

Anal. Chem.

R. L. Hansen and J. M. Harris, �??Total internal reflection fluorescence correlation spectroscopy for counting molecules at solid/liquid interfaces,�?? Anal. Chem. 70, 2565-2575 (1998).
[CrossRef] [PubMed]

T. Ruckstuhl, J. Enderlein, S. Jung, S. Seeger, �??Forbidden light detection from single molecules,�?? Anal. Chem. 72, 2117-2123 (2000).
[CrossRef] [PubMed]

Appl. Opt.

Biophys. J.

P. Schwille, �??TIR-FCS: Staying at the surface can sometimes be better,�?? Biophys. J. 85, 2783-2784 (2003).
[CrossRef] [PubMed]

T. E. Starr and N. L. Thompson, �??Total internal reflection with correlation spectroscopy: Combined surface reaction and solution diffusion,�?? Biophys. J. 80, 1575-1584 (2001).
[CrossRef] [PubMed]

A. M. Lieto, R. C. Cush, N. L. Thompson, �??Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,�?? Biophys. J. 85, 3294-3302 (2003)
[CrossRef] [PubMed]

Biosens. Bioelectron.

T. Ruckstuhl, M. Rankl, S. Seeger, �??Highly sensitive biosensing using a supercritical angle fluorescence (SAF) instrument,�?? Biosens. Bioelectron. 18, 1193-1199 (2003).
[CrossRef] [PubMed]

T. Ruckstuhl, A. Walser, D. Verdes, S. Seeger, �??Confocal reader for biochip screening and fluorescence microscopy,�?? Biosens. Bioelectron. (to be published).

ChemBioChem.

A. Krieg, S. Laib, T. Ruckstuhl, S. Seeger, �??Real-time detection of nucleotide-incorporation during complimentary DNA strand synthesis,�?? Chem BioChem. 4, 589-592 (2003).
[CrossRef] [PubMed]

Opt. Lett.

Science

T. A. Laurence and S. Weiss, �??How to detect weak pairs,�?? Science 299, 667-668 (2003).
[CrossRef] [PubMed]

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, W. W. Webb, �??Zero-mode waveguides for single-molecule analysis at high concentrations,�?? Science 299, 682-686 (2003).
[CrossRef] [PubMed]

Other

T. Ruckstuhl and S. Seeger, WO 9946596 (1999).

D. Axelrod, �??Total internal reflection fluorescence microscopy,�?? in Methods in Cellular Imaging, Ammasi Periasamy, ed. (Oxford University Press, New York, 2001), pp. 362-380.

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Figures (8)

Fig. 1.
Fig. 1.

Polar plots of the emission direction of isotropically oriented fluorophores with (a) a surface distance of zero and (b) a surface distance equal to a third of the emission wavelength in vacuum.

Fig. 2.
Fig. 2.

Comparison of the surface confinement achieved at the water/glass interface by the TIRF and SAF methods. All curves are equated at zero surface distance. At TIRF the decays of the excitation efficiency depend on the excitation angle θex. The decay of the collection efficiency of SAF is obtained for all supercritical angles. The fluorescence emission wavelength is assumed to be 5% greater than the excitation wavelength (Stokes shift).

Fig. 3.
Fig. 3.

Setup of the SAF microscope

Fig. 4.
Fig. 4.

(a) Required displacement of the beam expander lens in order to adjust the laser focus to the interface. (b) Spatial collection efficiency function of the low angle detection with the asphere. The shown contour plot is representative for all coverslip thicknesses within 150±10 μm.

Fig. 5.
Fig. 5.

Spatial collection efficiency functions produced by the parabolic element together with the large achromatic lens and a confocal aperture of 180 μm diameter. (a) For the standard coverslip thickness of 150 μm the collected volume element is aligned to the interface. Due to the collection of supercritical light modes the collection efficiency decays rapidly inside the water. The volume element of efficient collection inside the water is too thin to appear in the chosen scale. (b) Without an additional alignment the use of a 160 μm thick coverslip causes the collected volume to lay 10 μm lower than its designated surface position.

Fig. 6.
Fig. 6.

Corrected collection volumes for (a) 140 μm, (b) 150 μm and (c) 160 μm thick coverslips. The position of the confocal aperture was shifted by -1.8 mm, 0 mm and 1.8 mm from its home position respectively. The second column gives the collection efficiencies at the plane of the interface.

Fig. 7.
Fig. 7.

Lateral resolution of the microscope. (a) Confocal image of 20 nm fluorescence beads, (b) simulated PSF and (c) experimental PSF. The measurements were carried out at an excitation wavelength of 633 nm.

Fig. 8.
Fig. 8.

Detection volumes of (a) fluorescence collection at surface low angles (0°-24°) and (b) SAF collection (62°-75°). An excitation wavelength was set to 633 nm.

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