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Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability

Nature Nanotechnology volume 5, pages 127–132 (2010)Cite this article

Abstract

Although single-molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures (1.8 K)1, the field has since advanced to include room-temperature observations2, largely owing to the use of objective lenses with high numerical aperture, brighter fluorophores and more sensitive detectors. This has opened the door for many chemical and biological systems to be studied at native temperatures at the single-molecule level both in vitro3,4 and in vivo5,6. However, it is difficult to study systems and phenomena at temperatures above 37 °C, because the index-matching fluids used with high-numerical-aperture objective lenses can conduct heat from the sample to the lens, and sustained exposure to high temperatures can cause the lens to fail. Here, we report that TiO2 colloids with diameters of 2 µm and a high refractive index can act as lenses that are capable of single-molecule imaging at 70 °C when placed in immediate proximity to an emitting molecule. The optical system is completed by a low-numerical-aperture optic that can have a long working distance and an air interface, which allows the sample to be independently heated. Colloidal lenses were used for parallel imaging of surface-immobilized single fluorophores and for real-time single-molecule measurements of mesophilic and thermophilic enzymes at 70 °C. Fluorophores in close proximity to TiO2 also showed a 40% increase in photostability due to a reduction of the excited-state lifetime.

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Figure 1: Spherical colloids are efficient lenses for single-molecule imaging.
Figure 2: Single fluorophores in dry and wet samples can be imaged efficiently with colloidal lenses and a low light collection efficiency objective.
Figure 3: Cy3 photobleaching lifetimes increase in the presence of TiO2 colloids.
Figure 4: Single-molecule DNA polymerase activity can be observed with colloidal lenses and a ×20 0.5 NA air objective.

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Acknowledgements

The authors thank M. Brongersma, H.J. Lee and M.F. Yanik for discussions, and E. Townsend for writing the instrumentation software. This work was supported in part by the Department of Defense Advanced Research Projects Agency Optofluidics Center, the National Human Genome Research Institute (5R01HG003594-04), and the Howard Hughes Medical Institute. S.S. was funded by a Marie Curie fellowship (MOIF-CT-2006-0400320 FRETANDFORCE).

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  1. Jerrod J. Schwartz and Stavros Stavrakis: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, 94305, California, USA

    Jerrod J. Schwartz, Stavros Stavrakis & Stephen R. Quake

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Contributions

J.J.S., S.S. and S.R.Q. conceived and designed the experiments. J.J.S. and S.S. performed the experiments. J.J.S. and S.S. analysed the data. J.J.S., S.S. and S.R.Q. wrote the paper.

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Correspondence to Stephen R. Quake.

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We have filed a patent on colloidal lenses.

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Schwartz, J., Stavrakis, S. & Quake, S. Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability. Nature Nanotech 5, 127–132 (2010). https://doi.org/10.1038/nnano.2009.452

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