title: Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability.
abstract: Although single-molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures (1.8 K), the field has since advanced to include room-temperature observations, 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 vitro and in vivo. However, it is difficult to study systems and phenomena at temperatures above 37 degrees 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 TiO(2) colloids with diameters of 2 microm and a high refractive index can act as lenses that are capable of single-molecule imaging at 70 degrees 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 degrees C. Fluorophores in close proximity to TiO(2) also showed a 40% increase in photostability due to a reduction of the excited-state lifetime.
authors.affiliation: Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, California 94305, USA.
authors.forename: Jerrod J
authors.initials: JJ
authors.lastname: Schwartz
authors.forename: Stavros
authors.initials: S
authors.lastname: Stavrakis
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Quake
mesh_terms: D003102:Colloids / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006358:Hot Temperature; D007909:Lenses; D008832:Microchemistry / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D008863:Microspheres; D036103:Nanotechnology / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods; D014867:Water / Q000737:chemistry
chemical_list: D003102:Colloids; D005456:Fluorescent Dyes; D014867:Water