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)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.

Optofluidics incorporating actively controlled micro- and nano-particles

“…119,120 Individual particle lenses were demonstrated by several groups. 121,122 Such lenses have been used as manipulators of light exhibiting the ability to control the amplitude, phase, and polarization properties of light and its directions of propagation. [123][124][125][126][127] Beam control was demonstrated by displacing optically trapped particles such as an SiO 2 (Ø13 lm) microsphere through a light beam.…”

Ultra-high ordered, centimeter scale preparation of microsphere Langmuir films

“…This technology relies on the principle of optical near-field enhancement underneath a monolayer of nano-or micro-spheres that can lead to laser spots with size well below the wavelength, while the structures thereby obtained have features at the nanometer scale. Thus, it is possible to micro-and/or nanostructure the substrates of choice using a sphere-assisted photonic nanojet lithography technique [26][27][28][29][30][31][32]. These sub-diffraction lithography techniques find applications in areas related to optical devices, spectroscopy, optical microscopy, data storage, and imaging [27][28][29]33].…”

“…Thus, it is possible to micro-and/or nanostructure the substrates of choice using a sphere-assisted photonic nanojet lithography technique [26][27][28][29][30][31][32]. These sub-diffraction lithography techniques find applications in areas related to optical devices, spectroscopy, optical microscopy, data storage, and imaging [27][28][29]33]. Another alternative using microspheres is the so called ''colloidal lithography'' technique, where sphere monolayers are used as shadow masks in standard physical depositions.…”

Fabrication of microcraters on silicon substrate by UV nanosecond photonic nanojets from microspheres

“…Optical near-field enhancement underneath a monolayer of nanospheres can lead to laser spots with size well below the wavelength and the structures obtained thereby have features at the nanometer scale [2][3][4]. This technology involves focusing of light waves underneath the surface of a monolayer of particles to fabricate arrays of structures with micro-and nanometer size by reaching locally the fluence above the modification threshold of the material of interest [5][6][7].…”