Abstract
Optical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be imaged with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-contrast 3D microscopy.
Highlights
To apply force on a wide range of samples and measure the response down to single biological molecules, optical tweezers are widely used in physics, material science, and biology [1]
Using a light-emitting diode (LED) for interference-reflection microscopy (IRM) illumination, we show that single microtubules can be resolved with high contrast
Optical tweezers are often combined with various microscopy contrast methods ranging from bright-field, via differential interference contrast (DIC) [2, 3], or less common, phase-contrast [4] and dark field [5] to fluorescence including among others epi, TIRF, confocal, or STED configurations [6,7,8,9,10]
Summary
To apply force on a wide range of samples and measure the response down to single biological molecules, optical tweezers are widely used in physics, material science, and biology [1]. During an experiment, such filaments need to be imaged for extended periods of time To this end, optical tweezers are often combined with various microscopy contrast methods ranging from bright-field, via differential interference contrast (DIC) [2, 3], or less common, phase-contrast [4] and dark field [5] to fluorescence including among others epi-, TIRF, confocal, or STED configurations [6,7,8,9,10]. For near infrared trapping lasers, about 10 % of power is lost per prism Despite these limitations, DIC is often the technique of choice for microtubule-based, optical-tweezers assays. Our combination can be applied to a wide range of biological systems and allows versatile imaging and force spectroscopy with molecular resolution
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