Trapping nanoscopic objects to observe their dynamic behaviour for extended periods of time is an ongoing quest. Particularly, sub-100nm transparent objects are hard to catch and most techniques rely on immobilisation or transient diffusion through a confocal laser focus. We present an Anti-Brownian ELectrokinetic trap1–7 (pioneered by A. E. Cohen and W. E. Moerner) to hold nanoparticles and individual FoF1-ATP synthase proteins in solution. We are interested in the conformational dynamics of this membrane-bound rotary motor protein that we monitor using single-molecule FRET. The ABELtrap is an active feedback system cancelling the nano-object’s Brownian motion by applying an electric field. We show how the induced electrokinetic forces confine the motion of nanoparticles and proteoliposomes to the centre of the trap.
Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap — a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.
We present a holographic tweezers workstation to optically trap and spectroscopically characterise metal nanoparticles.
The versatility of the holographic tweezers allows us to create multiple traps manipulating several metal
nanoparticles simultaneously. We developed an imaging system to view the nanoparticles in a transmission darkfield
configuration without compromising the high numerical aperture of our trapping and viewing objective. In
addition we implemented single particle spectroscopy to interrogate the trapped particles' properties with the
ability to directly monitor the plasmonics coupling between particles while changing the distance between them.
We also demonstrate a laser based method to generate fixed arrays of nanoparticles.
Optical trapping is an established field for movement of micron-size objects and cells. However, trapping of metal nanoparticles, nanowires, nanorods and molecules has received little attention. Nanoparticles are more challenging to optically trap and they offer ample new phenomena to explore, for example the plasmon resonance. Resonance and size effects have an impact upon trapping forces that causes nanoparticle trapping to differ from micromanipulation of larger micron-sized objects. There are numerous theoretical approaches to calculate optical forces exerted on trapped nanoparticles. Their combination and comparison gives the reader deeper understanding of the physical processes in an optical trap. A close look into the key experiments to date demonstrates the feasibility of trapping and provides a grasp of the enormous possibilities that remain to be explored. When constructing a single-beam optical trap, particular emphasis has to be placed on the choice of imaging for the trapping and confinement of nanoparticles.