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Optical Entrapment (Laser Tweezing)

Principle

The phenomenon of laser tweezing relies on a difference in refractive index between an object (ie. particle, typically 1-2 microns in diameter) and the medium surrounding it. When the particle is placed at the focal point of a beam of incident laser light in a confocal microscope, some of the light is deflected (ie. refracted) as it passes though the particle and the rest is reflected. The photons of the refracted light emerge from the particle with a different  momentum from those incident on the particle due to the change in direction, and this creates a net force on the particle that 'pulls' it towards the objective lens. The reflected light on the other hand creates a force that 'pushes' the particle in the opposite direction from the pulling force due to the refracted light.  The net effect of the two forces is a trapping force that holds the particle in the focus of the laser beam.

Simple diagram showing the principles behind laser tweezing

Figure 1 A simple diagram to show the principles behind laser tweezing.
Forces due to reflected light are indicated in green. Forces due to refracted light are indicated in black.
The photons of the refracted light emerge from the particle with a different  momentum from those incident on the particle due to the change in direction, and this creates a net (‘gradient’) force on the particle that acts in the z direction towards the objective lens. The reflected light on the other hand creates a (‘scattering’) force acts in the z direction in the opposite sense to the gradient force. The net effect of the two forces is a trapping force that holds the particle in the focus of the laser beam. If the particle moves away from the z axis in the x-y plane, the net gradient force acts to move the particle back towards the z axis.

Typical Setup

The experimental setup used for tweezing multiple liposomes is outlined below:


A diagram of the experimental setup for tweezing multiple liposomes
Figure 2 A simplified diagram showing the basic experimental setup for laser tweezing of liposomes to enable both tweezing of multiple liposomes and observation of fluorescence emission.

Some movies showing trapping of liposomes are available
here

In order to trap liposomes, a solid-state Nd:YAG microwave laser is used as the source of the trapping beam. This is passed through a series of mirrors and lenses to condition the beam. One of the most important of these is the AOD device, which is able to move the focus of the laser precisely and rapidly rapidly between a number of different loci. This enables multiple particles to be trapped, providing that the diffusion coefficient of each particle is significantly longer than the time taken to switch between the different loci, which is usually the case. An Ar-ion laser is used as the light source for excitation of fluorescent markers, with a Uniblitz shutter included in order to allow the excitation to be pulsed (in order to reduce photobleaching). The whole apparatus is mounted on an air table to minimise disruption from vibrations.

Raman Tweezing

The basic Raman tweezers apparatus (Figure 3) as a little simpler than that required for trapping multiple particles, and uses a cw Argon ion laser of wavelength 514.5 nm and maximum power 2 W. The beam is passed through two sets of expansion optics (L1 to L4), reflected from a 33:67 beamsplitter and directed into the microscope. Here, a dichroic mirror is used to reflect the beam through the objective lens (x63) where the optical trap is formed. For trapping and acquisition of Raman spectra the laser is attenuated to a power of 12 mW, measured at the entrance aperture of the objective lens.

Experimental setup of Raman tweezers

Figure 3 A simplified diagram showing the basic experimental setup for laser tweezing of liposomes to enable observation of Raman scattering

The Raman light scattered from the focal point is collimated by the objective lens and passed back along the same optical pathway. A holographic notch filter is used to transmit only the Raman shifted light above 514.5 nm. The beam is then focussed on to the entrance slit of the spectrometer and detector. To allow observation of the trapped liposome, filtered light (>700 nm) from the microscope is used to image the particle onto a CCD camera.