Optical Tweezer with Femtosecond Laser Pulses

OBJECTIVE: When a light having a transverse Gaussian mode is focused, it generates a harmonic potential field that creates force of the ~pN range that can trap ~ micron sized particles. It has a wide range of applicability starting from physical sciences, biological sciences, chemical sciences etc.

THEORY: On tight focusing the harmonic potential, generated by the Gaussian laser beam, attracts the particles towards its focus. For this tweezing action, we need to have microscope objective with Numerical Aperture being greater than 1.2.The ray optics diagram of optical tweezer is given below

INSTRUMENTS REQUIRED:

1.      LASER, Mirrors, mirror holders, lenses, lens holders, irises, filter, LED light source.

2.      High numerical aperture objectives, Dichroic mirror.

3.      CCD camera, Photo Diode.

4.      Laser glasses for eye safety.

Optical Tweezer set up

SOFTWARE USED:

1. LabVIEW software for data acquisition.
2. Origin pro for data plotting and analysis.

Note: For user operation and usage no specific software needed.

EXPERIMENT PROCEDURE:

1.      Make a through hole on a microscopic slide.

2.      On one side of the hole stick a no.1 cover slip (0.13 – 0.17 μm thickness) such that it does not come out easily from the slide. Thus a well is being formed.

3.      Get ~ 1 μM concentration of ~1μm diameter polystyrene microsphere coated with fluorophores dispersed in water.

4.      Sonicate the sample in (3) for ~ 20 minutes on a bath sonicator.

5.      Turn on the trapping Laser.

6.      Turn on the ‘ML’ button to convert it to the femtosecond pulse Laser.

7.      Place one drop this sonicated said sample in (3) on the well.

8.      Place a drop of immersion oil on the oil immersion microscope objective with Numerical Aperture being greater than 1.2.

9.      Place the microscopic slide with the sample being placed on the well made onto it on the sample plane.

10.  Tighten it with the clamps.

11.  Move the objective such that the oil placed on it touches the bottom side of the slide.

12.  Let the femtosecond Laser pulses pass through the objective and get focused in the bulk sample (i.e., microscopic slide with the sample being placed on the well made onto it).

13.  Fix the power of the Pulsed Laser beam.

14.  Turn on the CCD camera to record the trapping event.

15.  When the ~1μm diameter polystyrene microsphere coated with fluorophores comes in vicinity of the focus, it feels the optical force field and jumps into the focus and as the microspheres are coated with flourophores, so the trapped microspheres generates the two-photon fluorescence denoting a microsphere is being trapped.

16.  CCD camera records the trapping event that is described in process (11).