Main Page/BPHS 4090/Optical Tweezers of Onions

Introduction

The Physics of Optical Tweezers

The principle of optic tweezers was first proposed and discovered by Arthur Ashkin in 1970. The principle relies on the refraction of light as it passes between media of differing indicies of refractions (symbolized by n), and the Gaussian profile of a trapping laser beam which is most intense at the central axis. Recall that a photon with wavelength L and moving along the z-axis has of momentum equal to: !!!equation!!! p= h/L z-hat As a photon passes through a bead of greater index of refraction than the surrounding medium, it will refract (change direction). This results in a change in momentum. As a reaction to this change in momentum, momentum is imparted to the bead in the opposite direction. The ray diagram foor this effect is given in Figure 1.

Figure 1 - In part a), there is more laser intensity on the right of the bead due to the gaussian profile of the laser beam, and hence there is a net force to the right. In part b), the bead is centered and the net force is zero.

Structure of an Onion Cell

Plant cells have the general properties of a rigid cell wall, a large open vacuole, a nucleus, and cytoplasm containing organelles in the spaces between the cell walls and vacuole. The onion cell is a classic and often-used example of this structure. The organelles in the cytoplasm are small (between 0.5 and 1 micron), and roughly spherical in nature- prime candidates for optical tweezing with a laser. Organelles move throughout the cyctoplasm either along action filaments, and along the the endoplasmic reticulum network.

Figure 1.1: The onion cell

Internal Structure of an Onion Cell[2]

The organelles move throughout the onion cell only in the cytoplasm and not through the vacuole. There are strand of cytoplasm located sporadically thoughout the cell. These strands themselves can be seem by the imagining optics when focused appropriately. In these stands of cytoplasm can be actin fibers along which organelles are transported. The process by which this occurs is demonstrated nicely in the following animation. [1]

Preparation of onion epidermis for optical trapping experiments

The common onion (Allium cepa) is often used to examine individual plant cells because of the ease of isolating sheets of cells that are one cell thick. The onion bulb can be sectioned into quarters or eighths. Then, the individual scale leaves can be separated.

The exposed concave surface can be scored with a sharp razor blade. With a very fine pair of forceps, pieces of the epidermis can be lifted (note the transparency of the peel). Before doing so, have a microscope slide ready with a drop of distilled water (or artificial pond water) so that the peel doesn’t become dehydrated. Unlike the photographs, a thin strip of Vaseline will be placed on the microscope slide in a rectangular shape slightly smaller in dimensions then the cover slip. Having placed the epidermal peel in the water inside the Vaseline ‘dike’, carefully (gently) place the coverslip on top, pressing to create a seal around the perimeter.

File:Onion5.jpg File:Onion6.jpg

Now ready to place in the holder on the optical bench (left), here is what the cells will look like (right, the nucleus and transvacuolar strands are indicated).

Lab Exercise: Optical tweezing of onion cells

Calibrate Optical Trap Depth

The goal of this experiment will be to determine the stength of the myosin motors which move the spherosomes along actin filaments. In order to achieve this goal you must first calibrate how strongly the spherosomes are trapped in the optical tweezers as a function of laser power. For this, we will use spherosomes moving in cytoplasmic medium near the cell walls, but not along actin fibers. The spherosomes are moving through the cytoplasm, adjacent to the endoreticulum network. Cell cytoplasm is a very complicated component of the cell, and can be characterized as a non-Newtonian fluid. The viscosity of the cytoplasm depends on the shear applied to the cytoplasm. For our purposes, we will take an educated guess of a viscosity of 20 times that of the viscosity of water. To determine the trapping force on the spherosome, we will move the spherosome through the cytoplasm as a constant velocity and see which velocity is great enough to dislodge the spherosome from the trap. The force exerted by the viscous cyctoplasm on a sphere of radius ,r is: In order to have nice repeatable motion with the piezo translators, it is necessary to start at 0V applied to the piezo controlling the direction in which you want to move.

Method

1. Determine the position of the laser. Notice that you can as you move the onion slide towards the microscope objecive using the z-xis manual control, the cover slip will come into focus first, and the laser position will be visible due to scattered light off the coverslip. Using the "region of interest" marker tool, place a ~2 micron circle around this position.
2. Focus on the top cell wall. As you continue moving the onion slide closer to the microscope objective, first the top cell wall will come into focus, then the middle parts of the cells, then, if the sample isn't too thick, the lower cell wall. You will see lots of organelles moving in many directions.
3. Adjust the half-wave plate for maximum transmission.
4. Trap one of these spherosomes in the optical trap.
5. Move at constant velocity
6. Adjust the laser power by rotating the half-wave plate. Repeat above step, and make a table of relative laser power and minimum velocity required to dislodge the spherosome from the trap.
7. From the above table calculate the force on the spherosome, and plot the data on a graph.

You now have a calibration graph that you can use in the next section to read off the force exerted on a spherosome for a given setting of the half-wave plate.