Ashkin was the first to show that laser tweezers or optical traps are a powerful tool for manipulating microscopic micronized living objects. In this video, we will show how we have applied this technology to capture and move neurons. This is the optical tweezers workstation.
The green arrows show the path taken by the laser beam through the microscopes optics. When a laser beam focused by the microscope objective lens is refracted by a cell. The force that changes the direction of the light ray acts on the cell moving the cell towards the center of focus.
This generates Pika Newton's of force sufficient to pull a cell through medium. In this video, we will talk about fabrication of dishes, preparation of retinal cells, plating of cells on cultured dishes, laser tweezer manipulation, and observation of cell grow. Because cells stick to glass, we needed to create a cell repellent area where cells can be easily trapped with the tweezers.
Half the dish was coated with poly hema, a cell repellent polymer. The other half was coated with cell one, A substrate that supports the growth of our cells. First, a cell is identified on the cell adhesive side of the dish.
Another cell is then trapped on the polymer side and transported to the adhesive side where both cells are paired. To prepare dishes, we use blow dusted sterilize polystar, 35 millimeter cultured dishes. First, we cut a one centimeter hole in the bottom with a small lathe or a drill press.
Take acid clean cover glasses and place them upright in a dust-free environment such as a hood. Take the polyus solution dissolved in 95%alcohol and place a few drops near the top of the cover glass on one half only Allow the poly hema to dry. Mark the polyus side of the cover glass.
Put S guard around the hole in the bottom of the cultured dish. Glue the cover glass to the dish with S guard. Make sure the polyamide is on the inside and also make sure that there are no leaks through gaps in the sill guard.
Place the cover glass so the edge of the poly hema coat runs down the center of the dish. Allow the dish to dry for two to three days, and then scratch the bottom of the dish with a diamond tip pen with marks, which will serve as reference points for positioning the dish on the microscope stage. Place the dishes in the hood and sterilize them under the ultraviolet light.
Place one or two drops of goat anti-US antibody solution on the non polyus side of the dish, taking care not to spill it on the poly hemic coating and leave for about three hours. Remove it and wash with sterile ringers. Place cell one antibody solution on the same area and incubate overnight ready for the experiment the next day.
The cells in our lab are derived from adult aquatic phase salamander retinas. The retinas are dissected and removed, digested with enzyme ated. The cultured dish can then be plated with isolated cells.
The eyes are removed. The cornea is dissected And removed. The The eye cup is placed in sterile ringers.
The retina is separated from the eye cup by scooping it out with the edge of a curved Forceps. Carbon dioxide and Oxygen is bubbled into a ringer solution containing propane and then sterilize through an ultra fine 0.2 micron Filter. The retinas are placed in the Enzyme solution and digested for about 40 minutes with agitation to Break up the tissue.
After washing three times, the retinas are dissociated by tri. Using a three millimeter ball pipette, the isolated cells are ready For plating before Plating. The cultured dish containing medium is placed on the microscope stage and oriented appropriately using the scratch marks on the bottom of the dish.
This allows the exact position of the dish to be duplicated at a future time. The cells are then plated on both poly hema and cell one Sides of the dish. The position of the dish on the microscope stage, as well as the laser power is controlled with the joystick.
First, we identified a cell adhered to the cell one side of the dish. This is a retinal multipolar cell identifiable by its dendrites. The coordinates of the cell's position is saved.
This is a rod photoreceptor cell on the polyus cell repellent side of the dish. The dish is lowered and the cell is captured by the laser and lifted clear of the bottom of the dish. The cell is now being transported to the other of the dish to the coordinates of the multipolar cell.
In reality, the cell is stationary. It is the dish that is moving. The cell is now passing over the edge of the poly hemo coating.
The protuberance extending from the cell is the axon terminal. The cell is well clear of any debris and other cells at the bottom of the dish, which may obstruct its movement. The defocus images of cells on the bottom of the dish are visible in the background.
The rod cell arrives at the multipolar cell and can be accurately positioned at the desired point and distance from that cell. The dish is raised and the cell adheres to the cell one substrate. The prime advantages to this procedure are that one can choose which cells to move and that the manipulations can take place in an enclosed sterile experiment.
Cell to cell interactions Can be monitored with video time lapse or by digital photography. Laser tweezers can be used with any biological technique Here, for example, a fluorescence technique demonstrates the presence of synaptic proteins at points of connection between cells moved by the Tweezers.
