Department of Neurology and Neuroscience, School of Medicine, University of Medicine and Dentistry of New Jersey - UMDNJ
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Clarke, R., Wang, J., Townes-Anderson, E. Using Laser Tweezers For Manipulating Isolated Neurons In Vitro. J. Vis. Exp. (19), e911, doi:10.3791/911 (2008).
In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.
Optical tweezers for manipulating isolated cells in culture
Trapping forces of optical tweezers are generated from the momentum of light (Ashkin, 1991; Ashkin et al., 1986). Although these forces easily trap cells in suspension, they are not able to move cells that adhere to a surface. Therefore, to reduce adhesion to the culture dish at the point where cells are trapped, the coverslip is coated with poly-2-hydroxyethylmethacrylate (poly-HEMA) (Sigma Chemical Co., St Louis, MO), a nontoxic compound with cell repellent properties previously employed for adhesion studies on endothelial cells (Folkman and Moscona, 1978). While poly-HEMA covers one half of the culture dish, the other half is coated with a substrate that supports cell growth. In our laboratory, Sal-1 is used as the substrate for culturing salamander retinal cells. The procedure for fabricating Sal-1 and poly-HEMA coated dishes is described below:
Fabrication of culture dishes
The composition of the amphibian Ringer’s solution, serum-free amphibian medium, and Ringer’s solution for enzyme digestion are found in MacLeish and Townes-Anderson (1988) and Mandell et al. (1993).
Preparation of cell cultures
The cells used in this video were derived from light-adapted, adult, aquatic-phase tiger salamanders (Ambystoma tigrinum), measuring 17–22 cm in length (Charles Sullivan Inc., Nashville, TN), maintained at 5°C on a 12 h:12 h light-dark cycle. The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Medicine and Dentistry of New Jersey in strict accordance with the guidelines from the National Institutes of Health. The procedures for preparing retinal cell cultures are described by Nachman-Clewner and Townes-Anderson (1996) and are as follows:
Laser tweezers manipulation of isolated neurons
Light has momentum, and when a light ray is refracted as it passes through a cell, a force is required to change the direction of the momentum. Because of the law of conservation of momentum, a force in the opposite direction must, in turn, react back on a cell. Ashkin (1991) showed that the force generated by a laser beam focused by a microscope objective lens will move a cell toward the center of focus. Even though a laser beam generates only a few piconewtons of force, this force is sufficient to pull a cell through medium. A near infrared wavelength that is poorly absorbed by water is used to avoid damage to the cell (Liu et al 1995). The laser-tweezer workstation used in our laboratory (Cell Robotics Inc., Albuquerque NM) uses a 1 W, continuous wave diode laser of 980nm wavelength. The laser light is transmitted to the cells via a 40x oil immersion plan neofluor objective lens of high numerical aperture (N.A.1.3) (Carl Zeiss Inc.) which focuses the beam at the same focal plane as the microscope.
Laser-tweezers-controlled micromanipulation presents a number of advantages over studies on randomly plated cells. For example, cells can be placed at a desired distance from one another which can be standardized for all manipulated cells. In addition, the laser beam passes through the transparent surface of the culture dish and, therefore, cell manipulation is done in an enclosed, sterile environment. The very small forces generated by the laser places a limit on what can be trapped. In our experience, adhesion and cell shape are the most important factors limiting successful trapping. In general, we trap isolated cells of 10-15μm in diameter and transport them distances of several millimeters across the culture dish to their destination. It is possible to move larger cells than these, e.g., Muller glial cells, which are 20-40μm in length and are the largest cells in our cultures.
Because trapping cells is a challenge when the cells adhere to the glass bottom of the culture dish and to each other, developing a cell-repellant surface on the coverglass using poly-HEMA proved invaluable to our success in manipulating cells. Even so, the cells on the poly-HEMA surface eventually became stickier and difficult to manipulate in cultures more than 1-2 hours old. In older cultures, the common practice in our lab was to try to pick up the cells with the laser using the maximum power setting, then turn the power down to transport it.
The ease with which small objects can be trapped in the laser means that debris can be pulled into the trap from distances well outside the cell. This proved to be a problem during the trapping of our cells. This can be avoided in most instances by turning down the power of the laser to the minimum possible that can hold the cell within the trap. The speed at which cells can be transported in the trap is limited by the viscosity of the medium. We normally set the speed at around 8-20 μm/second which was found to be within a safe range for transport.
The possibility that the laser has detrimental effects on the cells held in the trap must be considered. In our retinal cultures, we encounter dark objects such as pigment cells which are destroyed when captured in the laser beam. Clearly, therefore, these cells cannot be studied using laser tweezers. However, in previous studies on retinal neurons and photoreceptors carried out in our laboratory, we found no difference in neuritic growth and capacity to form connections between tweezer-manipulated cells and non-manipulated cells (Townes-Anderson et al 1998; Clarke et al 2008).
Research was supported by NIH Grants EY012031 and EY0182175 and the F.M. Kirby Foundation.
|25mm circle No.1 coverglass||VWR international||48380 080|
|poly-2-hydroxyethylmethacrylate (poly-HEMA)||Sigma-Aldrich||P-3932||Dissolve in 95% ethanol|
|Goat anti-mouse IgG antibody||Chemicon International||AP181||1mg in 1ml, dilute 10x for use|
|Sal-1 supernatant containing mouse anti-salamander antibody||generously provided by Dr. Peter MacLeish||Dr. Peter MacLeish, Morehouse School of Medicine, Atlanta, GA|
|3 mm bore 5ml pyrex disposable pipets||Corning||7078A-5|
|Cell culture dishes 35mm x 10mm||Corning||430165|
|Sylgard 184 silicone elastomer kit||Dow Corning|
|Optical tweezers-microtool or laser tweezers||Cell Robotics Inc.|
|1 W continuous wave diode laser of 980nm wavelength||Cell Robotics Inc.|
|Axiovert 100 inverted light microscope||Carl Zeiss, Inc.|
|40x oil immersion plan neofluor objective lens||Carl Zeiss, Inc.||Numerical aperture (N.A. 1.3)|
|Black and white CCD camera||Sony Corporation|
|Computer and joystick with software||Cell Robotics Inc.||for controlling a motorized stage|
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