Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Summary

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

Abstract

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.

Protocol

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)....

Discussion

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 m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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