Optical neuronal guidance in three-dimensional matrices

Abstract

We demonstrate effective guidance of neurites extending from PC12 cells in a three-dimensional collagen matrix using a focused infrared laser. Processes can be redirected in an arbitrarily chosen direction in the imaging plane in approximately 30 min with an 80% success rate. In addition, the application of the laser beam significantly increases the rate of neurite outgrowth. These results extend previous observations on 2D coated glass coverslips. We find that the morphology of growth cones is very different in 3D than in 2D, and that this difference suggests that the filopodia play a key role in optical guidance. This powerful, flexible, non-contact guidance technique has potentially broad applications in tissues and engineered environments.

Fig. 1

A schematic of the setup for optical guidance. Microscopy components: CCD: charge coupled device camera (including electron multiplied device(s); Obj: objective; Sample: the imaging sample; XYZ: motorized XY stage and piezoelectric Z control; Cond: condenser; Lamp lamp for brightfield and DIC. The guidance system consists of the following components: IR: infrared laser; ND: neutral density filter FSM: fast steering mirror; Tel: telescope for magnification and creating conjugate planes; SP: short pass mirror.

Fig. 2

Representation of laser beam positioning for optical neuronal guidance. The laser beam is placed so that the laser spot overlaps the leading edge of the growth cone, and is positioned at a point on the leading edge closest to the direction of the intended axonal trajectory. In the above illustration, the axon is extending straight down, and the direction of intended guidance is to the right. The beam is placed on the right edge of the growth cone, and oscillated to cover the largest area of the leading edge, resulting in an oscillatory direction about 45° from the intended axonal trajectory.

Fig. 3

Neurite trajectories in the absence of guidance. Shown above are eight of the ten control trials. The green areas represent the neurite at the beginning of the imaging period, and the magenta overlay shows the position at the end of the 30–40 min trial. The neurites typically extend without changing orientation or don’t extend significantly (scale bar: 10 µm) (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

Fig. 4

Optically induced turning. A 1064 nm laser beam was applied to the edge of an extending PC12 growth cone, causing the extension to grow into the focus of the beam. The growth cone was located about 50 µm above the coverslip. The red circle indicates the approximate position of the laser beam. The beam was oscillated at a rate of 0.1 Hz with a displacement of 1 µm in the x-direction. The laser power at the coverslip was 60mW. This cell extended out of the original focus window, and so the microscope stage was moved up during the capture (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

Fig. 5

Summary of Optically Guided Turns. The neurite at the beginning of the imaging period is shown in green, and the magenta overlays show the position at the end of the 30–40 min trial. At the top are the eight successful trials, which clearly exhibit a change in neurite direction. The bottom two trials did not show significant turning: one of these failed guided trials seemed attracted and/or attached to several collagen fibrils in a direction different from the intended direction, while the other failed guided trial did not produce significant advance. (Scale bar: 10 µm) (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

Fig. 6

Measurement of the change in neurite trajectory. For each trial, the initial and final images of the trial were extracted, and a line indicating the neurite trajectory was overlaid onto the image. The change in angle between these two lines was found to give a rough estimate of the change in neurite trajectory.

Fig. 7

Table of results for the change in neurite trajectory of guided trials. Eight of the ten trials showed a change in trajectory well above 5°. For the control trials, nine out ten trials showed changes in direction of less than 5°.

Fig. 8

Growth cone has very different morphology in 3D collagen matrix. Left: Actin (green) from growth cones on a coverslip coated with collagen monomers. Right: Actin (green) from growth cone growing in collagen (magenta). Note that cells in 2D have many more filopodia radiating in all directions, while cells growing in 3D have longer, more directed filopodia, with some very long extensions tightly associated with fibrils. Grid spacing is 5 µm (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).