The study of cellular responses to changes in the spatial distribution of molecules in development, immunology and cancer, requires reliable methods to reproduce in vitro the precise distributions of proteins found in vivo. Here we present a straightforward method for generating substrate-bound protein patterns which has the simplicity required to be implemented in typical life science laboratories. The method exploits photobleaching of fluorescently tagged molecules to generate patterns and concentration gradients of protein with sub-micron spatial resolution. We provide an extensive characterization of the technique and demonstrate, as proof of principle, axon guidance by gradients of substrate-bound laminin peptide generated in vitro using LAPAP.

Cells sense spatial distributions of molecules which trigger signal transduction pathways that induce the cell to migrate or extend by remodelling the cytoskeleton. However, the influence of local and small variations of extracellular protein concentration on chemotaxis is not fully understood, due in part to the lack of simple and precise methods to pattern proteins in vitro. We recently developed a new technology to fabricate such patterns which relies on photobleaching fluorophores to adsorb proteins on a cell culture substrate: laser-assisted protein adsorption by photobleaching (LAPAP). Here we report several key improvements to LAPAP: we created arbitrary patterns made of several different proteins simultaneously, we reduced the fabrication time more than one order of magnitude and we used secondary antibodies to significantly enlarge the spectrum of proteins that can be employed. As a result, multicomponent protein gradients can be produced using reagents that are typically available in life science research laboratories on a standard inverted microscope equipped with a camera port.

Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration.