Peligro

It has been shown hyphal tip growth inN. crassa can be redirected using optical tweezers with three different wavelengths of light (532 nm, 785 nm and 1064 nm), and the lower the wavelength used, the lower the laser power required to produce the changes.

Ranges, rather than average powers with associated errors have been quoted, at which hyphal growth alteration occurs for each wavelength. This is because, as is well known, biological systems have a large amount of variability and hence the resulting values are largely dependent on the size of the hyphal tips being studied and the patterns being produced. For a given run of experiments an effective power can quickly be found using trial and error with the aim of minimising the level of irradiation whilst still eliciting a response in the pattern of hyphal growth.

Assuming a diffraction limited focus, the range of laser intensities required at the focal plane of the tweezers to ellicite a response is lower for lower wavelengths, indicating a wavelength dependence. The cause of this is unknown. Possibilities include photoreceptors more sensitive to certain ranges of wavelength, preferential absorption of light by the medium and subsequent heating, or perhaps an optical repulsion, in the opposite sense to optical tweezers.

The results demonstrate that holographic beam shaping enables production of fixed pat- terns of light that can redirect or constrict hyphal growth over extended distances or initiate hyphal branching.

With all the optical methods employed to alter growth it is possible to use either too little or too much power. If too little power was used then hyphal tip growth was unaffected and the hyphae grew through the light. If the laser power was too high, then hyphal growth ceased, a response common in filamentous fungi exposed to stress. In the particular case of single optical tweezers, too high a power could cause hyphae to burst resulting in the cytoplasm leaking from the hyphal tip. A number of papers have assessed the damage caused to a variety of biological specimens by optical tweezers231–233. Potential sources for photodamage were cited as the generation of reactive oxygen species, two-photon absorption and transient local heating. It would be interesting to analyse the possible influence of these factors in future work.

It has previously been proposed that repulsion of the Spitzenk¨orper organelle complex by the optical trap is responsible for the variety of growth responses observed196,223,224_.

The work presented here provides further evidence to support this. It is only when the optical tweezers or light patterns were positioned at the hyphal apex that redirection of tip growth took place. Repeatedly switching the irradiation of the growing hyphal apex from one side to the other resulted in a repeated redirection of the growth axis giving rise to a zig-zag pattern of filamentous growth218,223. Figure 4.15 shows that even though the gaps between multiple optical traps were much smaller than the width of the hyphal tip it was not smaller than the Spitzenk¨orper which could therefore fit through the gaps without being influenced by the laser. Lines and ‘pseudowalls’ of light were shown to be much more effective at redirecting hyphal growth and when sufficient power was used, the hyphal tips were unable to grow across the light barriers.

It is not possible to say whether the effects of light at the green, near infrared and in- frared wavelengths are a physical phenomenon. Examples include optical repulsion of the Spitzenk¨orper224 or aversion to localised heating. Other possible phenomena include local intracellular generation of reactive oxygen species231 _{or a photoreceptor-mediated}

negative phototropism224.

It is believed that the growth alteration for both discrete and continuous patterns of light cannot be explained simply by the physics, i.e. optical forces, but must involve some unknown biological phenomenon.

The reason why hyphal branches were induced when hyphae became constricted by grow- ing into narrow channels bordered by light is unclear. It is possible that the initial per- turbation of hyphae by laser irradiation resulted in branch formation. Hyphal branch induction is often observed at the point of initial exposure to a laser trap223 _{and has}

also been initiated by using tweezers to apparently concentrate secretory vesicles within hyphae196.

The axial extent of the two dimensional continuous light patterns produced was very small compared to the actual vertical extent of the hyphal tips (<18 µm) so the alignment of the system and point of focus are thus highly critical. In order to increase the alignment tolerance, ‘pseudowalls’ of light were investigated and found to produce very consistent results in terms of redirecting hyphal growth. Also, the hyphae seemed less perturbed by the walls and followed the barriers much more closely than the single line of light. Two possible reasons for the improvement in results obtained with the three dimensional pseudowalls of light are as follows. Firstly, the same total power was used for both the pseudowalls and lines of light, but the pseudowall was spread between nine individual planes so at any one point the light was less intense, thus less damaging to cells. Secondly, because the pseudowalls were extended axially, given a misalignment in the system, there was a higher chance of the hyphal tip and Spitzenk¨orper being coplanar with the light field. It is foreseen that one of the main advantages of holographic beam shaping is its ability to produce light patterns with axial extent, unlike, for example, AODs. To increase the tolerance still further, the possibility of creating true three dimensional walls of light234

could be explored in future.

4.4.4 Conclusion

Optical tweezers provide a useful tool to manipulate the pattern of growth in filamentous fungi. More advanced techniques offer the potential to create artificial networks of hyphae in three dimensions by growing these filamentous organisms through light mazes. Fungal hyphae within mycelial networks sense and respond to each other in complex ways which regulates the morphology of the colony. Being able to precisely manipulate the three dimensional nature of these networks with light mazes may provide a useful experimental technique to analyse cell-to-cell communication in these complex systems.