Gaussian Beams
It has been established that both 785 nm and 830 nm simple optical traps can be used to manipulate hyphal growth and branching196,223,224,226. Here I examined the effects of 532 nm and 1064 nm light and included further experiments with 785 nm light for comparison.
The range of powers needed to cause a change in the pattern of hyphal growth varied largely with wavelength; for 1064 nm light 150−500 mW, for 785 nm 40−70 mW, and for 532 nm light 1.9−14 mW. For each range stated the minimum power was the lowest to cause an observable effect and the maximum was the highest to cause an effect without producing irreparable damage to the hyphae. The laser powers quoted are those incident on the back aperture of the microscope objective with the focus approaching the diffraction limit for each wavelength.
Examples of changed growth patterns for each wavelength, are shown in figure 4.14. The altered growth effect in each case was clearly associated with the proximity of the hyphal tip (and thus Spitzenk¨orper) to the optical trap. Once the hyphal tips had passed the trap, normal growth resumed back along its original direction, unless growth was stopped because a hyphal tip was exposed to too high a laser power. In all experiments it was important to judiciously use the minimum laser power necessary to cause a change in hyphal growth patterns otherwise it would commonly be stopped.
Unfortunately, the alteration of direction is short lived, both in time and distance, for all wavelengths studied. The usefulness of optical manipulation here would be greatly increased if the hyphae could be controlled over extended distances. To combat this the holographic methods are introduced.
Multiple Gaussian Beams
Figure 4.15 shows the result of allowing a hyphal tip to grow into a curve made from nine individual tweezers, produced using the holographic techniques already described.
The results were wildly inconsistent, rarely ending in a hyphal tip following the full pat- tern of multiple tweezers. These unsuccessful results generally occurred for one of two reasons. Firstly, the spacing of the tweezers often allowed hyphal tips to grow through the gaps between them without causing redirection. Secondly, if the hyphae grew through a tweezers their growth could cease from overexposure to the laser beam.
Figure 4.14: Examples of redirecting growth using 532 nm, 785 nm, and 1064 nm single Gaussian beam traps. The power required to cause the effect is between 1.9−14 mW at 532 nm, 40−70 mW at 785 nm and 150−500 mW at 1064 nm. The white circle indicates the position of the optical trap. Scale bar is 10µm.
Figure 4.15: Continuous redirection using nine 532 nm Gaussian traps produced with HOTs and objective A. The hyphal tip grew from bottom left to top right. White dots represent the position of each focussed trap. The laser power per tweezer focus was
≃2−3 mW. Scale bar is 10µm.
ation pattern. This even distribution is again possible through the use of holography to create continuous light patterns.
Continuous Light Patterns
Generating single, continuous, 30µm long lines of light at an angle≥50◦ to the direction
of growth consistently caused hyphal redirection as demonstrated in figure 4.16. The time stamps on subsequent figures are displayed in minutes:seconds.
Figure 4.16: Extended redirection using a single continuous line of 532 nm light pro- duced with objective A and introduced at 57◦ to the original axis of hyphal growth
at time 0 min. The final image in the sequence was captured after the laser filter was removed from its location in front of the camera, allowing visualisation of the laser pattern and position. The total laser power spread over the whole pattern was 8.1±0.1 mW. Scale bar is 10µm.
Producing a ‘channel’ of light made the hyphae ‘bounce’ down it to be guided along as shown in figure 4.17.
Figure 4.17: Guiding a hyphal tip through a 17µm wide channel bordered by 60µm long lines of light produced with objective B. The final image in the sequence was captured after the laser filter was removed to show the laser pattern and position. The white ‘smudge’ appearing in each image is an optical artifact. The total laser power spread over the whole pattern was 16.0±0.3 mW. Scale bar is 10µm.
Instances where the hypha was wider than the light channel resulted in width constriction as it entered the channel as exemplified in figure 4.18.
Additionally the first point of interaction between the hyphal tip and the light induced branch formation. In figure 4.18 this causes a hyphal branch to form at the lower left of the image and in figure 4.19 at the centre right of the last image. Hyphal branching never occurred within a channel bordered by light or from a hypha growing up against a line of light.
Switching between pre-calculated kinoforms on the SLM, hence optical fields in the sample plane, two sharp continuous redirections in hyphal growth can be produced as shown in figure 4.19. Having redirected the hyphae at an angle of 45◦ by time 4:44 minutes, the
Figure 4.18: Constriction and branching caused by a relatively thin channel (17µm wide) bordered by 60µm long lines of light produced with objective B. The final image in the sequence was captured after the laser filter was removed to show laser pattern and position. The white ‘smudge’ appearing in each image is an optical artifact. The total laser power spread over the whole pattern was 9.0±0.2 mW. Scale bar is 10µm.
kinoform was changed to produce a second line of light redirecting the hyphae by another 53◦ by time 10:27 minutes.
Figure 4.19: Multiple redirections of hyphal growth caused by switching between dif- ferent kinoforms produced with objective A. The switch in light patterns took place between 4:44 minutes and 4:49 minutes at which time the laser filter was removed to show laser pattern and position. The total laser power spread over each light pattern was 8.1±0.1 mW. Scale bar is 10µm.
As discussed in section 3.5.3 it is difficult to accurately measure laser power in holo- graphically generated beams. Here, the laser power input into the system was carefully selected by following a simple procedure. First, an estimate of the power required was made and then hyphal manipulation was attempted. If unsuccessful the power was either increased or decreased depending on whether the hyphae had been unaffected or overly affected respectively. It was also at times necessary to search and find a new hyphal tip to experiment on had the power needed been overestimated and caused damage.
A pseudowall of light, as described in section 4.4.1, improved the precision of growth redirection, an example of which is shown in figure 4.20. To produce the pseudowall of light the hologram used for figure 4.16 was axially stacked to produce nine axial planes of light, four each side of the normal focus, each separated by 0.5µm. This axial extension over 4µm makes the pattern more comparable in height to the hyphal tips (< 18 µm).
The hyphae consistently followed the edge of the pattern more closely with light sculpted in this way than with those used in the previous experiments.
Figure 4.20: Extended growth redirection using a pseudo 3D wall of 532 nm light produced with objective A. The final image in the sequence was captured after the laser filter was removed to show laser pattern and position. The total laser power spread over the whole pattern was 8.1±0.1 mW. Scale bar is 10µm.