Ytterbium-doped fibre femtosecond laser offers robust operation with deep and precise microsurgery of C. elegans neurons

Abstract

Laser microsurgery is a powerful tool for neurobiology, used to ablate cells and sever neurites in-vivo. We compare a relatively new laser source to two well-established designs. Rare-earth-doped mode-locked fibre lasers that produce high power pulses recently gained popularity for industrial uses. Such systems are manufactured to high standards of robustness and low maintenance requirements typical of solid-state lasers. We demonstrate that an Ytterbium-doped fibre femtosecond laser is comparable in precision to a Ti:Sapphire femtosecond laser (1-2 micrometres), but with added operational reliability. Due to the lower pulse energy required to ablate, it is more precise than a solid-state nanosecond laser. Due to reduced scattering of near infrared light, it can lesion deeper (more than 100 micrometres) in tissue. These advantages are not specific to the model system ablated for our demonstration, namely neurites in the nematode C. elegans, but are applicable to other systems and transparent tissue where a precise micron-resolution dissection is required.

Figure 1

Yb-doped fibre femtosecond laser integrated in epifluorescence microscope produces a small damage spot, comparable to Ti:Sapphire laser. (a) Lasers are integrated to an epifluorescence compound microscope through a beam-splitter or dichroic mirror. Note that some lasers require an external pulse picking device. (b) Damage spot on a thin layer of black ink is larger when induced with a nanosecond pulse laser (top) than with either of the femtosecond lasers (middle Ti:Sapphire; bottom Yb-fibre). Damage spots are not reduced in size two micrometres above or below the image focal plane for the nanosecond pulse laser, while undetected for the femtosecond pulse lasers. Scale bar = 5 μm. (c) Square diameter of damage plotted against log scale of the energy per pulse used to estimate theoretical minimum beam size (square root of slope) and the threshold energy (x-axis intercept) for Ti:Sapphire (red) and Yb-fibre (blue).

Figure 2

A single C. elegans dendrite can be injured without damage to neighbouring dendrites. A single dendrite located in a sensory bundle in the nose of the animals was successfully ablated (yellow chevron) using the Ti:Sapphire and the Yb-fibre lasers without collateral damage to adjacent dendrites. The nanosecond-pulse laser injured more than one dendrite. Scale bar = 2 μm.

Figure 3

Nanosecond laser induces larger gap at site of injury but injured neurons regenerate at similar rate. (a) C. elegans motoneuron axotomy. Commissures of D motoneurons in immobilized animals were axotomized, animals were recovered and axon found again next day to asses regeneration. Images taken before, 1 second after and 24 hours after injury. Sites of axotomy (yellow chevron) and regenerating branch (green chevron) are indicated. (b) Mean gap between injured tips was larger after injury with nanosecond laser. (c) Percentage of neurons that regenerated at 24 h was the same for the three types of lasers. Images in A for before and at 24 h are maximal projections of Z stacks while the images at 1 s are each a single frame, leaving some parts of the neuron out of the imaging plane. *p < 0.05 ***p < 0.001. Scale bar. = 5 μm.