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#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
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1. Introduction
Recent reports on using femtosecond (fs) lasers for conducting neuronal axotomy in C. elegans
have generated a lot of excitement in the field of neuroscience [1, 2, 3]. The ability to selectively
ablate an individual axon in a genetically tractable organism makes it an extremely powerful
system to study several aspects of neuronal function with an important one being regeneration.
Another fundamental cellular process, viz. transport of cargo, is essential in long polarized
cells like neurons since protein synthesis occurs in the cell body and synaptic transmission at
the end of the axon. Long term assays conducted over several hours to study transport were
introduced in and were till recently limited to larger organisms such as mice [4]. These are
usually carried out by tying a suture to compress long nerves such as the sciatic nerve and ex-
amining cargo accumulation at later time-points. Attempts to develop similar transport assays
in smaller genetic model organisms such as Drosophila have met with some success [5]. Post
axotomy imaging of transport in Aplysia neurons in culture has revealed that transport changes
occur rapidly and a microtubule-dependent vesicle trap is set up that optimizes growth cone
formation [6]. Since growth cone formation is an important step in axon regrowth it is critical
to develop assays in vivo that allow determination of changes to transport during this process.
We report the development of a transport assay in C. elegans using laser axotomy to physically
block transport. The nanosecond (ns) laser used in our studies reproduced earlier results using
fs systems for neuronal axotomy. By imaging accumulations of fluorescently labeled synaptic
vesicle proteins at the site of axonal microsurgery we can characterize anterograde and retro-
grade transport. This assay is simple in procedure and provides quick results (1 hour) and could
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
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be used to study several aspects of transport in neurons, some of which we discuss later. We
also studied the physical nature of the ablation process using time-resolved imaging to cap-
ture cavitation bubble dynamics in C. elegans and Drosophila larvae. Time-resolved imaging
of the cavitation bubble size provides a direct measure of the amount of damage during axo-
tomy procedures and can also provide estimates of the mechanical forces generated during the
process. We compare cavitation dynamics from our study with those reported in a recent study
on laser microsurgery in Drosophila embryos using 355 nm nanosecond laser pulses [7]. We
also discuss the advantages of nanosecond lasers as compared to femtosecond lasers for such
procedures.
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
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camera gain. Significance was tested using the student t-test with unequal variance.
3. Results
3.1. Nanosecond laser axotomy
In Fig. 1 we present results of axonal microsurgery using nanosecond laser pulses in C. elegans
larvae. A larva in which the PLM neuron was targeted is shown before ((a),(c),(e)) and after
((b),(d),(f)) the surgery. The damaged region or neuronal cut is shown magnified in the inset
in (b), (d) and (f). In all cases we observe a break in the axon at the site of laser focus. These
particular axotomies have been chosen to exhibit the range of damage that the laser microbeam
produced. For instance, in 1(b) and 1(d) we could observe clear breaks in the axon extending
over 3-5 μ m. In 1(f) however the visible break is barely discernable being < 1 μ m and the
only other evidence for axonal cutting is the displacement of the proximal end due to cavita-
tion forces generated by plasma formation. The average extent of damage in these axons was
measured to be 1.95 ± 1.81 μ m (n = 8). The large variation in extent of damage occurs due to
inclusion of all cut axons including those that move significantly due to the cavitation forces.
Fig. 1. Axonal microsurgery of PLM neurons in C. elegans larva with nanosecond laser
pulses. The axon is shown prior to (a,c,e) and immediately after surgery (b,d,f). Insets in
(b,d,f) show magnified regions around the cut site. Clear breaks can be seen in the axon
(b,d). In (f) the extent of damage is extremely small and is only noticeable as a dark band.
The proximal end of the axon was also displaced due to the cavitation forces generated
during the laser microsurgery. Pulse energy was 0.8 μ J with the laser being operated in
single shot mode.
We found that a pulse energy of 0.8 μ J was sufficient to cut axons in C. elegans with minimal
collateral damage. The laser was used in single shot mode and the operator manually fired the
laser till the axon was cut. The number of pulses varied between individuals but was always
< 20. In a majority of surgeries one laser pulse was sufficient to effect a complete axonal cut,
with the position of the laser spot being the major determinant of success. In a few cases we
also observed partial cutting of the axon effected by the first few pulses, following which a
succeeding laser pulse would fully cut it. Physical movement of the cut ends due to cavitation
forces could also be observed. Thus, the site of laser focus and physical movement influenced
the extent of damage observed immediately after the axotomy. Successful axonal cutting re-
sulted in a retraction of the two ends away from the site of laser irradiation. This retraction was
observable within 1 hr of microsurgery and has also been noted by other researchers [3, 13].
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
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We measured the extent of damage in C. elegans to be 4.65 ± 1.85 μ m (n = 17) at 1 hr after
laser microsurgery. To determine collateral damage during laser axotomy, we viewed animals
4, 6 and 8 hours post-surgery under DIC and noted scarring in only 8% of the animals (n = 24).
Animals undergoing laser axotomy showed viability levels > 95% after removal of anesthetic
and continued to undergo development and become fertile adults. Viability in control animals
(sham surgery treated) was 100% (n = 20).
Post surgery, C. elegans larvae were maintained at 16 ◦ C and followed in the 1-3 hr time
period to assess the time required for accumulation of vesicles and determine cellular out-
comes after damage. In Fig. 2(a,b,c) axons in 3 individual larvae are shown at different times
after surgery. One hour post surgery (Fig. 2(a)) we could observe robust accumulations of
GFP::RAB-3, a known synaptic vesicle marker. The accumulation of GFP::RAB-3 signal re-
sults from a block at the distal and proximal cut ends. At later time points we do not see any
accumulations. Instead we observed regenerative processes similar to those reported earlier for
fs laser surgery in C. elegans. At 3 hours, we could observe close proximity of the distal and
proximal ends (Fig. 2(b), inset). This maybe an attempted reconnection of the cut ends and
was observed in 46% of the animals (n = 13). Regrowth of a new axon from the proximal
end occurred in 54% of the animals (n = 13). At 9 hours, (Fig. 2(c)), regrowth was seen in all
individuals (n = 4). Similar putative reconnection events have been observed by others [3, 13].
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
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Fig. 3. Time-resolved imaging of cavitation dynamics in C. elegans. The bubble reaches its
maximum size within 20 ns of laser irradiation and the first collapse occurs within ≈30 ns.
At later times we observe a rebound and collapse. Arrows indicate the cavitation bubble in
different frames.
Fig. 4. Neuronal axotomy in Drosophila 1st larvae. (a) Two motorneuron bundles prior to
axotomy and (b,c) after successive cutting of each bundle. Arrows in (b) and (c) point to
the first and second axotomies respectively. Ventral ganglion is at lower left in each image
(marked with ∗). Pulse energy was 1.2 μ J and the laser was operated in single shot mode.
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
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was difficult to conclusively determine the bubble parameters in this time range due to large
variability.
4 7
(a) (b)
6
Bubble Radius (µm)
3 5
4
2
3
1 2
1
0 0
10 20 30 40 50 60 70 80 90 10 100 1000
Time (ns) Time (ns)
Fig. 6. Cavitation bubble dynamics in (a) C. elegans and (b) Drosophila. Bubble dynamics
are complete within 100 ns in C. elegans and 800 ns in Drosophila.
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6 times larger than in uncut axons (0.18 μ m 2 ) (n = 130 puncta, 10 individuals). We also found
that laser microsurgery did not significantly change the population of vesicles in regions away
from the cut (0.24 μ m 2 ) compared to uncut animals. The area-wise distribution of fluorescent
accumulations with distance is presented in Fig. 7(b). We observe that the area of accumulation
near the cut site is much larger compared with GFP::RAB-3 accumulations further away from
the cut site. Within the first micron of the retracted distal and proximal ends of the axon, greater
than 40% GFP::RAB-3 accumulations are larger than 1 μ m 2 and more than 20% of the accu-
mulations are between 0.5-1.0 μ m 2 . In uncut axons (Fig. 7(b), inset), fluorescent accumulations
with area ≥1 μ m2 are not observed while <20% of the puncta are between 0.5-1.0 μ m 2 in size
and the rest are smaller than 0.5 μ m 2 . A second vesicular marker, synaptobrevin::GFP (SNB-
1::GFP), was used to confirm the validity of the assay. Areas of SNB-1::GFP accumulations
1 hr post surgery at the proximal end (0.82 ± 0.15 μ m 2 ) and distal end (0.95 ± 0.17 μ m 2 ) of
the cut (n = 12 puncta or individuals) were on average 3 times larger than in uncut axons (0.27
± 0.05 μ m2 ) (n = 148 puncta, 24 individuals) (p< 10 −4 ). We also axotomized zdIs5 a strain
with cytoplasmic GFP marking the axon. Areas of fluorescent puncta in zdIs5 1 hr post surgery
were 0.44 ± 0.08 μ m 2 away from the cut site, 0.25 ± 0.12 μ m 2 at the proximal end and 0.46
± 0.16 μ m2 at the distal end (n = 9). Thus in zdIs5 there was no statistical difference between
the size of fluorescent puncta at the cut site and away from it.
Fig. 7. (a) Mean areas of GFP::RAB-3 puncta in axons of uncut and axotomized jsIs821.
Axons were cut between 10-20 μ m from the cell body. The area in (μ m2 ) of GFP::RAB-
3 accumulations at the distal and proximal ends of axotomized PLM axons 1 hour after
the cut (1.08) is 6 times larger than in uncut axons (0.18) (p< 10−4 ). In cut axons, the
mean area at regions away from the cut (0.24) is not statistically different from the uncut
axon. Inset shows an image of a cut axon used for quantification, with arrows pointing to
accumulations at the proximal and distal ends. ∗ points to cell body. (b) Size distribution of
fluorescent accumulations along the axon length. Within 1 μ m of the retracted distal and
proximal ends of the axon, ≥ 40% GFP::RAB-3 accumulations are larger than 1 μ m2 and
more than 20% of the accumulations are between 0.5-1.0 μ m2 . In uncut axons (Fig. 7(b),
inset), fluorescent accumulations with area ≥1 μ m2 are not observed while <20% of the
puncta are between 0.5-1.0 μ m2 in size and the rest are smaller than 0.5 μ m2 .
4. Discussion
Femtosecond laser pulses produce extremely localized damage zones as demonstrated previ-
ously in single cells [15, 16, 17] which makes them attractive for microsurgery in vivo. How-
ever, these laser systems still remain outside the reach of most researchers due to their cost
and complex operation. Nanosecond (ns) Nd:YAG pulsed lasers are the workhorses of the bio-
medical optics community and their simple operation and robustness combined with relatively
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
(C) 2008 OSA 23 June 2008 / Vol. 16, No. 13 / OPTICS EXPRESS 9891
cheap cost make them an attractive option to fs laser systems. Laser microsurgery with nanosec-
ond laser pulses has long been used in cell biology with exquisite precision. (See Ref. [18] for
a recent review). Nanosecond laser pulses at λ = 355 nm have also been used in vivo during
removal of the spindle in single-cell C. elegans embyros [19] and ablating cell junctions in the
aminoserosa in Drosophila embryos [20]. In the present study we extend the range of micro-
surgery procedures in vivo by demonstrating neuronal axotomy in C. elegans and Drosophila
larvae. Laser microsurgery relies on plasma formation, an intensity based phenomena for induc-
ing damage. The subsequent cavitation dynamics that are associated with plasma formation are
dependant upon the total energy provided to create the plasma. By using the 355 nm wavelength
we take advantage of higher photon energy to effect a reduction in the pulse energy necessary
for plasma formation and hence a reduction in the associated cavitation dynamics. We observe a
further reduction in plasma threshold in vivo due to linear absorption by endogenous molecules.
Hutson and Ma [7] have hypothesized that the absorber might be NADH, since it is present in
large quantities in developing organisms and has an absorption peak at λ = 340 nm. Due to the
reduction in threshold it was possible to successfully cut axons in C. elegans and Drosophila
with limited collateral damage and low pulse numbers. The extent of damage that we observed
in C. elegans after microsurgery (Fig. 1) was in a similar range to that noted previously using
femtosecond lasers. Specifically, Wu and co-workers report a gap length of 5 μ m (Fig. 5d, sup-
plementary information, Ref. [3]) and Bourgeois and Ben-Yakar report a damage range from
<0.5 μ m (4 nJ/pulse, 100 pulses) to 5.5 μ m (12 nJ/pulse, 1000 pulses, Fig. 8, Ref. [21]). Our
observed damage length of 1.95±1.81 μ m is within the limits provided by these data sets.
In C. elegans at 0.8 μ J pulse energy we noted internal wounding or scarring at the cut site in
2 out of 24 individuals (8 %). Animals with wounds developed into normal egg laying adults.
Using a higher pulse energy (1.5 μ J) larger wounds could be observed in all animals (n = 26).
Wu and co-workers also noted scarring in animals when using 4 ns pulses at λ = 440 nm to
conduct axotomies in C. elegans, while no scarring resulted during fs laser surgery [3]. This
indicates that while ns laser pulses produce axonal damage zones similar to fs laser pulses, in a
small number of animals collateral damage is larger with ns pulses.
Axotomy in Drosophila did not prove any different than in C. elegans, even though
Drosophila larvae are significantly larger and structures of interest are at least 5–10 μ m from
the coverslip (Fig. 4). Instead, we found that a small increase in pulse energy was sufficient to
perform microsurgies without removing the cuticle. The larger pulse energy caused a higher ex-
tent of damage in Drosophila motorneuron bundles (≈10 μ m). We also noticed larger physical
retraction of the axonal bundle upon cutting and this too contributed to the larger cut lengths.
Previously, in experiments studying nerve-muscle interactions, Fernandes and Keshishian had
used nanosecond laser pulses (λ = 440 nm, τ p = 3 ns, 30 μ J/pulse, 30 to 60 pulses at 15 Hz)
for ablation of Drosophila larval motorneurons [22]. A comparison with this data set is not
possible since the authors have not commented on the extent of damage caused by higher pulse
energy used during laser ablation, or on viability and development of larvae after ablation. In
Drosophila we did not conduct long term studies to determine the occurrence of wounds due
to laser irradiation. It is possible that damage due to cavitation effects although unseen could
contribute to changes in development programs resulting in the observed lethality prior to adult-
hood. However, grossly the development appears to be normal in 60% of larvae and we believe
this could be increased by careful selection of laser parameters.
Following microsurgery, axonal recovery was studied by imaging C. elegans larvae at dif-
ferent time-points (Fig. 2). We observed regenerative processes beginning at 3 hours after laser
microsurgery in the jsIs821 strain. The time-scale for regeneration was 3-9 hours in our exper-
iments. Interestingly, we observed shorter axonal regeneration time-scales as compared to the
6-24 hour recovery period previously reported for anesthetized animals [3, 13]. The previous
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
(C) 2008 OSA 23 June 2008 / Vol. 16, No. 13 / OPTICS EXPRESS 9892
data were generated using the zdIs5 or the muIs32 strains that have high levels of soluble GFP
marking mechanosensory neurons. Preliminary evidence from our lab using zdIs5 also gave us
longer recovery times than the 3-9 hour period observed for jsIs821. Further, recovery times
for usIs25 (data not shown), a strain with lower cytoplasmic GFP expression were faster than
for zdIs5. This suggests that altered recovery times are due to inherent differences in the strains
resulting from varied levels of GFP expression and unlikely to be due to differences in the
mechanism of damage or its extent produced by ns as compared to fs pulses.
Time-resolved imaging of cavitation dynamics in vivo provided a clear exposition of the
spatio-temporal scales of the damage process in our studies (Figs. 3 and 5). Bubble dynam-
ics proved reproducible in C. elegans with R max = 3.5 μ m and collapse within 100 ns. In
Drosophila, bubble dynamics were variable, due to the larger animal size and possibly different
microenvironments at the site of irradiation. R max in Drosophila was 4.5 μ m with a collapse
time of 215 ns. We also observed bubbles equivalent in size to R max in the time range of 30 to
40 ns. However, these were rare events and further studies are needed to conclusively determine
the complete bubble dynamics. Considering the large cavitation bubble size it was surprising to
note the limited extent of damage that we observed in these animals. It has been noted in vitro
that cells can withstand cavitation generated shear stresses several orders of magnitude higher
than physiological values due to short time-scales of exposure [23]. This fact coupled with the
damping of the bubble in vivo could account for the reduced collateral damage in the form of
scarring that was observed at the irradiation site. The time-resolved imaging also highlights the
regenerative capabilities of these organisms after facing large mechanical stresses.
Recently, Hutson and Ma also studied the cavitation dynamics during laser microsurgery of
Drosophila embryos using nanosecond pulses at 355 nm [7]. The dynamics were measured
by recording pressure transients associated with shock wave propagation and bubble collapse.
The time of bubble collapse was used to estimate R max using the Rayleigh equation. At a pulse
energy of 1 μ J, the authors observed an oscillation time (T osc ) slightly greater than 2 μ s with
an estimated Rmax of ≈11 μ m (Fig. 2 from Ref. [7]). In our experiments in C. elegans at
0.8 μ J, we imaged Tcol (= Tosc ) to be 30 ns with a R max of 3.4 μ m. To confirm the time-scales
of bubble dynamics in our experiments we also conducted time-resolved imaging in the 100-
1000 ns timescale in C.elegans and failed to observe any bubbles. Further, we undertook time-
resolved studies in Drosophila (Fig. 5). Due to the variability in bubble sizes at different time
points it was difficult to assign an exact collapse time. We only note that all dynamics were
complete by 800 ns, indicating that even in Drosophila cavitation was strongly damped. At
present we do not have an explanation for the difference in collapse times in our data set and that
reported in Ref. [7]. One possible reason could be the dissimilar sample geometries in the two
experiments. In our case the bubble expansion occurs in a 3D viscoelastic medium so the bubble
experiences a strong damping force from all sides. Earlier work has also reported the drastic
reduction in bubble size during expansion in tissue [24], while recent work from our lab has
shown that the bubble size is reduced by 54-59% for laser irradiation of rat corneas as compared
to the laser-induced lysis of adherent cells [12]. In the case of Hutson and Ma, the site of laser
irradiation is the amnioserosa, at the surface of the Drosophila embryo. Therefore, the bubble
experiences damping only at one surface, possibly causing it to expand into the medium and
collapse at longer time-scales. The above discussion highlights that there are still issues related
to cavitation damping in vivo that need to be resolved. While our time-resolved images provide
a first glimpse at laser microsurgery in vivo, detailed studies using supplementary methods such
as interferometry or hydrophone measurements are necessary.
It is also of interest to compare cavitation dynamics for ns and fs laser microsurgery. Re-
cently, Vogel and co-workers reported cavitation bubble sizes with an accuracy of ± 10 nm
for laser-induced plasma formation in water with 340 fs laser pulses at λ = 347, 520 and
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
(C) 2008 OSA 23 June 2008 / Vol. 16, No. 13 / OPTICS EXPRESS 9893
1040 nm [25]. The authors observed that at threshold, the cavitation bubble size was smaller
than the diffraction limited focus diameter. Specifically, at λ = 347 nm, R max increased slowly
with pulse energy and ranged from 0.19 μ m at 3.95 nJ to 1.8 μ m at 5.13 nJ. These bubble sizes
would be considerably smaller in vivo due to strong damping. The bubbles are smaller by a
factor of 2 to 20 as compared to the bubble sizes generated by ns laser pulses in C. elegans.
It is thus surprising to note that axotomy damage zones are comparable for ns and fs surgeries
and collateral damage in the form of scarring could be observed in only a small percentage of
animals.
Finally, we demonstrated the feasibility of neuronal axotomy for studying transport in vivo
using fluorescent proteins targeted to vesicles. Mean areas of fluorescent accumulations in C.
elegans at the proximal and distal ends in cut axons were 6 times larger for GFP::RAB-3 and
at least 3 times larger for SNB-1::GFP as compared to uncut axons. This indicated that vesicles
were transported to the site of the cut and remained trapped there. Such fluorescent accumula-
tions at the same time points were not observed for the zdIs5 strain since GFP is a cytoplasmic
marker. We only observed transient accumulation of GFP::RAB-3 (Fig. 2(a) and 7(a),inset).
This suggests that accumulation is due to a physical block to transport and unlikely to be due
to the formation of a growth cone. The growth cone should exist during the entire regenerative
process that has been reported to occur during a 6 to over 24 hour period. However no accu-
mulation of GFP::RAB-3 is seen at 3 hours or later in axotomized jsIs821 when regenerative
axon extensions are ongoing. The assay we have developed allows the study of several aspects
of transport such as: (1) determining whether a given protein is transported in both directions,
i.e. retrograde (away from the synapse) and anterograde (to the synapse) , (2) assessing the role
of specific transport mutants on longer time scales (1-2 hours, as opposed to a few minutes )
and (3) studying the dynamics of transport post complete or partial axotomy as pertaining to
regenerative processes. Some of these advantages apply equally to Caenorhabditis elegans and
Drosophila melanogaster.
5. Conclusion
We report the successful use of nanosecond laser pulses at λ = 355 nm to conduct neuronal
axotomy in C. elegans and Drosophila larvae. Axotomy in Drosophila could be used for stud-
ies comparing regeneration processes across model systems. A pulse energy in the range of
0.8-1.2 μ J and <20 pulses were sufficient to cut axons with high viability in both organisms.
Using time-resolved imaging we ascertained that damage occurs due to laser induced plasma
formation. The bubble expansion was significantly damped in C. elegans with bubble collapse
occurring within 100 ns. In Drosophila, while cavitation was variable, we observed that all
dynamics were complete within 800 ns. The bubble damping due to the viscoelastic medium
contributed to the reduced collateral damage that we observed. We also demonstrated the fea-
sibility of using neuronal axotomy as a means of creating a transport block in vivo and using
vesicular markers to characterize these blockages. We believe the results of our study opens up
this field to a wider community of researchers in neuroscience.
Acknowledgement
The authors acknowledge Nivedita Chatterjee for initiating this work. GNR is supported by a
Council of Scientific and Industrial Research (CSIR) fellowship. SSK is supported by a De-
partment of Science and Technology (DST) grant no. SR/SO/AS-67/2006 to SPK. KRR ac-
knowledges support from the National Institutes of Health (USA) via the Fogarty International
Research Collaboration Award. We thank CGC for the zdIs5 strain.
#93418 - $15.00 USD Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008
(C) 2008 OSA 23 June 2008 / Vol. 16, No. 13 / OPTICS EXPRESS 9894