Media 1: Fast monitoring of in-vivo conformational changes in myosin using single scan polarization-SHG microscopy

Originally published in Biomedical Optics Express on 01 December 2014 (boe-5-12-4362

Femtosecond Laser Axotomy in <em>Caenorhabditis elegans</em> and Collateral Damage Assessment Using a Combination of Linear and Nonlinear Imaging Techniques

<div><p>In this work highly localized femtosecond laser ablation is used to dissect single axons within a living <i>Caenorhabditis elegans</i> (<i>C. elegans</i>). We present a multimodal imaging methodology for the assessment of the collateral damage induced by the laser. This relies on the observation of the tissues surrounding the targeted region using a combination of different high resolution microscopy modalities. We present the use of Second Harmonic Generation (SHG) and Polarization Sensitive SHG (PSHG) to determine damage in the neighbor muscle cells. All the above is done using a single instrument: multimodal microscopy setup that allows simultaneous imaging in the linear and non-linear regimes and femtosecond-laser ablation.</p> </div

Comparison between PSHG and fluorescence for the muscle damage assessment of after the axotomy.

<p>Minimum collateral damage. Before the axotomy: a) TPEF image of YFP marked muscles and axons; b) SHG image of the muscles in the same region. After the axotomy: c) TPEF reveals a successfully cut axon, in the shape of a gap that interrupts the continuity of the axon, but no other damage is apparent; d) SHG image of the body wall muscle shows a small signal decrease at the targeted point on the axon. No other change or structural transformation is evident. PSHG analysis: c) Post-surgical pixel-resolution mapping of myosin <i>θ_SHG</i> at the muscle (color bar in degrees); d) <i>θ_SHG</i> (mean±1 standard deviation) for the muscles in the region surrounding the cut and in the control region(white squares). The control region was selected in the adjacent muscular cell far away from the axotomy. Unpaired two tailed <i>t</i>- test (n = 200 pixels for both sets) yields p>0.05 meaning that <i>θ_SHG</i> mean is not significantly different between the two regions. Arrows point to the place of the laser axotomy. Scale bar 10 µm.</p

TEM illustration of the anatomical region of <i>C. elegans</i> where the laser axotomy is performed

<p>. Body wall muscles are shown in green, axons in purple and cuticle in gray. Blue ellipsoid is the estimated full-width-at-half-maximum of the beam's point spread function. White line represents the limit of the maximum plasma density generated at the focal spot. Scale bar 300 nm.</p

Successful axotomies and associated damage assessment techniques.

<p>This table summarizes the total number of successful axotomies (n = 56 out of 61) and the techniques used for the damage assessment.</p

Damage assessment using linear imaging techniques.

<p>a) Confocal, b) LT and c) combined images of the region surrounding the axon before the laser dissection. d–f) show the same region after the surgery (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058600#pone.0058600.s001" target="_blank">Media S1</a>). Damage is evidenced by increased autofluorescence in the confocal image and a dark spot in the LT image. Both damage structures colocalize at the combined image. Excitation of the GFP labeled neurons was done at 488 nm. Arrows point to the place of the laser axotomy. Scale bar 10 µm.</p

Comparison between SHG and fluorescence for the muscle damage assessment after the axotomy. Large collateral damage.

<p>Before the axotomy: a) TPEF images of YFP marked muscles and axons; b) SHG signal of the same muscles; and c) merge of TPEF and SHG images for comparison. Post-surgical images: d) TPEF and e) SHG images showing the laser damage hat is evident over a larger region. c) Shows a merged TPEF and SHG images for comparison. Arrows point to the place of the laser axotomy. Scale bar 10 µm.</p

Collateral damage assessment using linear and nonlinear imaging techniques.

<p>Linear: a) confocal, b) LT and c) combined images of the region surrounding the axon before the laser dissection; d–f) show the same region after the surgery (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058600#pone.0058600.s002" target="_blank">Media S2</a>). Nonlinear: g) TPEF, h) SHG and i) combined images before the laser dissection; j–l) show the same region after the surgery. No collateral damage was observed in CFM while damage in muscle is evidenced with SHG microscopy. Arrows point to the place of the laser axotomy. Scale bar 20 µm.</p

Comparison between PSHG and fluorescence for the muscle damage assessment after the axotomy.

<p>Medium collateral damage. Before the axotomy: a) TPEF image of YFP marked muscles and axons; b) SHG image of the muscles in the same region. After the axotomy: c) TPEF reveals a successfully cut axon showing a gap that interrupts the continuity of the axon. No other damage is apparent; d) SHG image of the body wall muscle shows the change from SB to DB structure of the sarcomeres. PSHG analysis: c) Post-surgical pixel-resolution mapping of myosin <i>θ_SHG</i> at the muscle (color bar in degrees); d) <i>θ_SHG</i> (mean±1 standard deviation) for the muscles in the region surrounding the cut and in the control region(white squares). The control region was selected in the adjacent muscular cell far away from the axotomy. Unpaired two tailed <i>t</i>- test (n = 200 pixels for both sets) yields p<0.001 (****) meaning that <i>θ_SHG</i> mean is significantly different between the two regions. Arrows point to the place of the laser axotomy. Scale bar 10 µm.</p

Strain distribution in WS2 monolayers detected through polarization-resolved second harmonic generation

Two-dimensional (2D) graphene and graphene-related materials (GRMs) show great promise for future electronic devices. GRMs exhibit distinct properties under the influence of the substrate that serves as support through uneven compression/ elongation of GRMs surface atoms. Strain in GRM monolayers is the most common feature that alters the interatomic distances and band structure, providing a new degree of freedom that allows regulation of their electronic properties and introducing the field of straintronics. Having an all-optical and minimally invasive detection tool that rapidly probes strain in large areas of GRM monolayers, would be of great importance in the research and development of novel 2D devices. Here, we use Polarization-resolved Second Harmonic Generation (P-SHG) optical imaging to identify strain distribution, induced in a single layer of WS2 placed on a pre-patterned Si/SiO2 substrate with cylindrical wells. By fitting the P-SHG data pixel-by-pixel, we produce spatially resolved images of the crystal armchair direction. In regions where the WS2 monolayer conforms to the pattern topography, a distinct cross-shaped pattern is evident in the armchair image owing to strain. The presence of strain in these regions is independently confirmed using a combination of atomic force microscopy and Raman mapping.</p