Deep and Clear Optical Imaging of Thick Inhomogeneous Samples

Inhomogeneity in thick biological specimens results in poor imaging by light microscopy, which deteriorates as the focal plane moves deeper into the specimen. Here, we have combined selective plane illumination microscopy (SPIM) with wavefront sensor adaptive optics (wao). Our waoSPIM is based on a direct wavefront measure using a Hartmann-Shack wavefront sensor and fluorescent beads as point source emitters. We demonstrate the use of this waoSPIM method to correct distortions in three-dimensional biological imaging and to improve the quality of images from deep within thick inhomogeneous samples

Aphid adaptation to cucurbits: sugars, cucurbitacin and phloem structure in resistant and susceptible melons

Abstract Background Aphis gossypii, a strictly phloemophagaous aphid, colonize hundreds of plant families, and a group of clones formed a cucurbit-specialised host-race. Cucurbits are unique in having evolved a specific extra-fascicular phloem (EFP), which carries defence-related metabolites such as cucurbitacin, whereas the fascicular phloem (FP) is common to all higher plants and carries primary metabolites, such as raffinose-family oligosaccharides (RFOs). Both cucurbitacins (in the EFP) and galactinol (in the FP) have been suggested to be toxic to aphids. We investigated these hypotheses in cucurbit-specialized A. gossypii fed on melon plants with or without aphid-resistance conferred by the NLR gene Vat. We selected a plant-aphid system with (i) Vat-mediated resistance not triggered, (ii) Vat-mediated resistance triggered by an aphid clone adapted to the presence of Vat resistant alleles and (iii) Vat-mediated resistance triggered by a non-adapted aphid clone. Results We quantified cucurbitacin B, its glycosylated derivative, and sugars, in melon plants and aphids that fed on. The level of cucurbitacin in plants was unrelated to both aphid infestation and aphid resistance. Galactinol was present at higher quantities in plants when Vat-mediated resistance was triggered, but its presence did not correlate with aphid performance. Finally, we showed that cucurbit-specialized A. gossypii fed from the FP but could also occasionally access the EFP without sustainably feeding from it. However, the clone not adapted to Vat-mediated resistance were less able to access the FP when the Vat resistance was triggered. Conclusion We concluded that galactinol accumulation in resistant plants does not affect aphids, but may play a role in aphid adaptation to fasting and that Cucurbitacin in planta is not a real threat to Aphis gossypii. Moreover, the specific phloem of Cucurbits is involved neither in A. gossypii cucurbit specialisation nor in adaptation to Vat-dependent resistance

A comparative study of three-dimensional cone-beam CT sialography and MR sialography for the detection of non-tumorous salivary pathologies

International audienceBackground Imaging of the salivary ductal system is relevant prior to an endoscopic or a surgical procedure. Various imaging modalities can be used for this purpose. The aim of this study was to compare the diagnostic capability of three-dimensional (3D)-cone-beam computed tomography (CBCT) sialography versus magnetic resonance (MR) sialography in non-tumorous salivary pathologies.Methods: This prospective, monocenter, pilot study compared both imaging modalities in 46 patients (mean age 50.1 ± 14.9 years) referred for salivary symptoms. The analyses were performed by two independent radiologists and referred to identification of a salivary disease including sialolithiasis, stenosis, or dilatation (primary endpoint). The location and size of an abnormality, the last branch of division of the salivary duct that can be visualized, potential complications, and exposure parameters were also collected (secondary endpoints).Results Salivary symptoms involved both the submandibular (60.9%) and parotid (39.1%) glands. Sialolithiasis, dilatations, and stenosis were observed in 24, 25, and 9 patients, respectively, with no statistical differences observed between the two imaging modalities in terms of lesion identification (p 1 = 0.66, p 2 = 0.63, and p 3 = 0.24, respectively). The inter-observer agreement was perfect (> 0.90) for lesion identification. MR sialography outperformed 3D-CBCT sialography for visualization of salivary stones and dilatations, as evidenced by higher positive percent agreement (sensitivity) of 0.90 [95% CI 0.70–0.98] vs. 0.82 [95% CI 0.61–0.93], and 0.84 [95% CI 0.62–0.94] vs. 0.70 [95% CI 0.49–0.84], respectively. For the identification of stenosis, the same low positive percent agreement was obtained with both procedures (0.20 [95% CI 0.01–0.62]). There was a good concordance for the location of a stone (Kappa coefficient of 0.62). Catheterization failure was observed in two patients by 3D-CBCT sialography.Conclusions: Both imaging procedures warrant being part of the diagnostic arsenal of non-tumorous salivary pathologies. However, MR sialography may be more effective than 3D-CBCT sialography for the identification of sialolithiasis and ductal dilatations. Trial registration NCT02883140

Image quality improvement in depth.

<p>(a) Single planes images of nuclei and mitotic figures at various depths within the MCTS resolved with AO or not (w/o AO). Images are acquired by using the same excitation intensity at 595 nm and same exposure time (300 ms). Scale Bar 5 µm. (b) The corresponding <i>IG_ROI</i> Ratio image. (c) The corresponding intensity plots along the line indicated.</p

MCTS induced aberrations.

<p>(a) Transmitted light images of cells seed with 2.5 µm green beads (InSpeck Green, I-7219, Invitrogen) in a 96- well plate (bottom image). After 4 days in culture, these cells formed a MCTS incorporating the beads (below image). (b) Maximum projection of a three-dimensional stack of 100 images (z spacing 1 µm) of a MCTS expressing a fluorescent nuclear protein, H2B–HcRed and cultivated in presence of green fluorescence beads as shown in (a). Insets show magnified views of the bead outside (left) or inside (right) the spheroid. Scale bar, 2 µm. (c) Profile views of the maximum intensity projections x-y and x-z for beads either outside (top panel), or inside the MCTS (bellow panel) imaged at different depths. Images were acquired at fixed excitation intensity at 491 nm and a 100 ms exposure time. Images share the same intensity scale. Scale bar, 2 µm.</p

Non-common path optical aberrations.

<p>(a) Recorded wavefront in the imaging camera focal plane corresponding to the imaging path aberrations. (b) Reference recorded wavefront in the HSWF sensor path after correction of the imaging path aberrations corresponding to the differential aberrations. (c) Graph showing the 3^rd and 5^th order of Zernike coefficients of (a) and (b).</p

waoSPIM performance.

<p>Values obtained for the full width at half maximum (FWHM), the root-mean-square (RMS) and Strelh ratio from the bead images and wavefront map in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035795#pone-0035795-g002" target="_blank">Figure 2</a>. The FWHM was determined from the parameters retrieved from the fitted curve obtained from the plot intensity profile along the three axes respective to views in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035795#pone-0035795-g002" target="_blank">Figure 2 b and e</a>. na, not applicable.</p

waoSPIM performance.

<p>a, b and c correspond respectively to the sketch of the phantom beads geometry, the profile views and the wavefront maps for beads embedded in agarose 1% and imaged at a depth of 360 µm. d, e and f correspond respectively to the sketch of the phantom beads geometry, the profile views and the wavefront maps for beads embedded in 1% of agarose and imaged through the capillary glass at a depth of 350 µm. Images were acquired either without (w/o AO) and with AO (AO) at a fixed excitation intensity at 491 nm and a 100 ms exposure time. (a, d) In Sketches SH, sample holder; C, capillary. (b, e) Profile views without (w/o AO) and with AO (AO) of the maximum intensity projections x-y and x-z for beads either embedded in a cylinder of agarose (b), or in agarose imaged through capillary glass (e). Scale bar, 5 µm. (c, f) Wavefront maps corresponding to the raw wavefront minus reference wavefront recorded with the DM shape set to correct the optical setup aberrations (w/o AO) or with the AO closed loop (AO), the color scale correspond to the wavefront error in µm.</p

Scheme of the waoSPIM setup.

<p>In our experimental setup (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035795#pone.0035795.s005" target="_blank">Fig. S1</a>), a cylindrical lens focuses the light to a horizontal line (light sheet) that is imaged into the back focal plane of an illumination objective (10× NA 0.25) (yellow path). The sample is positioned in the light sheet inside a physiological chamber filled with aqueous medium. The emitted light is collected (red path) by an immersion objective (20× NA 0.5) fitted to the physiological chamber. AOTF, acousto-optic tunable filter; T1–T3, telescopes; M1–M5, mirrors; CL, cylindrical lens; DM, deformable mirror; D1 and D2, dichroic mirrors; HSWF, Hartmann–Shack wavefront sensor; B, Lens system for DM-HSWF pupil conjugation; TL, tube lens; CCD 1 and CCD2, coupled charged devices.</p

Signal and contrast improvement.

<p>Maximum projection of a 3D stack of 100 images (z spacing 1 µm) of a large MCTS expressing a fluorescent nuclear protein, H2B–HcRed, without (w/o AO) and with AO (AO). Scale bar, 50 µm. The asterisk marks the bead used as the point source emitter located at a depth of 150 µm. Both images were acquired by using the same excitation intensity at 595 nm and a 300 ms exposure time. (b) Magnified views of the bead. Scale bar, 5 µm. (c) Intensity profiles along the lines 1 and 2 indicated in (a). (d) FWHM (µm), RMS and Strelh ratio values for bead images in (b). (e) <i>IG_ROI</i> mapping images calculated from images in (a) and <i>IG_ROI</i> Ratio image calculated as</p><p></p>where is mapping images calculated from images obtained with AO and is mapping images calculated from images obtained without AO.<p></p