12 research outputs found
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
Using quantitative single molecule localization microscopy to optimize multivalent HER2-targeting ligands
IntroductionThe progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling.MethodsBy combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells.ResultsWe detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases.DiscussionCollectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics
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
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
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<sup>rd</sup> and 5<sup>th</sup> order of Zernike coefficients of (a) and (b).</p
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<sub>ROI</sub></i> Ratio image. (c) The corresponding intensity plots along the line indicated.</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
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
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<sub>ROI</sub></i> mapping images calculated from images in (a) and <i>IG<sub>ROI</sub></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
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Dynamic lateral organization of opioid receptors (kappa, muwt and muN40D ) in the plasma membrane at the nanoscale level.
Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOPwt ), and its naturally occurring isoform (MOPN40D ) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble-averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside-enriched domains and partial association with cholesterol-enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor-specific. KOP-containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOPN40D . Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOPwt , whereas this effect was not observed for MOPN40D