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

Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy

<p>Abstract</p> <p>Background</p> <p>Multicellular tumor spheroids are models of increasing interest for cancer and cell biology studies. They allow considering cellular interactions in exploring cell cycle and cell division mechanisms. However, 3D imaging of cell division in living spheroids is technically challenging and has never been reported.</p> <p>Results</p> <p>Here, we report a major breakthrough based on the engineering of multicellular tumor spheroids expressing an histone H2B fluorescent nuclear reporter protein, and specifically designed sample holders to monitor live cell division dynamics in 3D large spheroids using an home-made selective-plane illumination microscope.</p> <p>Conclusions</p> <p>As illustrated using the antimitotic drug, paclitaxel, this technological advance paves the way for studies of the dynamics of cell divion processes in 3D and more generally for the investigation of tumor cell population biology in integrated system as the spheroid model.</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

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

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

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

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

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