High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM

Today, Light Sheet Fluorescence Microscopy (LSFM) makes it possible to image fluorescent samples through depths of several hundreds of microns. However, LSFM also suffers from scattering, absorption and optical aberrations. Spatial variations in the refractive index inside the samples cause major changes to the light path resulting in loss of signal and contrast in the deepest regions, thus impairing in-depth imaging capability. These effects are particularly marked when inhomogeneous, complex biological samples are under study. Recently, chemical treatments have been developed to render a sample transparent by homogenizing its refractive index (RI), consequently enabling a reduction of scattering phenomena and a simplification of optical aberration patterns. One drawback of these methods is that the resulting RI of cleared samples does not match the working RI medium generally used for LSFM lenses. This RI mismatch leads to the presence of low-order aberrations and therefore to a significant degradation of image quality. In this paper, we introduce an original optical-chemical combined method based on an adaptive SPIM and a water-based clearing protocol enabling compensation for aberrations arising from RI mismatches induced by optical clearing methods and acquisition of high-resolution in-depth images of optically cleared complex thick samples such as Multi-Cellular Tumour Spheroids

Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer

International audienceUNLABELLED: ABSTRACT: BACKGROUND: The multicellular tumor spheroid (MCTS) is an in vitro model associating malignant-cell microenvironment and 3D organization as currently observed in avascular tumors. METHODS: In order to evaluate the relevance of this model for pre-clinical studies of drug combinations, we analyzed the effect of gemcitabine alone and in combination with the CHIR-124 CHK1 inhibitor in a Capan-2 pancreatic cell MCTS model. RESULTS: Compared to monolayer cultures, Capan-2 MCTS exhibited resistance to gemcitabine cytotoxic effect. This resistance was amplified in EGF-deprived quiescent spheroid suggesting that quiescent cells are playing a role in gemcitabine multicellular resistance. After a prolonged incubation with gemcitabine, DNA damages and massive apoptosis were observed throughout the spheroid while cell cycle arrest was restricted to the outer cell layer, indicating that gemcitabine-induced apoptosis is directly correlated to DNA damages. The combination of gemcitabine and CHIR-124 in this MCTS model, enhanced the sensitivity to the gemcitabine antiproliferative effect in correlation with an increase in DNA damage and apoptosis. CONCLUSIONS: These results demonstrate that our pancreatic MCTS model, suitable for both screening and imaging analysis, is a valuable advanced tool for evaluating the spatio-temporal effect of drugs and drug combinations in a chemoresistant and microenvironment-depending tumor model

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

Fluorescent organic ion pairs based on berberine: counter-ion effect on the formation of particles and on the uptake by colon cancer cells

International audienc

Mechanical Stress Impairs Mitosis Progression in Multi-Cellular Tumor Spheroids

<div><p>Growing solid tumors are subjected to mechanical stress that influences their growth rate and development. However, little is known about its effects on tumor cell biology. To explore this issue, we investigated the impact of mechanical confinement on cell proliferation in MultiCellular Tumor Spheroids (MCTS), a 3D culture model that recapitulates the microenvironment, proliferative gradient, and cell-cell interactions of a tumor. Dedicated polydimethylsiloxane (PDMS) microdevices were designed to spatially restrict MCTS growth. In this confined environment, spheroids are likely to experience mechanical stress as indicated by their modified cell morphology and density and by their relaxation upon removal from the microdevice. We show that the proliferation gradient within mechanically confined spheroids is different in comparison to MCTS grown in suspension. Furthermore, we demonstrate that a population of cells within the body of mechanically confined MCTS is arrested at mitosis. Cell morphology analysis reveals that this mitotic arrest is not caused by impaired cell rounding, but rather that confinement negatively affects bipolar spindle assembly. All together these results suggest that mechanical stress induced by progressive confinement of growing spheroids could impair mitotic progression. This study paves the way to future research to better understand the tumor cell response to mechanical cues similar to those encountered during in vivo tumor development.</p></div

Mechanically confined MCTS show bipolar spindle defects.

<p>(A) Maximal projection of two mitotic cells from two z-stacks of images of a cryosection from a mechanically confined MCTS (6 days in the PDMS microdevice) incubated with an anti-γTubulin antibody (green). Nuclei were stained with DAPI (blue) (scale bar, 5 µm). (B) Distribution (percentage) of mitotic cells as a function of the number of spindle poles in control MCTS (CTL, 37 mitotic cells analyzed from 10 MCTS from 3 independent experiment) and in the body of mechanically confined MCTS (Confined, 66 mitotic cells analyzed from 8 MCTS from 4 independent experiment). (C) Distribution of the pole-to-pole distance (in µm) in bipolar mitotic cells from the control (CTL) and mechanically confined (Confined) MCTS analyzed in (B). The lines correspond to the mean ± SD.</p

Mechanically confined growth impairs the regionalization of mitotic cells in MCTS.

<p>(A) Transmitted light images of a control MCTS and of a MCTS grown in a PDMS microdevice for 6 days. (B) Upper panels: Detection by immunofluorescence of mitotic cells (anti-phosphorylated Histone H3 antibody, pH3; in green) in cryosections of a control MCTS and a mechanically confined MCTS. The orientation of mechanically confined MCTS cryosections is parallel to the bottom of the channel (middle, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080447#pone-0080447-g001" target="_blank">Fig 1C</a>) and perpendicular to the bottom of the channel (right, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080447#pone-0080447-g001" target="_blank">Fig 1C</a>). Nuclei are stained with DAPI (blue). Lower panels: mean fluorescence intensity of pH3 staining in 8 cryosections from 6 control MCTS (3 independent experiments), 11 parallel cryosections from 11 mechanically confined MCTS (4 independent experiments) and 8 perpendicular cryosections from 6 mechanically confined MCTS (3 independent experiments). Dashed lines represent the walls of the PDMS channel. White lines indicate the width of the area where mitotic cells are localized (scale bar, 100 µm). (C) Percentages of mitotic cells (pH3-positive cells) in the peripheral (P) and the central (C) areas of control MCTS (n = 14 areas analyzed, from 7 MCTS from 3 experiments) and in the peripheral (P) and central (C) areas and the tips (Tips) of confined MCTS (n = 29 areas analyzed, from 12 MCTS from 6 experiments). The bars correspond to the mean ± SEM.</p

Growth-associated external mechanical stress leads to accumulation of cells arrested in mitosis.

<p>(A) Upper panel: Immunodetection of EdU incorporation (green) and mitotic cells (pH3-positive, red) in a cryosection from MCTS grown in PDMS microdevices for 6 days. Nuclei are stained using DAPI (blue). Lower panel: High-contrast image of the immunodetection of EdU incorporation (white) (scale bar, 100 µm). (B) Analysis of EdU incorporation in mitotic cells. Images correspond to magnifications of the regions indicated by the white squares in A from the tip (top panels) and the body (bottom panels) of a mechanically confined MCTS. The white arrow indicates a pH3- positive cell that is not EdU-positive. This cell is next to a pH3-positive/EdU-positive cell. (C) Percentage of pH3-positive/EdU-negative cells in the body (589 mitotic cells from 48 cryosections) and in the tips (331 mitotic cells from 95 cryosections) of mechanically confined MCTS (20 MCTS from 4 independent experiments) and in control (CTL) MCTS (358 mitotic cells from 34 cryosections from 15 MCTS from 4 independent experiment). Bars correspond to the mean ± SEM. (D) Map showing the localization of pH3-positive/EdU-negative cells in 8 cryosections from 8 mechanically confined MCTS from 4 independent experiments. The white line represents the outline of the MCTS and the red dots the localization of the pH3-positive/EdU-negative cells. The dashed lines indicate the microdevice PDMS walls.</p

Proliferation, hypoxia and apoptosis within MCTS grown in PDMS microdevices.

<p>(A) Transmitted light images of control and confined MCTS grown in a PDMS microdevice for 6 days. (B) Detection by immunofluorescence of Ki67 staining (marker of proliferative cells). Mean fluorescence intensity of 10 cryosections from 4 different control MCTS (from 3 independent experiments) and of 6 cryosections from 4 mechanically confined MCTS (6 days in the PDMS microdevice; from 3 independent experiments). (C) Detection by immunofluorescence of Cyclin A staining. Mean fluorescence intensity of 6 cryosections from 4 different control MCTS (from 3 independent experiments) and of 6 cryosections from 4 mechanically confined MCTS (from 3 independent experiments). (D) Detection by immunofluorescence of hypoxia (pimonidazole; in green). Mean fluorescence intensity of 8 cryosections from 8 different control MCTS (from 3 independent experiments) and of 8 cryosections from 6 mechanically confined MCTS (from 4 independent experiments). The grey circle indicates the MCTS margins. (E) Cleaved PARP staining (apoptosis marker). Mean fluorescence intensity of 6 cryosections from 6 control MCTS (two independent experiments) and of 6 cryosections from 6 mechanically confined MCTS (6 days in the PDMS device; 4 independent experiments). c-PARP, cleaved PARP. The grey circle indicates the MCTS margins. (A–E) The dashed lines indicate the PDMS walls. (A–D) The color scale indicates the fluorescence intensity (scale bar, 100 µm.). Nuclei are stained by DAPI (blue).</p