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Evaluation of 3D neurospheres with the IN Cell Analyzer 6000

Imaging and analysis of 3D biology on the IN Cell Analyzer 6000

Imaging and analysis of 3D biology on the IN Cell Analyzer 6000

The IN Cell Analyzer 6000 is a unique laser-based confocal high content imaging system with a novel and rapidly adjustable aperture inspired by the iris mechanism of the eye. Imaging thick tissues, such as neurospheres, requires confocal technology to reject background fluorescence and optically section the tissue. High quality images enable robust quantitative analysis and localization of proteins in complex 3D cellular systems.

Neurospheres are a way to grow 3D neural tissue in culture, enabling scientists to investigate neuroscience questions in a biological model that has advantages over traditional 2D cell culture models. Spheroid assays are advantageous as they recapitulate complex environments within tissues, creating a more physiologically relevant model for disease and drug discovery.

Experimental Design

Here, we have examined neurospheres treated with a cell proliferation inhibitor on an IN Cell Analyzer 6000 by acquiring images through Z. Neurospheres were immunostained with SOX1/(2)/3 as a marker for neural stem cells (green), ß-III tubulin as a marker for neurons (red), and Hoechst for a nuclear marker (Fig 1).

Figure 1. Confocal images of neurospheres at 40× magnification. Image A was taken at coverslip surface (bottom of plate) and subsequent images were taken at 10µm intervals towards the center of the neurosphere. Green: SOX 1/(2)/3. Red: ß-III Tubulin.

Figure 1. Confocal images of neurospheres at 40× magnification. Image A was taken at coverslip surface (bottom of plate) and subsequent images were taken at 10 µm intervals towards the center of the neurosphere. Green: SOX 1/(2)/3. Red: ß-III Tubulin.

Results

Developer Toolbox was used to determine the proportion of cells expressing SOX1/(2)/3 and ß-III tubulin in the neurosphere. Specifically, each cell in the neurosphere is recognized from the nuclear staining, and the amount of marker expression within each cell is assayed by calculating the intensity of fluorescence within that area. Visual examination through the Z stacks, taken at 10 µm intervals, suggest that SOX 1/(2)/3 is expressed more toward the exterior of the sphere and ß-III tubulin is expressed more toward the interior of the sphere (Fig 1). Segmentation analysis with Investigator confirmed the findings and quantified the expression of the markers throughout the spheroid (Fig 2).

Figure 2. SOX 1/(2)/3 expression and ßII tubulin expression were evaluated on a 2D intensity plot and 4 quadrants were created, where yellow represents high expression of both markers, blue represents low expression of both markers, green represents high SOX 1(2)/3 expression and low-III tubulin expression and red represents high -III tubulin expression and low SOX 1/(2)/3 expression. Then, the intensity values were plotted as a function of location where the X-axis represents the X-coordinates in the field of view and the Y-axis represents the Y-coordinates in the field of view.

Figure 2. SOX 1/(2)/3 expression and ß-III tubulin expression were evaluated on a 2D intensity plot and 4 quadrants were created, where yellow represents high expression of both markers, blue represents low expression of both markers, green represents high SOX 1(2)/3 expression and low ß-III tubulin expression and red represents high ß-III tubulin expression and low SOX 1/(2)/3 expression. Then, the intensity values were plotted as a function of location where the X-axis represents the X-coordinates in the field of view and the Y-axis represents the Y-coordinates in the field of view.

Summary

  • Imaging with an IN Cell Analyzer 6000 enables automated analysis of spheroids in round bottom wells via confocal imaging.
  • High quality images were obtained for analysis.
  • Investigator successfully segmented the neurosphere and evaluated expression level of markers throughout the spheroid.

Acknowledgments

Data and images are used with the kind permission of Dr. Satoshi Kawase, Dr. Takao Imai, and Dr. Hideyuki Okano at the Department of Physiology, Keio University School of Medicine.

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