About the quantitation of co-localized fluorescent signals
When it comes to high-content imaging of 3D spheroid models, one of the most common and yet underappreciated sources of optical aberrations and light attenuation is the spheroid culture plate. The natural evolution of spheroid culture methods has resulted in the generation of commercially available spheroid-dedicated microplates. These plates, while optimized for spheroid generation and culture, frequently lack two of the most important features of an imaging plate: planarity and transparency. These characteristics, along with a thin bottom and low skirt height, become even more important when high-content imaging involves high-resolution analysis of 3D cell models.
U-bottom ultra-low attachment plates are universally recognized as one of the easiest and most reliable plate formats to generate free-floating spheroids from seeded cell suspensions. While these plates may be well-suited for the generation and culturing of many types of spheroid cell models, they are sub-optimal for 3D imaging due to the optical aberrations generated by the curved surface of the well bottom. Microplates optimized for spheroid culture may also have thick plastic bottoms that are incompatible with high NA objectives and optical densities that can result in light scattering and attenuation.
Using human islet microtissues (spheroids) labeled with two nuclear markers, DAPI and anti-NKX6.1, we show the optical limitations of a commonly-used spheroid culture plate and the optical advantages of a first-in-class imaging-quality spheroid plate (featuring high planarity and ultra-thin optically pure bottom) by imaging the fluorescently labeled spheroids in both plate types using a confocal high content imaging instrument equipped with a dual-spinning disk with microlens, high NA objectives, and sCMOS camera.
Our results show that in Akura 384 well plates with flat bottom, the chromatic aberration is minimized compared to the u-bottom commercial spheroid culture plates. The images taken using 40x SAPO (Super Apochromat) objective displays below 1 µm spatial shift between blue and far-red fluorescent channels in Akura 384 plate whereas the spatial shift is above 10 µm in the u-bottom plate. The higher light attenuation in the u-bottom plate also prevents the accurate segmentation of the nuclei whereas in flat bottom plate we can segment and quantify the nuclei throughout the entire 150-µm diameter islet spheroid and perform a reliable co-localization analysis with DAPI and NKx6.1 signals.
By taking into consideration and controlling for all potential sources of optical aberrations and light attenuation, including those contributed by the imaging plate, it is possible to overcome many of the obstacles currently limiting the utilization of high-resolution imaging of 3D spheroid models.
Download InSphero's poster about the quantitation of co-localized fluorescent signals
