Scientists at the Foundation for Research and
Technology – Hellas (FORTH) in Heraklion, Greece have developed a significantly
improved methodology for the optical imaging of optically opaque 3D cell
cultures with enhanced resolution.
The so-called Phase-Retrieved Tomography (PRT) utilises
a Light Sheet / Selective Plane Illumination Microscope (SPIM), Optical Projection
Tomography and algorithms to solve challenges around autocorrelation. PRT’s ability to permit 3D image reconstruction
was tested on unfixed, uncleared multicellular tumor spheroids of 200 µm diameter
for early stage necrosis using a fluorescent viability dye as a representative and
practically relevant proof of concept for the technology.
In the model system individual tumor spheroids were
grown in hanging-drop cultures, and further incubated with the addition of the far-red
viability dye DRAQ7 for 24 hours to mark the presence of membrane-compromised dead
cells.
After a wash procedure a single spheroid was then
transferred into CyGEL Sustain, a thermo-reversible hydrogel mountant compatible
with living cells and tissues, and this combination placed in a FEP tube, which
was sealed and placed in a 37 °C waterbath for the duration of the imaging procedure.
Ancora, D., Di Battista, D., Giasafaki, G.,
Psycharakis, S. E., Liapis, E., Ripoll, J., & Zacharakis, G. (2017).
Phase-Retrieved Tomography enables Mesoscopic imaging of Opaque Tumor
Spheroids.
Scientific Reports, 7(1),11854 (open acess)
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