RESUMO
Tumor spheroids more faithfully mimic tumor biology than monolayer cultures and require three-dimensional microscopy. Our goal in this study was to overcome the limitations of signal to noise ratio that have traditionally limited three-dimensional imaging to depths of 100 microm or less. We studied the expression of hypoxia-inducible factor 1alpha (HIF-1alpha), the main regulator of cellular hypoxic response in C6 glioma spheroids. In our spheroids, red fluorescent protein is expressed constitutively and green fluorescent protein is expressed conditionally under control of a HIF-1alpha promoter. In this article, we show a series of optimizations that allowed us to obtain excellent quality confocal microscopy images at imaging depths of up to 320 microm. The combined use of special objectives, glass-bottomed culture dishes, and depth-dependent laser output modulation extended our depth range beyond previously accepted limits. This allowed us to image up to the equator of spheroids of 650 microm diameter, allowing interrogation of HIF-1alpha expression from the spheroid periphery to its hypoxic center.
