Projects

High-resolution 3D imaging large biological specimen such as spheroids or embryos is a tricky task because of light scattering insight the object, leading to a strong loss in image quality. The use of scanned Bessel beams has shown to increase the penetration depth into the specimen as well as resolution and contrast when using line confocal detection. However, image quality can be improved to a yet unknown level by using illumination beams with holographic feedback. Simply spoken, every beam illuminating one pixel line of the object will be optimized for the particular area of the specimen. Several variations of the illumination beam generated by a computer hologram (SLM) will be sent through the specimen at low laser power and at the emission wavelength of the fluorophores. Thereby no fluorescence will be excited and photobleaching will be minimized. The quality of the illumination beam will be analyzed by forward and sideward scattered illumination light and the result of the computed beam analysis will be delivered to the computer hologram. Thereby the quality of each illumination beam will be iteratively improved. The illumination time and computation time for each beam are expected to be only a few milliseconds. In this way we expect to achieve three dimensional image stacks as a function of time of an unequalled resolution and contrast all across the extent of the spheroid or the embryo having several 100µm in diameter.