The use of microscopy to examine macroscopic biological samples can present challenges due to interference caused by tissue itself, which can behave like a lens and cause misalignments that limit resolution when imaging large areas. These limitations can prevent researchers from getting a detailed view of the architecture of an intact sample, such as when studying the different regions of a whole brain. An international team of researchers from the University of Florence (Italy), University of Glasgow (UK) and European Molecular Biology Laboratory (Germany) recently developed a new tool that can correct some of the misalignments caused by biological tissues and produce high-resolution volumetric images of larger intact samples.
The team designed a system called rapid autofocusing via pupil-split image phase detection (RAPID) that automatically removes image degradation as it is introduced in real time. The design was inspired by the optical autofocus systems found in reflex cameras, which use a set of prisms and lenses to transform the blur of an image into a lateral movement. By stabilizing the microscope alignment in real time, the scientists were able to achieve subcellular resolution while studying the structures of a cubic centimeter-sized, cleared, intact mouse brain using light-sheet microscopy.
With the system, the researchers were able to examine the 3D spatial clustering of somatostatin-positive neurons across the entire brain, and also perform a brain-wide 3D morphological analysis of microglia, revealing significant differences between the different brain regions. The study was published in Nature Methods.
“While we originally invented RAPID for light-sheet microscopy, this autofocusing technology is actually suitable for all wide-field microscopy techniques. It is very versatile and sample agnostic with multiple applications beyond neuroscience,” said Caroline Müllenbroich, study co-author and lecturer at the University of Glasgow’s School of Physics and Astronomy.
The high resolution provided through the RAPID system can open up new avenues for quantitative analysis of large biological specimens as well as other automated microscopy tasks.
Photo: Whole-brain cell distribution analysis using RAPID-enabled LSM. Credit: University of Glasgow