Atacama Clear for Complex 3D Imaging of Organs

Abstract

3-dimensional (3D) imaging is a powerful tool for interrogation of intact tissues, but can suffer from poor resolution due to impediments such as high tissue autofluorescence that remains a significant challenge in imaging cleared samples, including human clinical specimens. We developed Atacama Clear (ATC), a 3D imaging technology that increases signal-to-noise ratios (SNRs) while simultaneously augmenting the capacity of tissue to be cleared. ATC exhibited SNRs that are up to 200% of widely used 3D imaging methods, potentiated all tested optical clearing solutions by up to 600%, decreased the time of optical clearing by up to a factor of 8, and enabled detection of poorly recognized antigens with a remarkable 4-fold increase in signal detection while using up to 10-fold lower antibody concentrations. Strikingly, ATC produced up to a 5x increase in transgenic fluorescent reporter protein signal detection, which is instead often diminished with currently used 3D imaging methods. This increased imaging efficacy enabled multiplex interrogation of tough fibrous tissue and specimens that naturally exhibit high levels of background noise, including the heart, kidney, and human biopsies. Indeed, ATC facilitated the use of AI based auto-segmentation with simple low tech stereo fluorescence microscopy, visualization of previously undocumented adjacent nephron segments that exhibit notoriously high autofluorescence, elements of the cardiac conduction system, and distinct human glomerular tissue layers, with cellular resolution. Taken together, these studies establish ATC as a platform for complex 3D imaging studies of basic and clinical specimens with superior resolution.