We describe a quantitative fluorescence projection tomography technique which measures the three-dimensional fluorescence spectrum in biomedical samples with size up to several millimeters. This is achieved by acquiring a series of hyperspectral images, by using laser scanning scheme, at different projection angles. We demonstrate that this technique provide a quantitative measure of the fluorescence signal by comparing the spectrum and intensity profile of a fluorescent bead phantom and also demonstrate its application to differentiating the extrinsic label and the autofluorescence in a mouse embryo.
There is increasing interest in the three-dimensional visualization and quantification of cellular circuits in the brain and therefore optical clearing methods are highly in demand for brain imaging. In particular, clarification without membrane damage is required to image lipophilic tracer-labeled neural tracts. However, previously reported DiI-compatible optical clearing methods are relatively slow and can hinder transparency for imaging. Here, we present DAS, a new, convenient, inexpensive and reproducible aqueous clearing reagent that can efficiently clarify tissues with minimal volume enlargement and reliably preserves emission from fluorescent proteins and lipophilic dyes in membrane integrity preserved tissues.
Optical clearing methods are highly in demand in organism-level biomedical system research since they can facilitate deep optical imaging by reducing light scattering in tissue and then enable three-dimensional signal visualization and quantification of tissues. While the previously reported optical clearing methods have addressed some of six key issues (i.e. transparency, efficiency, reproducibility, preservation of emission from fluorescence proteins, preservation of membrane integrity, and the ease of operation), none has yet addressed all of them. Here, we present a new, convenient, inexpensive and reproducible approach to optical clearing, termed UbasM, providing unprecedented performance in terms of clearing rate, the ease of operation and satisfactory fluorescence protein/membrane integrity preservation while achieving sufficient transparency to permit 3D volumetric imaging.