True isotropic resolution
Optically, there are two main categories into which we divide biological samples: (nearly) transparent and (partially) opaque. Transparent samples present relatively low scattering/absorption, so that the image resolution does not vary significantly with depth in the sample. In such cases image distortions are primarily due to the optical system, i.e. the image is blurred by the point-spread function (PSF) of the microscope. These images do have anisotropic resolution (most optical microscopes have poorer axial than lateral resolution), but it is spatially invariant (the degree of blurring does not depend on the location within the sample). Since each angle has its best resolution along a different direction (e.g. vertical and horizontal), a fusion of images in different angles can achieve isotropic resolution by combining the best information from each view.
In a partially opaque sample the primary problem is not anisotropic resolution, but light quality degrading as one images deeper into the sample. In this sort of specimen, the resolution will also be anisotropic (when imaged with a traditional single-objective microscope), but when the absorption is high this anisotropy is often a relatively minor effect. In this case the goal of multi-view fusion is to combine the information from the different regions of the sample, where there are the best imaging data.
One question that arises when doing multi-view fusions is: What is the optimal number of views to combine? It is proposed that for mostly transparent samples, as a rule of thumb, the optimal number of views can be estimated using Noptimal ~ 360°/(α/2) where α is the detection half-angle and can be found from NA = ni·sin(α), where ni is the refractive index of the medium*.
For large cleared samples the typical NA of the detection objectives are between 0.2-0.4. That means that for a typical organic clearing agent with 1.47 refractive index, in order to achieve isotropic resolution the system should acquire data from 92 to 46 angles, respectively.
Our QLS-scope is the only system in the market that can acquire data from up to 500 different angles with an angular resolution of 0.7 degrees and, through reconstruction of the final 3D image, provide true isotropic resolution of large cleared samples.
* Stelzer et all, Optics Express, June 2007 / Vol. 15, No. 13, 8029