F8789

For bead experiments for color-classification (for both the training of the ANNs and the analysis of unknown samples), 40 nm dia. fluorescent beads from Invitrogen (F10720; yellow and orange FluoSpheres with emission peaks at 515 and 560 nm, respectively) were diluted in Dulbecco’s phosphate buffered saline (DPBS), mixed, and sealed between a glass slide and a pre-cleaned #1.5 thickness coverslip, and imaged with the above optical setup. The 488-nm and 561-nm lasers were used to excite the two types of beads to similar levels of brightness. To record images at different axial positions, the objective lens was scanned by the built-in motor over a range of −400 to + 400 nm of the focal plane in 50 nm steps. To compare the performance of the axial-localization ANN with cubic-spline MLE, 40 nm dia. fluorescent beads from Invitrogen (F8789; dark red FluoSphere with an emission peak at 680 nm) were similarly prepared as described above, excited with the 644-nm laser, and scanned from -400 nm to + 400 nm in 50 nm steps.