4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues
Figure 1
Schematic depicting optical setup for ratiometric localisation imaging.
A. A single laser at 671 nm is used to provide excitation and the collected light is split into two bands using a dichroic mirror. The bands are imaged side by side on an electron multiplying CCD (EMCCD). An optional cylindrical lens allows astigmatism based 3D localisation. B. A vector field that shows the distribution of lateral chromatic shifts between the channels measured with a bead calibration sample. This vector field was used for chromatic shift compensation during fitting of the single molecule events as detailed in the Methods. The longest arrows shown correspond to a shift magnitude of ∼90 nm. C. Single molecule events are observed as flashes with an intensity component in each channel. When these intensities are plotted against each other, discreet populations emerge corresponding to each fluorochrome in the sample. One such plot, obtained from a sample in which neurons had been transfected with GFP-alpha-sap97 and subsequently labeled with antibodies against GFP (Alexa 647 secondary) and synapsin (Alexa 750 secondary) is shown. Inset: Recorded emission spectra of Alexa 647 (green) and Alexa 750 (red), the black trace is the transmission curve of the dichroic mirror in the splitter device.