Central mask filtering – SAF only detection
A first approach to discrimante between supercritical angle fluorescence (SAF) emission and undercritical emission (UAF), is to introduce a cnetral blocking mask in a conjugated plane of the BFP of a commercial objective to remove UAF components.
We observed a slight loss in the lateral resolution that is due to the larger PSF of the fluorophores with emission dipoles orthogonal to the surface.
Nevertheless, the quality of the images permits us to easily resolve the structures of the membrane. Besides, image quality could be further improved by using deconvolution techniques. The axial sectioning is of the order of 100 nm. Moreover, penetration depth and lateral resolution performances are maintained
throughout the whole image. The central mask-SAF technique allows one to perform the observation of the whole cell and its membrane simultaneously at low cost using a standard
epifluorescence microscope and an incoherent source that provides an homogeneous illumination all over the sample.
fig. Images of HEK cells in (a) UAF + SAF mode and in b) Central mask-SAF mode.
This work has been developped during Thomas Barocca thesis.
Related publication : Full-field supercritical angle fluorescence microscopy for live cell imaging T. Barroca, K. Balaa, J. Delahaye, S. Leveque-Fort, and E. Fort Optics Letters Volume: 36 Issue: 16 p: 3051-3053 Aug 15 2011