Virtual SAF (vSAF) has been developed during the PhD work of Thomas Barroca.
In vSAF, supercritical angle information is retrieved by subtracting two images associated to two different filtering in the back focal plan (BFP). By inserting a bertrand lens (BL), the conjugated plan of the back focal plan can be easily imaged on the camera. The experimental setup is based on a standard fluorescent microscope equipped with a white lamp, a large NA objective lens (1.49), and an EM-CCD camera are used. The vSAF optical system is mounted between the microscope and the camera. A dual path system or a single one with a filter wheel, allows one to take one image with the whole BFP (a) and a second with only the UAF components (b)–(c) is the result of the subtraction (a)–(b) to obtain the virtual mask where only the SAF components are conserved. (d)–(f ) show the corresponding images of an N2a cell in the image plane. vSAF configuration offers lateral resolution (h) similar to classical epifluorescence (UAF+SAF).