Figure 1
SR-SIM/AFM system. (a) Simplified schematic set-up of the SR-SIM/AFM system. In this case, structured illumination is generated using a grating (G). Collimated and expanded laser light illuminates the grating, resulting in the diffraction of multiple orders. Only 0th and ± 1st orders are allowed into the illumination path and focused on the back focal plane of the objective and the created stripped illumination pattern excites the sample. Collection of the fluorescence signal is achieved by a high aperture microscope objective, a dichromatic mirror (DM) and a tube lens (TL). The AFM laser is reflected by a mirror (M) to a high-dynamic range camera coupled to a microscope port for cantilever laser spot alignment. At the same time, nanomechanical mapping of the sample is performed by a probe consisting of a flexible cantilever and a tip. After simultaneous SIM and AFM images acquisition, they are combined into a single image. (b) Image of the SR-SIM/AFM system. The N-SIM illuminator module provides the structured illumination. AFM scan head (XYZ) and sample positioning stage (XY) are mounted on the inverted optical microscope. Laser spot alignment of the AFM is accomplished using a standard monochrome CCD camera while fluorescence signal is acquired with a scientific CMOS camera. (c) Schematic illustration of the simultaneous SR-SIM/AFM imaging conditions within the sample region. SR-SIM allows to observe the fluorescence signal with a two-fold enhancement in lateral and axial resolutions compared to widefield microscopy. 3D-SIM mode generates an illumination pattern on the sample that is very favourable for integration with AFM without inducing cantilever distortions. Under this illumination, AFM quantitative imaging mode measures a force-distance curve at every pixel of the image.
