Spatially-incoherent annular illumination microscopy for bright-field optical sectioning

Abstract

Numerous advanced microscopic imaging techniques have been proposed for optical sectioning, but they generally employ a complex and costly optical system. Here we report a microscopy termed spatially-incoherent annular illumination microscopy (SAIM). It allows for simple, effective, non-fluorescence, and bright-field optical sectioning. The proposed technique is implemented by installing an annular array of light emitting diodes (LEDs) on a standard bright-field microscope for illumination. The LED array produces distinctive illumination, that is, each LED provides coherent, large-angle oblique illumination while all LEDs generate spatially-incoherent annular illumination. Such a distinctive illumination can improve both lateral resolution and axial resolution. The improvement of lateral resolution is due to the coherent and large-angle oblique illumination. The spatially-incoherent annular illumination can improve the axial resolution. It is because, for defocused structures, each LED results in a blurred image with a different lateral shift and all LEDs result in an incoherent stagger superposition of the defocused images. The superposition looks much more blurred, which improves the contrast of the in-focus image remarkably. We experimentally demonstrate that SAIM is able to provide bright-field optical sections with 600-nm axial resolution and 150-nm lateral half-pitch resolution by using a 525-nm wavelength LED array and an objective with 100X, numerical aperture (NA) 1.25.