Spatially Incoherent Annular Oblique Illumination Microscopy

Currently,many advanced optical microscopies have been proposed,such as super-resolution microscopy.But due to the simple equipment and the convenience of operation,conventional bright-field microscopy is still widely used in pathological diagnosis and other fields related to life science.Additionally,the pathologic confirmation under the bright-field microscope remains the gold standard for the clinical diagnosis of many diseases,such as cancer.Therefore,one of the microscopists' research goals will still be to improve the imaging performance of the bright field microscope for satisfying the increased demands in these fields.This paper presents a spatially incoherent annular oblique illumination microscopy(SIAOIM)in order to improve the lateral and axial resolution.SIAOIM can achieve optical sectioning and three-dimensional(3D)imaging in the bright field microscope.This will provide more useful information for clinical pathological diagnosis and other applications related to life science.First,we study the characteristics of the contrast and lateral resolution for imaging under spatially incoherent annular oblique illumination(SIAOI)in theory.According to the angular spectrum theory,we analyze imaging characteristics of SIAOI and K?hler illumination by simulation.Compared to K?hler illumination the image under SIAOI has a higher visual lateral resolution.Then according to geometrical optics,we model a multilayer sample to analyze the axial resolution characteristics.The axial resolution under SIAOI is also higher.Finally,by the Ewald sphere theory,we analyze the 3D resolution.It also proves that SIAOI can improve the lateral and axial resolution.In order to verify the theoretical analysis and to evaluate the performance of optical sectioning and 3D imaging,we design and fabricate several LED annular oblique illumination(LAOI)sources with different properties.The LAOI microscopic system is designed and built.Using our microscopic system,we study imaging characteristics of LAOI experimentally.The results show that the proposed method can resolve Element 5 in Group 11(bar or space width 154 nm)of a resolution target(USAF)with an objective lens(NA=1.25,100×)and a 525-nm wavelength source.This proves that our method can improve the lateral resolution.To further evaluate optical sectioning performance,we provide the monochrome and colorful optical section images of biological samples,and the 3D image of the chromosome of an onion root-tip cell is reconstructed.According to the localization method of single molecules in super-resolution fluorescence microscopy,we further present a centered localization method for axial super-resolution imaging for sparse and strongly scattered samples with the prior knowledge,such as the nanowires.By this method,the axial resolution of the 3D result of nanowires is approximate to 100 nm.It is significant for life science research to image unstained and weak absorbing samples.Based on our microscopic system,we report a label-free 3D microscopy.By 3D gradient operation,the background with out-of-focus images is eliminated and a series of background-free sectioning images is obtained.Only the edge structure of the sample is visible.The 3D skeleton structure of the sample is reconstructed.We also use high-NA LAOI and the low-NA objective to obtain the dark-ground image of weak absorbing samples.Because the visual resolution under SIAOI is still lower than that of oblique illumination,according to the Fourier slice theorem,we present a synthetic aperture microscopy based on intensity constrained iteration with our system.We obtain multiple images under symmetrical oblique illuminations by LEDs with different illumination azimuth angles;then the gradient descent optimization algorithm is used to synthesize isotropic high-resolution image.The experimental results show that the proposed method can resolve Element 6 in Group 11(bar or space width 137 nm)of the USAF test target using an objective lens(NA=1.25,100×)and a 520-nm wavelength sources.In the same case,the highest distinguishable elements for K?hler illumination are Element 2 in Group 11(bar or space width 218 nm).This proves that the proposed method can provide higher resolution.To improve the imaging speed we present a deconvolution microscopy.Using the obtained convolution kernel of our system,the resolution of an image captured under LAOI can be enhanced directly.The proposed method without phase involvement has higher robustness,and provides a new way to improve imaging resolution.Due to its simplicity of the setup required,its convenience of operation and good compatibility with conventional bright-field microscopes using K?hler illumination,SIAOIM is suitable for large-scale promotion and has great value in the biological medical field,such as pathological diagnosis,etc.