Abstract: A system and method for optical sectioning in bright field microscopy (OSBM). The system includes a bright field optical microscope having automated change of focus, a substage condenser fitted with an adjustable aperture iris diaphragm, a digital camera that records the microscope image of samples, and one or more digital computers to perform digital image processing. The OSBM method comprises operating the microscope to Kohler illumination, using the iris diaphragm of the condenser to generate contrast in images, acquiring a Z-stack of images of the unstained sample, and applying a sequence of digital image processing filters to the Z-stack, resulting in optical sections from where the final three-dimensional (3D) image of the sample can be reconstructed by computational device. The final 3D images produced by this invention present quality comparable to that of available optical sectioning techniques that require sample labeling, such as light sheet fluorescence microscopy.

Abstract: A system and method for optical sectioning in bright field microscopy (OSBM). The system includes a bright field optical microscope having automated change of focus, a substage condenser fitted with an adjustable aperture iris diaphragm, a digital camera that records the microscope image of samples, and one or more digital computers to perform digital image processing. The OSBM method comprises operating the microscope to Kohler illumination, using the iris diaphragm of the condenser to generate contrast in images, acquiring a Z-stack of images of the unstained sample, and applying a sequence of digital image processing filters to the Z-stack, resulting in optical sections from where the final three-dimensional (3D) image of the sample can be reconstructed by computational device. The final 3D images produced by this invention present quality comparable to that of available optical sectioning techniques that require sample labeling, such as light sheet fluorescence microscopy.

Abstract: The herein invention discloses and claims a quantitative image restoration in bright field (QRBF) method that faithfully recovers shape and enables quantify size of individual unstained samples. The QRBF method restores out-of-focus BF images of unstained samples by applying deconvolution, which enhances contrast and allows for quantitative analysis. To perform deconvolution our procedure uses a point spread function modeled from theory. Image deconvolution can be performed even from a single input image in two dimensions (2D-QRBF), and quantitative information such as size and shape of samples can be determined from restored 2D-QRBF images. Application of 2D-QRBF in a high-throughput screening process to assess shape changes in cells during hyperosmotic shock shows that the described digital restoration approach is suitable for quantitative analysis of unstained BF images in high-throughput image cytometry.

Abstract: The herein invention discloses and claims a quantitative image restoration in bright field (QRBF) method that faithfully recovers shape and enables quantify size of individual unstained samples. The QRBF method restores out-of-focus BF images of unstained samples by applying deconvolution, which enhances contrast and allows for quantitative analysis. To perform deconvolution our procedure uses a point spread function modeled from theory. Image deconvolution can be performed even from a single input image in two dimensions (2D-QRBF), and quantitative information such as size and shape of samples can be determined from restored 2D-QRBF images. Application of 2D-QRBF in a high-throughput screening process to assess shape changes in cells during hyperosmotic shock shows that the described digital restoration approach is suitable for quantitative analysis of unstained BF images in high-throughput image cytometry.