Separation of transmitted light and scattering components in transmitted microscopy

In transmitted light microscopy, a specimen tends to be observed as unclear. This is caused by a phenomenon that an image sensor captures the sum of these scattered light rays traveled from different paths due to scattering. To cope with this problem, we propose a novel computational photography approach for separating directly transmitted light from the scattering light in a transmitted light microscope by using high-frequency lighting. We first investigated light paths and clarified what types of light overlap in transmitted light microscopy. The scattered light can be simply represented and removed by using the difference in observations between focused and unfocused conditions, where the high-frequency illumination becomes homogeneous. Our method makes a novel spatial multiple-spectral absorption analysis possible, which requires absorption coefficients to be measured in each spectrum at each position. Experiments on real biological tissues demonstrated the effectiveness of our method.