Correction and quantification of MTF in scanning laser microscopy.

Abstract

Observations using conventional microscopy often lack information related to the fine structures of an object, such as intensity and phase, because of limitations in the lens aperture. In this study, the intensity and phase information are appropriately converted into an electrical signal using laser scanning and photodetectors. Intensity and phase are completely separated, and the missing information is restored based on the frequency of the electrical signal. Using this method, the original intensity and phase information of the object to be observed can be correctly restored. Therefore, we propose a novel method to calculate the degree of intensity and phase modulation by calculating the direct and alternating current components obtained from the output of the sum and difference of the two photodetectors. The degree of spatial frequency modulation is corrected according to the electrical signal frequency to detect transparent or unstained cells. We first performed laser scanning of an object. Then, signals were detected using two photodetectors placed in the far-field, separated by the optical axis as the boundary. The output signals of the photodetectors were processed and the intensity and phase were unambiguously separated, thus allowing the visualization of the phase information of the transparent bodies and unstained cells. Spatial frequency correction was performed to correct the modulation. Our method successfully separated the information related to the intensity and optical path difference (OPD). In future work, by accurately correcting the intensity and OPD, it will be possible to separate the absorption rate from the ratio of the irradiation light intensity to the observed intensity and to separate the OPD into the refractive index and the thickness information. This method allows the accurate determination of these parameters in a noninvasive manner.