Transmission-type high-resolution laser scanning microscope

To visualize the microscopic internal structure of cells, some conventional methods exist in the literature, such as irradiating fluorescent substances with light that maintains activation and light that suppresses activation to reduce the effective spot diameter or irradiating samples with moiré stripes of irradiating light and analyzing the pattern of the moiré stripes. This study proposes a high-resolution method to detect the intensity and optical path difference (OPD), representing the phase distribution reflecting the internal structure of the object, in a transmission image without using pigmentation for observing near-transparent objects such as cells or soft materials in liquids. First, the object is scanned using a laser beam. Second, the intensity owing to the interference between the 0th- and 1st-order diffracted light is detected using two photodetectors as electrical signals. The signals are separated into DC and AC components, with the intensity and phase separated through signal processing using their ratio. Two tilting optics are inclined toward each other at a symmetrical position with respect to the optical axis of the objective lens. The arrangement ensures that the incident diffracted light becomes part of the 0th-order diffracted light and ± 1st-order diffracted light at high spatial frequency. Thus, in contrast to conventional optical systems, high-resolution transmitted images can be obtained by acquiring high spatial frequency diffracted light. Moreover, the lens is placed close to the glass slide to inject the diffracted light with high spatial frequency into the lens of the oblique optical system. The proposed method obtains images of OPDs with a resolution higher than the reciprocal of the cutoff frequency of the numerical aperture of the objective lens used in the actual optical system.