| dc.description.abstract | Structured illumination microscopy produces periodic fringes in the image, and then obtains higher-frequency spatial information through mathematical operations, improves the lateral resolution of the optical system, and exceeds the limit of diffraction. However, in the past literature, structured illumination microscopy is rarely used for two-photon fluorescence imaging.
Compared with the single-photon fluorescence microscope, the excitation laser has a better penetration depth to the sample, causing less damage to the sample, and has a higher optical sectioning ability and better longitudinal resolution. However, according to the diffraction limit, two-photon fluorescence microscopy is inferior to single-photon fluorescence microscopy in lateral resolution.
To apply the structured illumination microscope to the two-photon fluorescence microscope, the two-photon fluorescence image needs to be spatially modulated to form stripes. If the ground-state depletion principle can make the fluorescent molecules enter the dark state without emitting two-photon fluorescence, then Stripes can be formed on two-photon fluorescent images by modulating the depletion laser.
The ground-state depletion principle has not been used to suppress the two-photon fluorescent signal. This paper selected four samples for experiments, namely R6G-PVA, Eosin Y-PVA, fluorescent beads F8800 and F8801. First, the experiment of suppressing single-photon fluorescence was carried out, and the past literature was used as a reference to confirm that the sample has a dark state and can suppress the single-photon fluorescence signal. Then, the experiment of suppressing single-photon fluorescence was carried out to observe whether the two-photon fluorescence could be suppressed by depletion laser (Green laser, wavelength of 532 nm), and compare the degree of suppression of the four samples. In order to apply to two-photon structure illumination, the signal-to-noise ratio of the two-photon fluorescent signal, influence of integration time on two-photon fluorescence, the effect of the interval time between the depletion laser and the excitation laser (Infrared laser, wavelength of 1064 nm) on the suppression of two-photon fluorescence. | en_US |