結合結構式基態耗乏之掃描式雙光子結構照明顯微術(1/2)

In order to improve the resolution of optical microscopy, a variety ofsuperresolution techniques has been developed to break the boundary ofdiffraction limit. Among them, structured illumination microscopy makes theresolution improvement by shifting the high spatial frequency componentsoriginally unreached into the passband of the optical transfer function. Tointegrate the structured illumination with two-photon fluorescence microscopy toget simultaneously lateral superresolution and optical sectioning power, our teamhas developed a system combining point-scanning geometry and temporalintensity modulation to achieve structured illumination. However, since thefluorescence images are acquired by imaging the signals onto a 2D camera, thepatterns’ visibility is suppressed by the scattered fluorescence signals. And thus,the resolution improvement and penetrability are limited. In order to break thislimitation, our team proposes a whole new system configuration. In this system,structured light is used to deactivate the fluorophores through ground-statedepletion and the images are acquired by using laser point-scanning along withphotomultipliers. Because the photomultiplier can integrate all the signals comingfrom excitation spot, in contrast to the former way of imaging, the backgroundnoises due to scattered fluorescence signals can be avoided and the collectionefficiency of signals can be increased. Therefore, the signal-to-noise ratio andpattern’s visibility will be improved to get better resolution improvement andpenetrability. In addition to the fundamental structured illumination, if increasingthe depletion light’s intensity to achieve saturated ground state depletion,saturated structured illumination microscopy can be performed to get muchhigher resolution improvement.