Enhanced axial resolution of lattice light sheet microscopy by fluorescence differential detection.

Lattice light-sheet microscopy (LLSM) is promising in long-term biological volumetric imaging due to its high spatiotemporal resolution and low phototoxicity. However, three-dimensional (3D) isotropic spatial resolution remains an unmet goal in LLSM because of its poorer axial resolution. Combing LLSM with fluorescence differential detection, namely LLSDM, has been proposed to improve the axial resolution of LLSM in simulation. It demonstrates the possibility of further enhancing the axial resolution in 3D volumetric imaging with LLSM by specifically discarding the off-focus photons captured using a complementary optical lattice (OL) profile generated with additional 0-π phase modulation at the objective pupil plane. The direct generation of the complementary lattice profile using the binary phase modulator conjugated to the sample plane for amplitude modulation, as used in LLSM, is also permittable. Nevertheless, the previously proposed configuration fails to provide a symmetric complementary lattice pattern along the axial axis, thus leading to the imbalanced off-focus photon suppression in the reconstructed images after subtraction [Opt. Lett.45, 2854 (2020)10.1364/OL.393378]. Here, we modified the LLSDM theory which can produce an ideal complementary lattice pattern with central zero intensity and symmetrically distributed sidelobes. We also analyzed the impact of numerical aperture matching between the original and complementary lattice patterns and presented the consistency between the simulated and experimental results. As demonstrated by imaging the distribution of fluorescent beads and microtubules in fixed U2OS cells, as well as the dynamics of filopodia in live U2OS cells, LLSDM provides about 1.5 times improvement in axial resolution, and higher imaging contrast compared with traditional LLSM.

Label-free difference super-resolution microscopy based on parallel detection.

In this paper, a new method is proposed for super-resolution imaging of non-fluorescent samples. This approach is based on the intensity difference between confocal image and negative confocal image, which are simultaneously acquired at one sample scanning. In order to get these two different images simultaneously, the sample was illuminated by two different focused spots from the same laser source: the doughnut spot and the solid spot. The effectiveness of the label-free difference microscopy based on parallel detection was validated by experiments on some samples including 80 nm gold beads, 100 nm silver nanowires, and Blu-ray DVD without fluorescent dyes. By subtraction of the reflected light intensity from the sample, the final resolution of the image without deconvolution was enhanced about 1.6 times compared with confocal imaging. This technique can be applied to surface topography detection of metallographic or other non-fluorescent materials.