Super-resolution two-photon microscopy via scanning patterned illumination

We developed two-photon scanning patterned illumination microscopy (2P-SPIM) for super-resolution two-photon imaging. Our approach used a traditional two-photon microscopy setup with temporally modulated excitation to create patterned illumination fields. Combing nine different illuminations and structured illumination reconstruction, super-resolution imaging was achieved in two-photon microscopy. Using 2P-SPIM we achieved a lateral resolution of 141 nm, which represents an improvement by a factor of 1.9 over the corresponding diffraction limit. We further demonstrated super-resolution cellular imaging by 2P-SPIM to image actin cytoskeleton in mammalian cells and three-dimensional imaging in highly scattering retinal tissue.

Figure 1

(a) Scanned patterned illumination acquired from a fluorescein solution phantom. The phase (φ) was shifted in equal increments three times along a single direction before the incident angle (θ) was shifted by $\frac{2\pi }{3}$ radians. This process was repeated to acquire nine images. (b) The phase and angle movement information corresponding to the Fourier components were calculated from each incident angle (imposed circles) and the sequence was reconstructed to achieve super-resolution. (c) Schematic of 2P-SPIM experimental setup. (d) Imaged 110-nm fluorescent nanosphere by direct 2P microscopy (top) and 2P-SPIM (middle) images used to extract the corresponding PSFs (bottom).

Figure 2

(a) Comparison between direct 2P (gray scale, left) and 2P-SPIM (pseudocolor, right) images of same fluorescence nanosphere phantom. A region within the sample as highlighted by the yellow box was magnified to compare (b) direct 2P microscopy and (c) 2P-SPIM. The monolayer of the 200-nm fluorescent spheres can be resolved using 2P-SPIM. Smaller clusters of spheres were also imaged using (d) direct 2P microscopy and (e) 2P-SPIM to compare structures visualized by (f) SEM microscopy.

Figure 3

(a) Direct 2P microscopy (gray scale, top) and 2P-SPIM (pseudocolor, bottom) images of the actin cytoskeleton in the same HeLa cell stained with Alexa Fluor 546 phalloidin. (b) Magnified direct 2P microscopy image of the region within the box. (c) Magnified 2P-SPIM microscopy image of the region within the box. (d) One-dimensional profiles of imaged microtubules at the location highlighted by the lines. Resolution is 180 nm in 2P-SPIM image.

Figure 4

Volumetric imaging using 2P-SPIM. (a) Two-dimensional 2P-SPIM reconstructed image of ganglion cell dendrites in ground squirrel retina at a depth of 100 µm from the vitreal surface. A magnified comparison (within the box) of (b) direct 2P microscopy and (c) 2P-SPIM images. The 2P-SPIM reconstruction can resolve the object with a size of ∼145 nm, which is still much beyond its diffraction limit, at the depth of 100 µm. Three-dimensional image stacks at depths from 94 to 110 µm in the retinal tissue were created using both (d) direct 2P microscopy and (e) 2P-SPIM. A total of 16 slices acquired at 1-µm depth interval were stacked to form the volumetric images. The cell soma, at top, is removed. Z-axis resolution is limited by the 2P point spread function.