Mirror-enhanced super-resolution microscopy

Abstract

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.

Keywords: confocal; interference; point spread function; super-resolution.

Figure 1

Schematic diagram of MEANS (a) and correlative images of a single cell acquired in TIRF (b) and MEANS (c) microscopy imaging modes. MEANS microscopy can be easily realized with a confocal microscope and takes advantage of axial interference between the incident and reflected electromagnetic field to generate an axially confined PSF ~100 nm above the reflective mirror surface. The merged image (d) of TIRF (green) and MEANS (red) shows that the MEANS approach complements the oblique illumination-based TIRF modality by optically sectioning a cell at a different axial layer which, in this case, is close to the mirror surface. The sample here is a Vero cell immunostained for tubulin using an AlexaFluor-488 secondary antibody. Scale bar=10 μm.

Figure 2

Theoretical simulation results of the focal intensity profiles of: (a) confocal excitation, (b) 4Pi excitation, (c) MEANS excitation, (d) STED depletion and (e) MEANS-STED depletion. The origin of the z-axis denotes the center of PSFs in a–c and the mirror position in d and e. Whereas confocal microscopy can generate a PSF with 700 nm axial thickness, 4Pi and MEANS can generate PSFs with ~110 nm axial thickness, benefitting from axial interference. Objectives with n=1.5, NA=1.4, and λ_ex=488 nm and λ_dep=592 nm are used for simulation. The local maximum intensity for MEANS d is approximately four times that of conventional confocal microscopy a and two times that of 4Pi b because in 4Pi, the beam is split into two and then recombined, whereas MEANS takes full advantage of the incident intensity through reflection. Because the intensity of depletion is improved by 3.6-fold, close to two-fold resolution enhancement over conventional STED can be obtained for MEANS-STED, as shown in (f).

Figure 3

Imaging of nanodiamond particles embedded in agarose with different excitation wavelengths. Excitation with longer wavelength results in a further PSF relative to the mirror. In the lower RGB image, the fluorescence excited by 470–490, 550–570 and 650–670 nm is mapped to the B, G and R channels, respectively. Arrows show beads with different colors, which indicates that they are at different depths and are differentiated by MEANS-excitation scan optical nanosectioning (MEANS-ESON). Scale bar=5 μm.

Figure 4

Confocal and MEANS image of the dual-stained Vero cell. The microtubules of the cell are stained with Dylight 650 (pseudo-colored red), and the nuclear pore complex of the cell is stained with Alexa 488 (pseudo-colored green). (a–h) Image series taken from a confocal microscope (Olympus FV1200) with axial step of 0.5 μm. As observed, MEANS forms at 2.5 μm depth in f, in which the nuclear pore proteins are shown clearly as grains. Scale bar=5 μm.

Figure 5

Imaging of the actin filaments with different thickness of silica spacer. Adjusting the spacer thickness can obtain the cross-sectional imaging of different layers of the cell specimen. For MEANS constructive interference, the maximum spacer thickness is 100–150 nm. The images are all of the same size. Scale bar=10 μm.

Figure 6

The NPC of a Vero cell (a–c) imaging using confocal (a: upper right) and MEANS-STED (a: lower bottom) modalities, and the hRSV viral filaments imaging via conventional STED (f–i) and MEANS-STED (l–o) modalities. MEANS-STED gives clear lateral resolution enhancement in a. (b) Magnification of the boxed area in a, in which the porous structure of the NPCs can be observed. (c) Magnified individual NPCs. (d) Plot of the intensity distribution along arrows in c. To demonstrate the advantage of the axial confinement of MEANS-STED, the hRSV filaments are imaged with conventional STED (on coverglass, f–i) and MEANS-STED (on mirror, l–o). The simulation of the convolution of conventional STED PSF vs MEANS-STED PSF with the filament structure is shown in (e) and (k), respectively. The central hollow structure is hard to visualize due to the uniform intensity distribution in e, but in k it can be observed. (i,o) Magnifications of the white box areas in h and n, respectively. The hollow structure of the hRSV-F can be visualized, taking advantage of the optical sectioning of MEANS-STED. (j, p) Intensity plots of the line indicated by the yellow arrows in i and o, respectively. Gaussian fitting of the data in j only shows one slope with width of 214 nm, whereas the fitting in p indicates that the width of the hRSV-F is ~56 nm, and the distance between the split peaks is ~119 nm. Scale bar=500 nm (f, l).