Nanometer Axial Resolution by Three-Dimensional Supercritical Angle Fluorescence Microscopy

We report a noninvasive fluorescence microscopy method and demonstrate nanometer resolution along the optical axis. The technique is based on the influence of the microscope slide on the angular intensity distribution of fluorescence. Axial positions are determined by measuring the proportion of light emitted below the critical angle of total internal reflection, which behaves in a classical way, and light emitted above the critical angle, which is exponentially dependent on the distance of the fluorophore from the microscope slide.

Figure 1

Principles of 3D-SAFM. (a) Radiant flux into angles $\theta$ from the optical axis of a fluorophore with randomized orientation at different distances $z$ from a water-glass interface (${\theta }_c=61.1°$, dashed line) in fractions of $\lambda$. SAF emission is significantly reduced already for small distances, while UAF emission is unaltered. The indicated areas represent the portions captured by the objective. (b) Optical setup. (c) Simulated $z$ dependence of the relative detection efficiencies of SAF and UAF for Cy5 ($\lambda =670\mathrm{nm}$) for water-glass. The moderate decline of UAF follows the laser intensity distribution near the surface. (d) Enlarged view of the optical paths.

Figure 2

3D-SAFM of a fluorescence coated microsphere of $5\mu \mathrm{m}$ diameter. (a) Schematic of the experiment. (b) Raw UAF image. (c) Raw SAF image. (d) 3D image of the bead surface-contact region. Pixels with SAF intensities below a threshold are shown in black. (e) $z$ profile through the center of the bead fitted to a sphere of $5.1\mu \mathrm{m}$ diameter. The sample was scanned with a pixel size of 156 nm and 10 ms integration time per pixel. $\sim 1100–2600$ counts per pixel were detected in both channels together for determining the $z$ positions. Scale $\mathrm{\text{bars}}=1\mu \mathrm{m}$.

Figure 3

3D-SAFM of 36 nm diameter fluorescent nanospheres. (a) Raw UAF image, (b) Raw SAF image and (c) 3D image of beads adsorbed at the water-coverslip interface. (d) Raw UAF image, (e) Raw SAF image and (f) 3D image of beads embedded in agarose gel. The sample was scanned with a pixel size of 156 nm and 10 ms integration time per pixel. Scale $\mathrm{\text{bars}}=2\mu \mathrm{m}$.

Figure 4

Axial localization accuracy of 3D-SAFM. (a) The $z$ dependency of the relative SAF/UAF ratio at a water-glass interface for $\lambda =670\mathrm{nm}$ fitted by a monoexponential function (solid line). (b) The relative error of the distance measured between two points separated axially by $\Delta z_1=10\mathrm{nm}$, $\Delta z_2=50\mathrm{nm}$, and $\Delta z_3=200\mathrm{nm}$ as a function of the error $\Delta z_0$ in establishing the $z$ origin. Even for a very large $\Delta z_0$ of 100 nm the relative error of the distance measured between two points is below 8%.

Figure 5

3D-SAFM of the microtubule network of a mouse embryonic fibroblast cell. Microtubules further away than 640 nm from the coverslip surface (corresponding to $<2\%$ of the normalized SAF/UAF ratio) are shown in gray. (Inset) 3D representation of the pixels in the region outlined by the box. The sample was scanned with a pixel size of 156 nm and 3 ms integration time per pixel. Scale $\mathrm{\text{bar}}=10\mu \mathrm{m}$.