Resolution doubling in 3D-STORM imaging through improved buffers

Abstract

Super-resolution imaging methods have revolutionized fluorescence microscopy by revealing the nanoscale organization of labeled proteins. In particular, single-molecule methods such as Stochastic Optical Reconstruction Microscopy (STORM) provide resolutions down to a few tens of nanometers by exploiting the cycling of dyes between fluorescent and non-fluorescent states to obtain a sparse population of emitters and precisely localizing them individually. This cycling of dyes is commonly induced by adding different chemicals, which are combined to create a STORM buffer. Despite their importance, the composition of these buffers has scarcely evolved since they were first introduced, fundamentally limiting what can be resolved with STORM. By identifying a new chemical suitable for STORM and optimizing the buffer composition for Alexa-647, we significantly increased the number of photons emitted per cycle by each dye, providing a simple means to enhance the resolution of STORM independently of the optical setup used. Using this buffer to perform 3D-STORM on biological samples, we obtained images with better than 10 nanometer lateral and 30 nanometer axial resolution.

Figure 1. Improving photon counts for STORM with COT.

(a) Mean photon counts measured for three different STORM buffers using different thiols (MEA and/or BME) and the same oxygen scavenging system (glucose oxidase/catalase, see Table S1 & Methods for details on composition); number indicates the corresponding fold-increase upon addition of 2 mM COT (red bars) compared to no COT added (black bars) (pH ∼7.5) (b) Normalized photon count distributions for the “MEA+BME” buffer as a function of COT concentration (pH 8). (c) Mean photon count as a function of COT concentration for the buffer “MEA+BME” (pH 8).

Figure 2. Influence of pH on STORM imaging.

(a) Decrease in pH as a function of time for the “BME+MEA” buffer containing glucose oxidase/catalase as oxygen scavenging system, (buffer #3 in Table S1) both with and without the addition of 2 mM COT. (b) Average photon counts per molecule as a function of pH for the “BME+MEA” buffer both with and without the addition of 2 mM COT. (c) pH as a function of time using the PCA/PCD oxygen scavenging system both with and without the addition of 2 mM COT (d) Normalized photon count distribution for the PCA/PCD buffer with and without COT at pH = 8 (mean photon count is 8,700 without COT and 32,000 with 2 mM COT).

Figure 3. Buffer-Enhanced 2D STORM imaging of microtubules.

(a) Widefield, and (b) STORM image of a COS-7 cell stained with alpha-tubulin primary and Alexa-647-F(ab’)2 secondary antibodies, imaged in Buffer #4 (see Table S1). (c) Zoom on the ROI (red box) defined in (b), number of localized molecules = 1960. (d) Lateral profiles taken either from a 200 nm-wide region (violet box) and corresponding curve shown in violet, or averaged over seven 200 nm-wide regions (inside the dashed red box) highlighted in (c) and the corresponding curve in red. (e) Averaged lateral profile shown in (d) fitted with a double Gaussian. (f) Model of the stained microtubule (see also Figure S4). The labeled antibodies are expected to form a ring around the microtubule with an inner diameter of ∼25 nm and an outer diameter of ∼50 nm. (g) STORM image of COS7 cells strained with Cep152 primary and Alexa-647 F(ab’)2 secondary antibodies, imaged in Buffer #4 (see Table S1). The top panel shows two tori from a side view, the bottom panel one torus in side views and one torus in cross-section. Scale bar is 1000 nm for (a–c) and 500 nm for (g).

Figure 4. Buffer-Enhanced 3D STORM imaging of Microtubules.

COS-7 cells were stained with alpha-tubulin primary antibodies and Alexa-647 F(ab’)2 secondary antibodies and imaged. 3D-STORM images are color coded by depth.(a–c) Astigmatic 3D-STORM with an oil objective and PBS-Glycerol buffer (Buffer #5, see Table S1): (a) 3D-STORM image and corresponding axial profiles from 300×300 nm-wide regions taken (b) on the edge of the cell (dashed box) or (c) in a denser central region with microtubules crossings (full box).(d-f) Astigmatic 3D-STORM with a water objective and an index-matched buffer (Buffer #4, see Table S1): (d) 3D-STORM image and corresponding axial profiles from the 300×300 nm-wide regions taken (e) on the edge of the cell (dashed box) or (f) in a denser central region with microtubules crossings (full box). For each axial profile, positions of the fitted Gaussian peak maxima (green) as well as FWHM (blue) are indicated. Scale bar is 5 µm.

Figure 5. Buffer-Enhanced 3D STORM imaging on a biplane astigmatic microscope.

(a) Schematics of the SR-200 inverted microscope (Vutara, Salt Lake City, UT). D: Dichroic mirror, F: Fluorescence filter, T: Tube lens, B: 50/50 Beamsplitter. Fluorescent light from a single molecule is collected by the objective and is imaged onto two different planes located on distinct parts of the EMCCD camera. The distance between the two planes is set by the optical path difference, and results in a measured axial shift of 780 nm. Optical aberrations are represented by in the schematics by the lateral shift in the position of the tube lens. (b) Measured PSF in the two planes which are used for 3D localization, shown at three different depths. (c) Biplane-Astigmatic 3D STORM image of a cell stained with Alexa-647-conjugated alpha-tubulin antibodies, color-coded by depth. Buffer #4 is used (See Table S1) (d,e) Lateral and axial profiles were measured and averaged from five 200-nm wide regions. The blue line corresponds to a Gaussian fit, and the corresponding FWHM value is indicated. (f) Estimation of the resolution performed by convolving the projection of the known structure with a 40 nm wide Gaussian function, and the resulting expected distribution. Scale bar is 1 µm for (b) and 2.5 µm for (c).