Stochastic Optical Reconstruction Microscopy (STORM)

Abstract

Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc.

Keywords: single molecule localization microscopy (SMLM); stochastic optical reconstruction microscopy (STORM); super-resolution fluorescence microscopy.

Figure 1

The illustration of how to select parameters for (d)STORM super-resolution image reconstruction. (A) The representative raw image from a single frame. (B) The image after background removal. (C) The illustration of how to estimate PSF width. A line (thickness = 2 pixels) across the yellow area in the image is plotted, shown in (D). The full-width at half maximum (FWHM) is shown in about 2.5 pixels. (E) The illustration of how to estimate standard deviation of the background. The histogram of the background in the yellow box of (E) is shown in (F) and standard deviation is about 8.5 pixels, shown in red box.

Figure 2

A snapshot of “Run Analysis” of ThunderSTORM ImageJ plugin.

Figure 3

A simplified schematic of the (d)STORM instrument setup.

Figure 4

Comparison of SNR performance for the commonly used cameras at different signal levels. Here, only quantum efficiency (QE) and read noise are considered because the dark current noise can be ignored under deep cooling. The SNR is defined as below. $$\begin{array}{l}\mathit{SNR}=\mathit{Signal}·QE/\sqrt{\mathit{ExcessNoise}·\mathit{Signal}·QE+{\mathit{ReadNoise}}^2}\\ \{\begin{array}{c}\mathit{ExcessNoise}=\mathit{1},\mathit{EMCCD}\\ \mathit{ExcessNoise}=\mathit{0},\mathit{other}\mathit{cameras}\end{array}\end{array}$$

Figure 5

Super-resolution dSTORM images of nucleosomes (H2B immunostained with Alexa Fluor 647) in the cell nucleus (A) before and (B) after the drift correction. The focal plane of the imaging object is ~2–3μm above the surface of the coverslip. (C–D) The zoomed-in region of the white box in in A and B.

Figure 6

Effect of different background levels on the localization accuracy. (A, C) The raw image and localized (B, D) super-resolution image of a single fluorescent emitter at a low background level of 1000 photons per pixel and a high background level of 1000 photons per pixel, respectively, at a total photon number for a single fluorescent emitter set to 5000 to mimic the emission properties of Alexa Fluor 647. The final localization precision, measured by the standard deviation of localized positions, is reduced by over a factor of 2 at a high background level compared to a low background level. Total photon number of a single fluorescent emitter: 5000. Pixel size of the raw image: 100 nm; pixel size of the super-resolution image: 5 nm.

Figure 7

Effect of non-uniform background on the localization accuracy. A non-uniform background leads to significant localization bias. (A) From left to right, the background is simulated as 100 to 500 photons per pixel. (B) The localized position carries a bias of over 15 nm at a localization precision of ~10 nm. Total photon number of a single fluorescent emitter: 5000. Pixel size of the raw image: 100 nm. Pixel size of the super-resolution image: 5 nm.

Figure 8

Effect of overlapping molecules on the localization accuracy. (A) Simulated raw image with overlapping molecules, defined as the molecules with a distance less than the diameter of the PSF. (B) The localized positions exhibit a significant bias of over 10 nm, compared to the ground truth. Total photon number of a single fluorescent emitter: 5000. Pixel size of the raw image: 100 nm.

Figure 9

The reconstructed dSTORM images of H2B in the case of (A–B, E–F) low labeling density and (C–D, G–H) high labeling density. (A–B) The dSTORM images of H2B in the case of low labeling density before and after applying a Gaussian smoothing filter (σ = 10 nm). (C–D): The reconstructed dSTORM images of H2B in the case of high labeling density before and after applying the same Gaussian smoothing filter as (B). The figure insets of (A, C) are the corresponding wide-field image of H2B. (E–H): The zoomed-in region of the white boxes shown in (A–D).

Figure 10

The dSTORM images of microtubules immuno-stained by Alexa Fluor 647 reconstructed by different numbers of imaging frames. The insets show the magnified region.

Figure 11

The dSTORM of nucleosomes which is labeled by histone H2B immuno-stained via Alexa 647 and reconstructed by different numbers of imaging frames. The right two figures show the magnified structure as indicated in the white boxes. Number in the right top of the figure insets is the localization number in the selected area for the same single cluster.

Figure 12

(A–B) The conventional wide-field and reconstructed super-resolution image of microtubules from MCF10A cells, immuno-stained by Alexa 647 using dSTORM imaging and reconstructed. (C–D) The zoomed-in region of the white boxes shown in (A–B), respectively. (E) The cross-sectional profile from a marked region in (D), together the fitted full-width at half-maximum (FWHM). Illumination laser with a wavelength of 642 nm (VFL-P-1000–642-OEM3, MPB Communications) at a power density of 3 KW/cm² is used, a total of 40,000 image frames at a speed of 50 Hz (acquisition time of 20 ms) are recorded on a sCMOS camera (pco.edge 4.2, PCO-Tech) with a pixel size of 130 nm on the sample plane. The super-resolution image is reconstructed using the least-square single-emitter Gaussian fitting method. The extracted molecules are fitted with least-square single-emitter Gaussian function model. Those candidate molecules that meet the following criteria are rejected: (1) total photon number less than 100; (2) the FWHM of PSF with 50% larger or smaller than that of the ideal PSF; (3) position with more than 2-pixel distance from the region center; (4) peak intensity vs. background intensity less than 0.5. The final super-resolution image was reconstructed by accumulating all molecules that meet the above criteria, with a pixel size of 10 nm followed by a Gaussian smoothing filter (σ=10 nm).

Figure 13

Two-color super-resolution imaging of methylated (H3K4me3) and acetylated (H3K9Ac) histone proteins by dSTORM. (A–B) The dSTORM images of H3K4me3 and H3K9Ac. H3K4me3 is labeled with Cy3B and H3K9Ac is labeled with Alexa Fluor 647 in the cell nucleus of MCF-10A cells. (C–D) The representative raw images of Alexa Fluor 647 channel (H3K9Ac) and Cy3B channel (H3K4me3), respectively. Continuous illumination with a 642 nm or 561 nm laser are used in the two-color dSTORM imaging. The two channels are imaged sequentially at the exposure time of 20ms, for 30000 imaging frames using 642 nm excitation, followed by 30,000 imaging frames using 561 nm excitation. Fluorescent beads (0.1μm diameter, F8803, excited using 488 nm laser) are used as fiduciary markers on the coverslip to correct for 3D system drift every 200 frames.

Figure 14

Two-color super-resolution imaging of methylated (H3K4me3) and acetylated (H3K9Ac) histone proteins by STORM based on dye-pair photo-switchable fluorophores. (A–B) Reconstructed STORM image. H3K4me3 is labeled with Cy2-Alexa Fluor 647 and H3K9Ac is labeled with Alexa 405-Alexa 647. (C1–C3) The representative raw images of the three consecutive frames after the activation of 488 nm for Cy2-Alexa Fluor 647 pair for one cycle. (D1–D3) The representative three consecutive frames after the activation of 405 nm for Alexa Fluor 405-Alexa Fluor 647 pair for one cycle. The samples are periodically activated with a sequence of 405nm, 488nm laser pulses and then imaged with a 647 nm laser. In each switching cycle, one of the activation laser is turned on for 1 frame, followed by 3 frames of illumination with 647 nm imaging laser. The imaging frame that immediately after an activation pulse is recognized as a controlled activation event and a color is assigned accordingly. A total of 40000 frames include 10,000 activation frames and 30,000 imaging frames for each channel, acquired at the exposure time of 15ms. Cross-talk subtraction algorithm (described in the protocol) is used to subtract the non-specific activation signal.