Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)

Abstract

We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

Figure 1

Stochastic optical reconstruction microscopy (STORM) with photo-switchable fluorophores. (a) The diagram shows a STORM imaging sequence using a hypothetical hexameric object labeled with red fluorophores that can be switched between a fluorescent and a dark state by a red and green laser, respectively. All fluorophores are first switched to the dark state by a strong red laser pulse. In each imaging cycle, a green laser pulse is used to switch on only a fraction of the fluorophores to give an optically resolvable set of active fluorophores. Next, under red illumination, these molecules emit fluorescence until they are switched off, allowing their positions (indicated by white crosses) to be determined with high accuracy. The overall image is then reconstructed from the fluorophore positions obtained from multiple imaging cycles. (b) A single Cy5 switch on DNA can be turned on and off for hundreds of cycles before being permanently photobleached. A red laser (633 nm, 30 W/cm²) is used to excite fluorescence (black line) from Cy5 and to switch Cy5 to the dark state. A green laser (532 nm, 1 W/cm²) is used to return Cy5 to the fluorescent state. The alternating red and green line indicates the laser excitation pattern. The recovery rate of Cy5 depends critically on the close proximity of Cy3.

Figure 2

The high localization accuracy of individual switches during each switching cycle defines the intrinsic resolution of STORM. (a) The point spread function (PSF) of the emission from a single switch on DNA during a single switching cycle. Fitting the PSF to a 2-dimensional Gaussian (not shown) gives the centroid position of the PSF. (b, c) The centroid positions of an individual switch determined in 20 successive imaging cycles before (b) and after (c) correction for sample drift. Scale bars: 20 nm. (d) A histogram of the standard deviation of centroid positions. The standard deviation is determined as (σ_x + σ_y)/2 for each switch using 20 imaging cycles, where σ_x and σ_y are the standard deviations of the centroid positions in the x and y dimensions. This histogram was constructed from 29 switches.

Figure 3

STORM can resolve structures with sub-diffraction-limit resolution. (a) STORM cleanly resolves two switches separated by a contour length of 46 nm on double-stranded DNA. The STORM images show two clearly-separated clusters of measured switch positions (crosses), each corresponding to a single switch. The center-of-mass position of each cluster is marked by a red dot. The inter-switch distances are 46 nm, 44 nm and 34 nm for these three examples. Scale bars: 20 nm. (b) Comparison between the inter-switch distances measured using STORM (grey column) and the predicted distance distribution considering the flexibility of DNA (dashed blue line). (c) STORM images of four switches attached to a double-stranded DNA, pair-wise separated by a contour length of 46 nm. The measured switch positions are clustered by an automated algorithm (see Supplementary Methods online) and different clusters are indicated by different symbols. Scale bars: 20 nm. (d) STORM images of RecA-coated circular plasmid DNA. The top panels show indirect immunofluorescence images with switch-labeled secondary antibody taken by a total internal reflection microscope. The bottom panels are the reconstructed STORM images of the same filaments. Scale bars: 300 nm.