Sub-diffraction-limit imaging by Stochastic Optical Reconstruction Microscopy (STORM) Michael J. Rust, Mark Bates, Xiaowei Zhuang Harvard University Published Online August 9, 2006 Nature Methods Vol.3 No.10 Presented by Artie Wu

STORM High-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores Imaging resolution of 20nm

Outline Background Motivation Fluorescence Microscopy Alternatives diffraction Motivation Fluorescence Microscopy Alternatives STORM Results Conclusions

Background Nuclei rearranges itself Stoke’s shift – diff between excitation peak and emission peak occurs bc molecule loses small amount of absorbed energy before re-releasing the rest of the energy as fluorescence. Energy lost as thermal energy

Background Resonant state, electron transferred to triplet state and flipped Fluorescence lifetime ~1-10 nsec Phosphorescence probability ~0.1% Phosphorescence lifetime msec bc can’t easily flip again

Motivation I Resolution limit set by diffraction of light Fluorescence microscopy widely used in molecular and cell biology Widely used for noninvasive, time-resolved imaging with high biochemical specificity Drawback: standard fluorescence microscopy not useful for ultra-structural imaging Theoretical resolution limit of conventional optical microscopy is 200-300nm for visible light

Fluorescence Microscopy Alternatives Lateral resolution of 10s of nanometers Near-field scanning optical microscopy (NSOM) Multiphoton fluorscence Stimulated emission depletion (STED) Saturated structured-illumination microscopy (SSIM)

Near-field scanning optical microscopy (NSOM) Image at interface due to evanescent field Study what goes on near membrane Exocytosis & endocytosis Build up point by point Drawback: low imaging depth Near-field (or evanescent) light consists of a nonpropagating field that exists near the surface of an object at distances less than a single wavelength of light. Light in the near-field carries more high-frequency information and has its greatest amplitude in the region within the first few tens of nanometers of the specimen surface. not diffraction limited and nanometer spatial resolution is possible This phenomenon enables spectroscopy of a sample that is simply not possible with conventional optical imaging techniques. Near-field imaging occurs when optical probe is positioned a very short distance from the sample and light is transmitted through a small aperture at the tip of this probe or through prism

Motivation II Single-molecule detection leads to sub-diffraction-limit spatial resolution Stochastic optical reconstruction microscopy (STORM) Fluorescence image constructed from high-accuracy localization of individual fluorescent molecules Imaging resolution: ~20nm using TIRF and photoswitchable cyanine dye, Cy5

STORM Cy5: fluorescent and dark state using different λ Cy3: secondary dye Series of imaging cycle In each cycle Only 1-3 switches in FOV are switched ON Stochastically different subset of fluorophores are ON Red: 633nm, 30W/cm2, 2s Green: 532nm, 1W/cm2, 0.5s Photobleaching: 230s Red laser light produces fluorescent emission from Cy5 can also switch dye to stable dark state Green laser light converts Cy5 back to fluorscent state, but recovery rate depends on proximity of secondary dye Cy3, to increase speed of switching from dark to fluorescent Only a fraction in ON, so each of active fluorophores is optically resolvable from rest -> No overlap Rate constants for turning on and off has a linear dependence on laser intensity Switch from trans-cis isomer of dye (cis is nonfluorescent)

Resolution Limited by accuracy of localization of switches 2d Gaussian fit to PSF used to find centroid position of switch To determine localization accuracy, a switch was attached to short dsDNA that was surface-immobilized at low density Fluorescence image from singe switch gave PSF. Gaussian fit to this image was used to get centroid position of switch Histogram of standard deviation of centroid positions FWHM: 18 +/- 2nm

Centroid position Fit to pixelated Gaussian function A: background fluorescence level Io: amplitude of peak a,b: widths of Gaussian distribution xo,yo: center coordinates of peak δ: fixed half-width of pixel in object plane Fluorescent structures were first isolated in 13x13 pixel square fitting window data analysis. Criteria to ensure high accuracy localization of single switches: Switch has to be of at least 3 frames Images were fit by nonlinear least-squares regression to continuous ellipsoidal Gaussian, ellipicity <15% Photoelectrons in peak has to be greater than 2,000 Final centroid coordinates obtained from this fit were used as one data point in final STORM image

Results I Linear, dsDNA with 2 switches separated by 135 bps (46nm) Theoretical dist = 40nm Experimental dist = 41nm Linear, dsDNA labeled with multiple switches separated by a well-defined number of base pairs Immobilized DNA by labeling DNA w/ multiple biotins and attached them to high-density streptavidin layer - > increase likelihood of multiple attachments between DNA and surface 46 nm, 44 nm and 34 nm for these three examples. Scale bars, 20 nm Theoretical mean of 40 nm determined using known contour and flexibility of DNA Column = STORM measurements, dashed line = predicted distance distribution

Results II Longer DNA with 4 switches spaced 46 nm apart Localize large number of switches within diffraction-limited spot by cycling switches on/off 4 clusters of switch positions following bent contour consistent with predicted length of DNA Can use STORM as general biological imaging technique Circular DNA plasmid coated with biotinylated RecA protein and imaged using indirect immunofluorscence with switch-labeled secondary antibody taken by TIRF. RecA used for repair and maintenance of DNA Better resolution when compared to wide-field image

Conclusions STORM capable of imaging biological structures with sub-diffraction-limit resolution Resolution limited by # photons emitted per switch cycle Cyanine switch ~3000 photons/cycle Theoretical localization accuracy of 4nm Corresponds to imaging resolution of ~20nm Imaging speed improved by increasing switching rate Stronger excitation or fluorophores with faster switching kinetics Valuable tool for high-resolution in situ hybridization and immunofluorescence imaging