1. 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

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

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

4. 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

5. 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

6. 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 nm for visible light

7. 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)

8. 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

9. 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

10. 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)

11. 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

12. 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

13. 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

14. 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

15. 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