1. PALM/STORM How to get super-resolution microscopy.
Nanometer-scale instead of micron-scale
FIONA & Turn on/off dye
(accuracy and resolution)
SHRIMP – Super High Resolution IMaging with Photobleaching
2a. PALM – Photoactivated Localization Microscopy
b. STORM – Stochastic Optical Reconstruction Microscopy

2. How fine can you see? The Limits of Microscopy
Ernst Abbe
For visible microscopy,
Resolution is limited to ~250 nm
Ernst Abbe & Lord Rayleigh
Recent microscopy: nm,
Here we present techniques which are able to get
super-accuracy (1.5 nm) and/or super-resolution (<10 nm, 35 nm)

3. FIONA: Diffraction limited spot: Single Molecule Sensitivity
Accuracy of Center = width/ S-N
= 250 nm / √104 = ~2.5 nm = ± 1.25nm
Width of l/2 ≈ 250 nm
center
width
Enough photons (signal to noise)…Center determined to ~1.3 nm
Dye lasts 5-10x longer -- typically ~30 sec- 1 min. (up to 4 min)
Start of high-accuracy single molecule microscopy
Thompson, BJ, 2002; Yildiz, Science, 2003

4. Nanometer Distances between two (or more) dyes
Super-Resolution:
Nanometer Distances between two (or more) dyes
SHRImP
Super High Resolution IMaging with Photobleaching
In vitro

5. Nanometer Distances between two (or more) dyes
Super-Resolution:
Nanometer Distances between two (or more) dyes
SHRImP
Super High Resolution IMaging with Photobleaching
Distance can be found much more accurately than width (250 nm)
Resolution now:
Between 2-5 molecules: <10 nm
(Gordon et al.; Qu et al, PNAS, 2004)
Next slides
gSHRIMP: > 5-15 molecules
~ nm
Via 2-photon: ~ 35 nm (next time)
132.9 ± 0.93 nm
8.7 ± 1.4 nm
72.1 ± 3.5 nm
In vitro

6. Regular Microtubules (In vitro) Image
Take regular Image.
Then one fluorophore photobleaches.
Subtract off, get high resolution, repeat. 60
Imaging resolution
300 nm
1000 nm
Rhodamine-labeled microtubules, TIR
Actual 24 nm; Measured 300 nm

7. Microtubules in a COS-7 cell
Regular- resolution image
500 nm
Super-
gSHRImP FWHM: 96.5 ± 1 nm
Microtubules in a COS-7 cell
Spots localized versus frame
Labeling: 1:100 dilution of DM1A primary antibodies and 1:100 dilution CF633-labeled secondary antibodies
Photostability: PCA and PCD were added
Power: OD 1 on MegaMan 1 orange line (594 nm)
Camera: 50 em gain, 0.05 s per frame, using Andor iXon+ (Subzero)
Data were taken on March 17, 2010, file 19to20b.tif.
2 um
500 nm
Standard FWHM: 560 ± 20 nm

8. STochastic Optical Reconstruction Microscopy
STORM & PALM Most Super-Resolution Microscopy Inherently a single-molecule technique
PhotoActivation Localization Microscopy
Zhuang, 2007 Science
Betzig, 2006 Science
Huang, Annu. Rev. Biochem, 2009
2-color
secondary antibodies
Cy2-Alexa 647
Cy3-Alexa 647

9. Make dyes blink with dye-pairs
Photo-switching of Cy3-Cy5, 5.5, 7
532 nm
657 nm always on– excites Cy-dye &
turns it off
Cy3-Cy5 on DNA, antibody

10. 3-D (z) resolution

11. Fig. 2. Three-dimensional STORM imaging of microtubules in a cell.
Conventional indirect immunofluorescence image of microtubules
3D section
(color coded)
Three-dimensional STORM imaging of microtubules in a cell. (A) Conventional indirect immunofluorescence image of microtubules in a large area of a BS-C-1 cell. (B) The 3D STORM image of the same area, with the z-position information color-coded according to the color scale bar. Each localization is depicted in the STORM image as a Gaussian peak, the width of which is determined by the number of photons detected (5). (C to E) The x-y, x-z, and y-z cross sections of a small region of the cell outlined by the white box in (B), showing five microtubule filaments. Movie S1 shows the 3D representation of this region, with the viewing angle rotated to show different perspectives (27). (F) The z profile of two microtubules crossing in the x-y projection but separated by 102 nm in z, from a region indicated by the arrow in (B). The histogram shows the distribution of z coordinates of the localizations, fit to two Gaussians with identical widths (FWHM = 66 nm) and a separation of 102 nm (red curve). The apparent width of 66 nm agrees quantitatively with the convolution of our imaging resolution in z (represented by a Gaussian function with FWHM of 55 nm) and the previously measured width of antibody-coated microtubules (represented by a uniform distribution with a width of 56 nm) (5).
C-E zoom in of box in B
B Huang et al. Science 2008;319:
Published by AAAS

12. 3D Movie
B Huang et al. Science 2008;319:

13. Fig. 3. Three-dimensional STORM imaging of clathrin-coated pits in a cell.
Magnified View
Conventional direct immunofluorescence image of clathrin
2D STORM of same region
(all z’s)
x-y cross section
Magnified View
Three-dimensional STORM imaging of clathrin-coated pits in a cell. (A) Conventional direct immunofluorescence image of clathrin in a region of a BS-C-1 cell. (B) The 2D STORM image of the same area, with all localizations at different z positions included. (C) An x-y cross section (50 nm thick in z) of the same area, showing the ring-like structure of the periphery of the CCPs at the plasma membrane. (D and E) Magnified view of two nearby CCPs in 2D STORM (D) and their x-y cross section (100 nm thick) in the 3D image (E). (F to H) Serial x-y cross sections (each 50 nm thick in z) (F) and x-z cross sections (each 50 nm thick in y) (G) of a CCP, and an x-y and x-z cross section presented in 3D perspective (H), showing the half-spherical cage-like structure of the pit.
B Huang et al. Science 2008;319:
Published by AAAS

14. Use photoswitchable GFP
PALM
Use photoswitchable GFP
Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated,
localized (to ~2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a super-resolution image.
Fig. 1. The principle behind PALM. A sparse subset of PA-FP molecules that are attached to proteins of interest and then fixed within a cell are activated (A and B) with a brief laser pulse at λact = 405 mm and then imaged at λexc = 561 mm until most are bleached (C). This process is repeated many times (C and D) until the population of inactivated, unbleached molecules is depleted. Summing the molecular images across all frames results in a diffraction-limited image (E and F).
The principle behind PALM. A sparse subset of PA-FP molecules that are attached to proteins of interest and then fixed within a cell are activated (A and B) with a brief laser pulse at λact = 405 mm andthenimagedat λexc = 561 mm until most are bleached (C). This process is repeated many times (C and D) until the population of inactivated, unbleached molecules is depleted. Summing the molecular images across all frames results in a diffraction-limited image (E and F). However, if the location of each molecule is first determined by fitting the expected molecular image given by the PSF of the microscope [(G), center] to the actual molecular image [(G), left], the molecule can be plotted [(G), right] as a Gaussian that has a standard deviation equal to the uncertainty σx,y in the fitted position. Repeating with all molecules across all frames (A′ through D′) and summing the results yields a superresolution image (E′ and F′) in which resolution is dictated by the uncertainties σx,y as well as by the density of localized molecules. Scale: 1 × 1 μm in (F) and (F′), 4 × 4 μm elsewhere.
E Betzig et al. Science 2006;313:
Published by AAAS

15. Correlative PALM-EM imaging
1 mm
TIRF
PALM EM
Mitochondrial targeting
sequence tagged with mEOS
Patterson et al., Science 2002

16. G. H. Patterson et al., Science 297, 1873 -1877 (2002)
Photo-active GFP
G. H. Patterson et al., Science 297, (2002)
We report a photoactivatable variant of GFP that, after intense irradiation with 413-nanometer light, increases
fluorescence 100 times when excited by 488-nanometer light and remains
stable for days under aerobic conditions
Native= filled circle
Photoactivated= Open squares
Wild-type GFP
T203H GFP: PA-GFP

17. Photoactivation and imaging in vitro.
G. H. Patterson et al., Science 297, (2002)

18. “Regular” dyes blink (Cy5,…) simultaneously excited with 514, 647

19. The End