PALM/STORM
How to get super-resolution microscopy.
Nanometer-scale instead of micron-scale
FIONA & Turn on/off dye
(accuracy and resolution)
1.
SHRIMP – Super High Resolution IMaging with Photobleaching
2a. PALM – Photoactivated Localization Microscopy
b. STORM – Stochastic Optical Reconstruction Microscopy
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: 1-100 nm,
Here we present techniques which are able to get
super-accuracy (1.5 nm) and/or super-resolution (<10 nm, 35 nm)
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
Prism-type TIR 0.2 sec integration
center
280
240
Photons
200
160
120
width
80
40
0
5
10
15
Y ax
is
15
10
20
20
25
25
5
0
ta
X Da
Enough photons (signal to noise)…Center determined to ~1.3 nm
Z-Data from Columns 1-21
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
Super-Resolution:
Nanometer Distances between two (or more) dyes
SHRImP
Super High Resolution IMaging with Photobleaching
In vitro
Super-Resolution:
Nanometer Distances between two (or more) dyes
SHRImP
Super High Resolution IMaging with Photobleaching
Distance
can
be0.93
found
much more
132.9
nm
8.7
±±
1.4
nmnm
72.1
±
3.5
accurately than width (250 nm)
Resolution now:
600
600
700
Between 2-5 molecules: <10 nm
500
500
600
400
400
500
(Gordon et al.; Qu et al, PNAS, 2004)
300400
300
Next slides
gSHRIMP: > 5-15 molecules
~ 20-100 nm
Via 2-photon: ~ 35 nm (next time)
200300
200
100200
100
In vitro
100
0
0
0
-100
-100
1000
-100
1000800
1000
800600
800
400
600600
200
400400
10001200
800 1000
1000
600 800800
400 600600
200 400400
200 0
0
200200
Regular Microtubules (In vitro) Image
•Take regular Image.
•Then one fluorophore
photobleaches.
•Subtract off, get high
resolution, repeat.
Imaging resolution
300 nm
1000 nm
Rhodamine-labeled microtubules, TIR
60 nm
Actual 24 nm; Measured 300
Microtubules in aRegularCOS-7
cell
Super-
resolution image
Spots localized versus frame
2 um
500 nm
Standard FWHM: 560 ± 20 nm
gSHRImP FWHM: 96.5 ± 1 nm
500 nm
STochastic Optical
Reconstruction Microscopy
Zhuang, 2007
Science
Localization
STORM & PALM PhotoActivation
Microscopy
Most Super-Resolution Microscopy
Betzig,
2006
Inherently a single-molecule technique Science
Huang, Annu. Rev. Biochem, 2009
Cy3-Alexa 647
Cy2-Alexa 647
2-color
secondary
antibodies
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
3-D (z) resolution
Fig. 2. Three-dimensional STORM imaging of microtubules in a cell.
Conventional indirect
immunofluorescence
image of microtubules
C-E zoom in
of box in B
B Huang et al. Science 2008;319:810-813
Published by AAAS
3D section
(color coded)
3D Movie
B Huang et al. Science 2008;319:810-813
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
B Huang et al. Science 2008;319:810-813
Published by AAAS
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 diffractionlimited image (E and F).
E Betzig et al. Science 2006;313:1642-1645
Published by AAAS
Correlative PALM-EM imaging
1 mm
TIRF
1 mm
PALM
1 mm
EM
Mitochondrial targeting
sequence tagged with mEOS
Patterson et al., Science 2002
Photo-active GFP
G. H. Patterson et al., Science 297, 1873 -1877 (2002)
We report a
photoactivatable
variant of GFP that,
after intense
irradiation with 413nanometer 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
Photoactivation and imaging in vitro.
G. H. Patterson et al., Science 297, 1873 -1877 (2002)
“Regular” dyes blink (Cy5,…)
simultaneously excited with 514, 647
The End