Review of FIONA/PALM/STORM
How to make photoactivatable fluorophores
How to get 3-D Measurements
Intro to STED (?)
Super-Accuracy:
Nanometer Distances w Single Molecules
W.E. Moerner,
Crater Lake
Fluorescence Imaging with
One Nanometer Accuracy
1.5 nm accuracy
1-500 msec
Center can be found much
more accurately than width
center
Prism-type TIR 0.2 sec integration
Betzig, Zhuang
Resolved!
280
240
200
Photons
Super-Resolution: PALM/STORM.
between (activatable) molecules
160
Width
(250 nm)
120
80
40
0
5
10
Y ax
15
10
15
20
is
20
25
25
5
0
ta
X Da
Dxcenter = width /√N
≈ 250/√10k = 1.3 nm
Z-Data from Columns 1-21
Good for dynamics
Yildiz et al, Science, 2003
Normal- vs. Super- resolution
Let’s say you have a single fluorophore
(a few nanometers in size, much less than the diffraction limit)
~ 250 nm
In a microscope, what does its emission look like??
Does it matter if your excitation light is focused to a (diffraction-limited spot)
or you are exciting with wide-field? No, either one produces a diffraction-limited spot.
PALM (STORM)- Photo-activated localization
super-resolution microscopy
~ 250 nm
A.
B.
The PALM cycle
You have PALM spelled out with really tiny molecules
separated by a tiny distance—such that each letter is
less than a diffraction limit apart.
How to see what is written?
First you try regular fluorescence, labeling it with
some fluorescent dye and shine light to make it
fluoresce. What do you see?
Each dye emits with a diffraction-limited (i.e., about
250 nm) size. The result is B. It’s not well resolved.
However, if you can make each fluorescent molecule
emit one at a time, then you can determine where
the dye is by doing FIONA—taking the SEM (instead
of the Standard Deviation), where you can determine
it’s position to within a few nanometers. Then you
repeat this measurements many many times, until
you get the entire image. See next page.
Betzig et al. Science 2006
10 - 20 nm resolution (localization precision)
PALM (STORM)- Photo-activated localization
super-resolution microscopy
Weak near-UV light
Read out with
visible light
After
many
cycles
Activate with weak near UV-light;
Once activated, shine visible
light to get out fluorescence.
Locate each fluorphore to within
a few nanometers by taking the
center of the emission (rather
than the diffraction-limited
width). Record the position of
these molecules, Then repeat,
until you get all of the position
of all of the fluorophores.
The PALM cycle
Betzig et al. Science 2006
10 - 20 nm resolution (localization precision)
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
PhotoActivation Localization Microscopy (F)PALM
(Photoactivatable GFP)
1 mm
TIRF (reg. diff’n limit)
1 mm
PALM
1 mm
TEM
(Transmission Electron Microscopy)
Mitochondrial targeting
sequence tagged with mEOS
(an photoactivatable Fluorescent Protein)
Patterson et al., Science 2002
The Wild Type Green Fluorescent Protein (wtGFP)
consisting of a cyclized tripeptide made of Ser65, Tyr66, and Gly67.
Jennifer Lippincott-Schwartz, and George H. Patterson
Science 2003;300:87-91
Published by AAAS
Photoactivatable GFP
Thr203 → His203
Absorbance spectra of purified wtGFP before (A) and after (B) photoactivation with ∼400-nm light.
After activation
Before activation
A cell expressing PA-GFP
was imaged with the use
of 488-nm excitation (Pre)
before and after ∼1-s
photoactivation of the
nuclear pool with 413-nm
laser light.
Jennifer Lippincott-Schwartz, and George H. Patterson
Science 2003;300:87-91
Published by AAAS
Comparison between regular- and super-microscopy
Pre-synaptic Bouton
Regular mscopy
STORM mscopy
Synapse (30 nm)
PSD
Post-synaptic Spine
Valtschanoff and Weinberg, 2003
Zhuang, Neuron, 2010
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
“Regular” dyes can be made to blink
They are off; then can be made to come
on.(Cy3B, Cy5, Alexa 647…)
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)
(Notice
box)
3D Movie
B Huang et al. Science 2008;319:810-813
The End