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Martes Cuantico

h"p://martescuan/co.weebly.com

4 de Noviembre a las 3:30,
El premio Nobel de Química de 2014
ó
viendo lo que no se puede ver:
microscopios ópticos con resolución nanométrica

Luis Martín-Moreno
lmm@unizar.es

Zaragoza, 4/11/2014

          Optical

Instruments.

∼1660

∼1590

1665. Hooke:
cork cells
1670-80‘s van Leeuwenhoek:
protozoa, bacterias,
spermatozoids

Heliocentrism,
expansion of universe,
extrasolar planets,
dark energy&matter...

1950

The Nobel Prize in Physics 2014 was awarded jointly to
Isamu Akasaki,

Hiroshi Amano and Shijo Nakamura

"for the invention of efficient blue light-emitting diodes which
has enabled bright and energy-saving white light sources".

The Nobel Prize in Chemistry 2014 was awarded jointly to
William E. Moerner,

Stefan W. Hell and Eric Betzig

"for the development of super-resolved fluorescence microscopy".

Special thanks for some viewgraphs

Triple-label imaging with combined techniques.

Fig. 5. Triple-label imaging with combined techniques. (A) Overlaid DIC and
TIRF (yellow) images of paxillin and vinculin coexpressed in an HFF-1 cell. (B)
Diffraction-limited epi-fluorescence image of mCerulean-tagged actin (blue)
overlaid with PALM images of Dronpa-tagged paxillin (green) and tdEostagged vinculin (red) shows adhesion complexes at the periphery of the cell
aligned with the termini of actin bundles. An expanded view (C) of the boxed
region in B reveals parallel arrays of interwoven paxillin and vinculin aggregates along the length of each AC, as well as possibly nascent adhesion
complexes consisting of adjacent paxillin (arrowheads) and vinculin aggregates (arrows). Further magnified views (D and E) of the boxed regions in C
indicate other examples of adjacent aggregates of either paxillin (arrowheads) or vinculin (arrows) within larger adhesions.

Shroff H et al. PNAS 2007;104:20308-20313

velocity per track (µm/s)
1.5

er

k
ac

r
ER itot
m 2B
H
0
0.1

probability

t = 0 min

0
mCherry-H2B
GFP-EB1

interphase
prometaphase
anaphase
cytokinesis
0

0.5
1.0
1.5
velocity per track (µm/s)

2.0

3.04 min

GFP-Scr1 (scramblase)

t = 0 ms

314 ms

628 ms

942 ms

1256 ms

1570 ms

1884 ms

2198 ms

Lattice light-sheet microscopy: Imaging molecules to embryos at
high spatiotemporal resolution
Bi-Chang Chen et al.
Science 346, (2014);
DOI: 10.1126/science.1257998

Fig. 4. Intracellular dynamics in three dimensions. (A) Cells in prophase (left)
and anaphase (right), showing histones and 3D tracks of growing microtubule ends,
color-coded by velocity. Color-coding of each track by height (movie S11) or growthphase lifetime (movie S12) is also possible. Each image in (A) represents a distillation
of a few time points from a 4D, two-color data set typically covering hundreds of time
points per cell (compare with Movie 5). Graph shows the distribution of growth rates
at different stages of mitosis, averaged across 9 to 12 cells (compare with figs. S8

6.02 min

9.51 min
and S9). (B) The 3D spatial relationship of histones (green), mitochondria (yellow),
and ER (magenta) at four time points during mitosis in a slab extracted from a larger
4D, three-color data set of HeLa cells imaged for 300 time points (compare with
Movie 6). (C) Volume renderings at eight consecutive time points of a single specimen of the protozoan T. thermophila taken from a 4D data set spanning 1250 time
points (compare with Movie 7). Imaging at 3 ms per frame in a single plane
(compare with movie S13) reveals the motions of individual cilia.

            The

diffraction limit.
Screen

x

∆xscreen

∆x

z

            The

diffraction limit.
Screen

x

∆xscreen

∆x

z

∆k∆x ≈ 2

            The

diffraction limit.
Screen

x

∆xscreen

∆x

z

~ r) = ~eσ eıkx x eıkz z
E(~
|k| =

p

kx2 + kz2 = ω/c

kz2 ≥ 0 → |kx | < |k|

∆k∆x ≈ 2

            The

diffraction limit.
Screen

x

∆xscreen

∆x

z

~ r) = ~eσ eıkx x eıkz z
E(~
∆kscreen

|k| =

p

∆xscreen

kx2 + kz2 = ω/c

kz2 ≥ 0 → |kx | < |k|
∆kscreen = 2

∆k∆x ≈ 2

ω
c

∆xscreen ≈ λ/2

            The

diffraction limit: influence on spacial resolution

Eexc

α
M ∆r

∆r

object plane

image plane

λ
M in[∆rn ] =
2n sinα
Principles of Nano‐op0cs, L. Novotny and B. Hecht

            The

diffraction limit: influence on spacial resolution

n

(n/n') M 2∆r

n'

Eexc
∆r

α

object plane

image plane

λ
M in[∆r⊥ ] = 2
2
n sin α
Principles of Nano‐op0cs, L. Novotny and B. Hecht

500 nm

α
200 nm

Wavelength

Lens

E. Abbe (1873), Arch. Mikroskop. Anat. 9, 413.

resolution: confocal microscopy.

Total detected signal does not 
depend on z ‐>  I
nz
n's

θ

object
n
f

f'
sin θ
=
sin θ ' f

θ' z
n'

f'

image

detector

ns

µ

Just placing a pinhole in front of 
the detector  ‐> I=I(z)
ns

µ

n's

θ

object
n
f

Principles of Nano‐op0cs, L. Novotny and B. Hecht

nz

f'
sin θ
=
sin θ ' f

θ' z
n'

f'

image

detector

     Axial

     4Pi-

Microscopy: resolution improvement in Z
70 - 140 nm

Coherent illumination and/or fluorescence detection
S.W. Hell (1990), Europ. Patent OS 0491289.
S.W. Hell, et al. (1992), Opt. Commun. 93, 277.
M. Schrader, et al. (1998), Biophys. J. 75, 1659.
H. Gugel, et al. (2004), Biophys. J. 87, 4146.

Commercial 4Pi-microscope

Z- resol < 90 nm (Live cells /aqueous cond.)
H. Gugel, et al. (2004), Biophys J 87, 4146.

Z

Microtubules, mouse fibroblast
Immunofluor, Oregon Green

2 µm

X

2 µm

Confocal

4Pi
S.W. Hell, et al. (1992), Opt. Commun. 93, 277.
M. Schrader, et al. (1998), Biophys. J. 75, 1659.
H. Gugel, et al. (2004), Biophys. J. 87, 4146.

            Nanophotonics

λ
∆x ≥
2

and the diffraction limit.

Minimum lateral size of light

- For nm resolution we need λ in the nm range -> X rays
- X rays are intensively used for this reason, but have problems
(too much energy, so they damage matter)

With light λ is 500-900nm, so ∆x ≈ 250nm
Poor resolu)on

It is not possible to address 
single molecules separately

Revisi+ng the 
diffrac+on limit 
(I)

     Beating

the diffraction limit: retrieving Fourier components

x
z
However, if we could play with kz2<0...
as large as we want
arbitrarily small

     Beating

the diffraction limit: retrieving Fourier components

x
z
These waves can not exist
in uniform media, but…

exist if there are interfaces
Problem:
the field is only intense close to the interfase
-> near-field optics

Scanning probe techniques

1. Images of SiC islands
(metallic at 10 µm)

2. Fields in metallic environments

Revisi+ng the 
diffrac+on limit 
(II)

But,
Abbe’s limit is related to two 
simultaneously emi;ng emi<ers. 

If one could scan them 
independently...

26

     Beating

the diffraction limit: measuring 1 emitter at the time
nz

ns

µ

n's

θ

object

f'
sin θ
=
sin θ ' f

n
f

Point spread funcGon
1

(a)

image

n'
f'

lim
θmax ≪π/2

"
"
"E(x, y, z = 0)"2 =

(b)

z =0

θ' z

$
#
J1 (2π ρ)
˜ 2
π 4 µ2x NA4
2
,
(2π ρ)
˜
ε02 n n ′ λ6 M 2

NAρ
.

(4.14)

(c)

r =0

ρ˜ =

z =0

µz

2

| E|

0
–1

–0.5

0

r NA / M λ

0.5

1

–1

0

1

z NA2 / (M 2 l 2n')

2

–1 –0.5

Spa+al resolu+on is NOT posi+on accuracy!
Principles of Nano‐op0cs, L. Novotny and B. Hecht

0

0.5

1

1.5

r NA / M l

λ

∆x =
2n sinα N

     Fluorophores

Fluorescence is the emission of light by a substance
that has absorbed light or other electromagnetic radiation.

28

     Fluorescence

excitation
in dilute samples
cm
/

WAVER(. BRIBER

LETTERS
PHYSICAL REVIEW
16883
16886

VOLUME 62, NUMBER 21

Optical Detection and Spectroscopy of Single Molecules in a Solid

30

GHz

W. E. Moerner and L. Kador '
IBM Research DivisionA, lmaden Research Center, San Jose, California 95120
(Received 17
March 1989)
LETTERS
REVIEW
PHYSICAL

VOLUME 65, NUMBER 21
two diA'erent

crystal at 1.8 K. The nearly Gaussian bands result from
that the spectrum presents the expected irregular distrithe superposition of a large number of the much narseveral arguments
bution.
we examine
Hereafter,
rower homogeneous lines of single molecules. The resostrengthening our assertion.
is determined by the
of each molecule
frequency frequency
22 MAYnance
1989
2 shows that
The expanded
scale of Fig.
in its surroundings.
is illustrated in
defects
crystal
dots resolve
into well-shaped This
most
of the
(approximately
excitation
a subliB, by
Fig. 1, curve
Lorentzian)
of the spectrum
Thethewidths
peaks rangeof from
peaks.
and was
bands 25are
mation-grown
The O~ flux
02 about
mWmuch
10
to 15 MHz. flake.
The excitation
19 NOVEMBER 1990
i.e. , below the saturation flux 71 mWcm, and

double-modulation

we have observed the optical-absorption

techniques,
spectrum
Using
Excitation in a p-Terphenyl
Detected
Single Pentacene
by Fluorescence
host crystal at liquid-helium temperatures.
To
molecules of pentacene
in a p-terphenyl
of single dopant Molecules
achieve this, frequency-modulation
spectroscopy was combined either with Stark or ultrasonic modulaM. Orrit
3. Bernard
tion to remove interfering background signals
from and
residual
amplitude modulation, and the number of
in resonance was reducedHerztienneC,
to one by operating
molecules
in the wings
line. etTriplet
inhomogeneous
Centre de Physique
Moleculaire
entre National
Recherche
de of
ia the
Universite
Scientiftc
Optique et
bottleneck saturation appears to be suppressed in the single-molecule regime.

cm,

Crystal

WAVER(. BRIBER
500
de Bordeaux I,

78.50. —
w, 33.20. Kf, 33.70.3g

16883

16886

351, Cours de Ia Liberation, F-33405 Talence CEDFL;, France

PACS numbers:

/ cm

(Received 9 July 1990)

30

GHz

thatoptical
narrowdetecExperiments
the linewidth.
fluorescenceTheexcitation
clearlyinterest
of were
a pentacenepeaks in ous
prove in
spectrum
observations
of SFS
There has been
much recent
performed
stem infrom
claim is supported
the distribution,
molecules.
single
doped p-terphenyl
width,
envivarious
absorbers
near This
the inhomogeneous
line bycenter
with relatively
of single crystal
tion and spectroscopy
large
of theionspeaks,
as well confined
as by the incorrelation
the SMD,
sudden wedrops
and to
height
(=10 ).lightTo and
achieve
values of
in vacuum
of the
ronments. and
atomic
proceed
Single
NH emitted
GHz
of the
of certain
We attribute
the byhole
surgestraps
molecule. to
burningout ofin alaser
singlewavelength
& 1 to
the NHthese
and peaks.
cooled and
haveemission
been detected
limit
electromagnetic
moving
resultsof show
the feasibility
the optical
a single
molecule
and its localline
environment.
and study
the ofwings
of the
where NH can be
have shown These
interesting
quantum ofjump'
inhomogeneous
a variety
/
For example, if one assumes a
made arbitrarily small.
phenomena, that test our underphoton antibunching
78. 55. Kz,
numbers:
50.—
61.70.Rj
p,
In 42.
Gaussian shape for the inhomogeneous line with a stanmedia,
single
liquid
standing ofPACS
quantum
physics.
dard deviation of 0.02 nm [full width at half maximum
viruses and bacteria and single proteins with multiple
detected using
42 GHz], ' then the SMD spectra reported
been detection
optical absorbing
have
traps
(FWHM)
chromophores
hole burning.
Nondestructive
of single
optical
from thephotons
respectively.
line center
and hydrodynamic
focusing in
techniques,
are detected
which emitted
The rate at ata distance
(iii) observed
centers
such as molecules
a solid would
provide new below were
proin a solid
The detection
to 6-8
deviations.
value ofofthe
at
single absorbersscience
equalmust
N& detector.
As
be standard
the darkThecount
and would
higher than
for both offundamental
applications.
prospects
the line center was approximately 10 -10 in our samnew tool for the study of local
vide an important
~
\a
the
bottleneck
of the
limited
is
the
A molecule could serve in a microphysics
intensity
usually
experiment
as
by
~
~ ~
absorber-host interactions that wouldgO be uncomplicated
ples and thus in the SMD region the simple Gaussian
M~we
~~gT~ ~r~p
~ ~
"macroscopic"
rate of
the maximum
metastable
state T l,-10
average
~
tripletNB=10
addressable
the
a trulythe local
probe
~
~
by
10'
10
to
model
would
over
as
as
It
is
not
.
normal
too
ol
predict
~
~
averaging
many
by
~
0
be the
absorption
large
enough (qr is the inSingle-molecule
detection
the ulti- surprising
field.absorbers.
rr )long' must
(SMD),
light
(qr are
at liquidin a solid
that there
tails on
similar
Furthermore,
inhomogeneous
the triplet lifelimit
in
chemical
in
crossing
mate
be
of
interest
tersystem
might
eA'ectively
analysis,
Sl and
line that extend out furtheryield
than from
helium temperatures the absorbing centers are
predicted
by therTGaussian model,
show no
thereforefundamentally,
the host matrix
ultrasensitive
time). corresponding to more and more improbable,
trapped by
sensors.and More
designing
fluorescence
or recoil
the
demonstrated
and Kador
of eA'ects.
free experiments,
theDoppler
a single Inmolecule,
from the usual highly strained
Recently,sites.Moerner
spectroscopy
D
aba singleaccess
from direct
the problem
detecting
FM spectroscopy
signal
prinquantum
specof the inabsorption
first optical
SMD in theis far
to I Although
wings limited
over of
would
sites, the
provide
averaging
many
sorber in a transparent host is complicated by Rayleigh
the true sensitivity limit in many cases is controlled
ob- ciple,trum
of pentacene (Pc) in a para-terphenyl (p-TP) crysthe distributions of microscopic parameters. Recent
and/or Raman background signals from the large num'
by interfering background signals from residual ampliatoms'
bear
same
on the this,
tal.
Our experiments
using
servation
or
atomic
ions
in
beams
or
of
single
utiber (10' -10'
tude modulation
To
overcome
we system,
) of host molecules or ions in the probing
(RAM).
C
modulated abof doubly
excitation
has revealed
electromagnetic
five abas few asphenomena,
from several
volume. To date,traps
the fluorescence
lized fluorescence
two new techniques:
FM instead
modulation
Stark double
'
(Pc-p- TP) system
been detected
sorption.
77 K
arehasconcealed
on averaging
such
as quantum
ions in a jumps,
crystal atthat
using
and The
sorbing
ultrasound
(FMS)
FM pentacene-p-terphenyl
double
modulation
'' The0
more information
a fixed
Bothextensively
on direct
methods relystudied
authors.
several modulacould be (FMUS).
has been
individuals.
over
ThisFarfascinating
many frequency
physics
by internal
1.a laser.
theabout
excitation
fluorescence
region
spectra
perand the possible
the line shape
of
single absorber
tion Ol
of the
with
an external
field ideally
and a the three
answer
and absorption
of this
to big
molecules
in a solid.
0.05 GHz
system
generalized
02 sites line
~
could be obenvironment p-terphenyl
turbations due to the local
demodulation
remove
RAM.
~
step toA,
~
~
~ ~
different
thick
Curve
crystals.
above
requirements.
and trapping insingle
molecules in a solid are second
Coolingpentacene
gO ~
~
~
absorption
tained if the actual frequency-dependent
The measurements were performed on the inhomo~ ~
~
~
~
of
we
for
~ ol
used
one
not problems,
in contrast
to
the
case
confined
ions.
~ ~
~
The
was
the
basically
optical
setup
~
0
melt-grown
crystalcentershowing
Gaussian
inhomogeneous
be
in a solid couldthe
spectrum of a single absorbing
geneously broadened
O~ (592.32 nm) and Oq (592. 18
EXCITATION
UENC
Y
However,
excitation
a single-mode ring
Our
source,
burning.
recorded. other difficulties arise from the complexity of nm) hole
site zero-phonon
of pentacene
absorptions
origin
bands.
sublimation
flake
bands
andp-terphenyl
presenting
CR-699, single
the system,
instant resoluRadiation
ofof molecular
laser (Coherent
by the dopantdyenarrower
g. , the shift
fine structure
Recent e.
statistical resonances
measurements
molecules
in Bridgman-grown
transihost's
crystals'
broadened
on an inhomogeneously
optical
(SFS)
immersed
helium
at 1.6byK.an electro- Shape
detection
and the
superAuid
of the
dynamics
tion 0.
stabilized
5 MHz) inwas
signal
intensityC,
substructure
dueweak
cooling-induced
presumably
a single molecule's excitation peak at
an alSingle-molecule
provide
in p-terphenyl Here,
tion ofthe
were observedLass
crystalswe use
pentacene
signals (Conoptics,
both The
the red
on II).
D
host background.
fluorescence
against
samples were
optic modulator
a
small
volume
spectrum
a
sublimation
flake.
The
different
single-molecule
detection
ternative
(SMD)
very
The bottom spectrum is approxiscales.
to
of
approach
and
the
blue
the
of
frequency
edge
although
edge
only
Oi
02,
excitation; i.e. , we monitor the emitted intensity as a
prepared from zone-refined p-TP (250 passes). Pc (Al1
I
and spectroscopy. The SFS experiments used a powerresults will be described here. Total
long-wavelength
of
further purification
because
sensitive
was used without
drich)
function
of are
the excitation
This very
frequency. laser
dotslow-background
the narrow
individual
molecules.
Lorentzian
with
The vertical
peaks concentrations
about 12
mately
frequency1 x 10
technique, excitation
and 2x
10
ful,
ranged between
method
can be
in varineeded.
the low
used
to study hole
Bridgman oncrystal was
samples mol jmol,
burning
the
to detect
modulation
and concentration
the measurements
(FM)
only
were Aperformed
spectroscopy,
calibration
an
etalon.
is
in
scale
counts/channel.
spectrum
D,
with
mol/(mol
from 100-200
the meltpmat in10thickness.
a low
Pc), from which
density.coefficient on a frequency scale
grown
cleaved
ations
in optical
the absorption
To prevent
samples
in this system
regime for thermal
less than
drift of
the be
volume However,
in
frequencya host-guest
of
focal
Let
cleaved.
the concentration
us trytheto modulating
could
during acquisition
optimize ideally
samples
29
where NH is the average number
scales
SFSby
as (NH) excitation.
the spectra,
a small
4.2- Pc was reaperture lens
SMD
fluorescence
known accurately
as of
some
the crystal
was0.85
notnumerical
region
within
homogenevolume
one
of
absorbers
in
the
an electromagnetic
probed the interaction with light, the mm jected
focal length
was
to pre(i) In order to increase
The same positioner
crystal served
duringon growth.

Inhomogeneous broadening:
linewidth associated to emission of
MANY fluorophores 
in different local environments

I

~

~

~

~

~

~

~

~

~

~

~

~4

~

I

1

E-EXCITATIOX FREQI EX CY
Ol, g.

FIG.

"

~

~

of

of

~

l

~

FREQ

~

B,

to

of

defects.

FIG. 2.

of

of

I

of

',

of
v;

FWHM

MHz.

E-EXCITATIOX FREQI EX CY

FIG. 1. Ol, g.

of the fluorescence excitation2717
spectra

to rationalize if one assumes
overall detection well-shaped
estimate
enon is ratherto adifficult
single molecule.
smolecule. We
resolve into yield to be
most
of theourdots
(approximately
We also studied the temperature dependence of a few
several molecules, but it can be un
one peak involves
few tenths of a percent, so that the measured signal
peaks. For most molecules we observed a broadening
widths
from
Lorentzian)
The
of
the
peaks.
peaks
range
      a(5000
isolated burn
4 K. In an"hole
which increasedassuming
stood very simply
countss ') is consistent with that from a single
by markedly abovetransient
case, we first saw broadening from 2. 5 to 3 K, then narexcitation
flux was about 25 mW
10
to
15
MHz.
The
it depends
of a single molecule.
4 K.partic
above the
molecule.
broadening on
rowing, followedSince
by renewed
This behavior could be an example of motional narrowflux
71
and
i.
below
the
saturation
e.
mWcm,
,
we assume that this hole burning h
molecule studied,
At high excitation flux, the molecule spends an avering in the optical domain. It also demonstrates the adspectroscopy for singling
vantage of individual-molecule
age time rr "shelved" in the triplet state T~, between
out anomalous cases that would otherwise disappear on
emission sequences when it carries out excitation/
400 averaging.
500
While scanning the excitation across certain peaks, we
emission cycles between So and S~. Therefore, the emisobserved the sudden disappearance of the fluorescence
signal. Most often the emission resumed after an intersion is interrupted by "dark periods"; i.e. photons are
ruption lasting a few seconds. Figure 4 shows an inSuch
emitted in "bunches.
stance of this behavior, where both spectra were taken
cm, an eA'ect can only be obnote photons/
mation-grown
flake. The O~ ofand 02 bands are much
Pc molecules.
of single
are the absorption
emission
of aboutWe5X10
successively on the same resonance peak. This phenomaverage
give an lines
(orrateatirregular
at Thea single
system
quantum
served when
looking (iii)
detected
distri- yield to be
at
the expected
that the
spectrum
presents
detection
overall
enon
estimate
our
is rather difficult to rationalize if one assumes that
We
smolecule.
detector.
of
500
bution.
we of examine
Hereafter,
random
because
the measured signal
overlapping
that arguments
one peak involves several molecules, but it can be undersoof
a percent, several
them),
a few oftenths
very few of"macroscopic"
of
state T
WAVER(. BRIBER / cm
our rate
assertion.
strengthening
GHz
') is consistent with that from a single
"hole burning"
countss
stood very simply by assuming transient
(5000
1st scan
(SMD),
(qr
(qr rr )
and
bunches
blurs
several
quickly
The
scale ofdark
Fig. 2 shows that
frequency
16886 such 16883
of signals
Sl expanded
of a single molecule. Since it depends on the particular
molecule.

Fluorescence from the tiny excited volume was collectlaser beam, the absorption
excitation is spectrum
at variancewillwith width
as this or
hypothesis
focused excitation.
Figure 1, curve 2, shows the broad excitation specgrown crystal with However,
By further reduced by a small parabolic mirror (25 mm diameter, N. A.
ofvolume,
the number
statistical fluctuations
display the measurements
statistical fine
themoleof of
autocorrelation
eiby
um of the O~ and 02 sites of Pc in a melt-grown p-TP
in
the
excited
the
number
molecules
of
ing
fo'
custom-made by Soptel) inside the cryostat
and
This
statistical
cules
at
fine the peaks'
a
absorbing
1,
given
frequency.
establish
needed to
moreat work
structure,
will absorb
ther one or no molecule
a givenis frequency.
ystal at 1.8 K. The nearly Gaussian bands result from
co-workwas
outside the
and
on the of
described
Moerner
structureis then
photomultiplier
by
ir-widths
and their small
of
setThe
of
distributed
narrowlines
of acryostat
ecused
the much nar- (RCA
The spectrum
afirst
width.
peak
sharp
range
superposition
large number
'
Pc-p31034
laseroflight
filtered out
a Schottregularlyers across
A02). Thelines
We
observe
this molecules
inthevalues
could
a inhomogeneous
sample.
resomolecules.
The by
each
wer
homogeneous
singlewas
profile,
favor
of single
incorreareTPstrong
arguments
red-pass
The
autocorrelation
fluorescence
either (like
RG630
function
of
eflect
a few
spectra
glass.
is determinedREVIEW
claim
thatofthe
nce frequency
each 21molecule PHYSICAL
sponding to anin individual
by the LETTERS
isotopic subweak
evenexcitation
perturbations
because molecule.
19 NOVEMBER
1990
NUMBER
very We
VOLUME 65, of
home-made
recorded on aThis
the intensity
tensspectrum
aissublimation
flake excited
of
of thousands
curve
its surroundings.
is illustratedcorrelator,
in
excitation
of Fig.of1, pm
an example
defects inwas
ystal
could remove
neighborhood
oneofC,molecule's
in
stitutions)
Fluorescence
Molecules
Detected byof
a, p-Terphenyl
Crystal
thata most
of accidental
but hundreds
dots of a meltthe excitation
reproducibleof pm
a subli- Excitation
the isolated
spectrum
this, i.ine.with
g. 1, curve
B, byPentacene
forSingle
devised
measurements.
few
light-scattering
broad
or a degeneracy.
an beam
We
bands
are
note
lineswith
Pc molecules.
ation-grown
much
are the grown
of single
absorption
O~ andthe
02 broad
focused
curve The
excitation
excitation.
Figure 1, flake.
crystal
2, shows
By
M. Orrit
and 3. Bernardspecheight. Study
the peakreducThe next useful parameter is further
distri- volume, eithat Scientiftc
theingspectrum
the
expected inirregular
presents
entre National de p-TP
ia Recherche
Universite
et HerztienneC,
etthe
de Bordeaux
Physique
Optique
I,molecules
of
excited
trum ofCentre
and Moleculaire
number
Pc
in a melt-grown
the deO~
02 sites351,
of excitation intensity
function
as the
a
the ofpeak
of
heights
Cours de Ia Liberation, F-33405 Talence CEDFL;, France
several
we
examine
bution.
Hereafter,
arguments
1990) from
result
bands 9 July
ther one or reveals
will saturation'
no molecule
absorb at a given
crystal at 1.8 K. The nearly Gaussian (Received
in the frequency.
range of 100 mW
a clear
cm
WAVER(. a
our assertion.
ir-determinalines distributed
the superposition of BRIBER
The spectrum
a set of sharp
is then
large number of the much nar-strengthening
accurate
an
However,
cm
as
expected.
that narrow peaks in the fluorescence excitation
Experiments clearly prove
of a pentaceneThespectrum
expanded
frequency scale of Fig. 2 shows that
16886
correeach
across
the
The
inhomogeneous
rower homogeneous
lines crystal
of 16883
profile,
single
stem from molecules.
claimresois supported by regularly
the distribution, width,
doped p-terphenyl
single molecules. This
is difficult with our setup.
absolute intensity
tion of
intothewell-shaped
of the dots resolve
(approximately
and height of the peaks, as well as by the correlation of the emitted most
and the sudden drops and
individual
molecule.
Werate
molecule is determined by the lightburning
claim
that bythe
toAtanhigh
nance frequency
ofof each
sponding
30 surges
GHz
is fixed
the intersysthe
emission
the emission of certain peaks. We attribute these to the hole
of a single
molecule.
powers,
The widths
of
the
Lorentzian)
peaks.
peaks
range
, Fig. 1, curve C, isfrom
resultssurroundings.
show the feasibility of the
of a single molecule
environment. spectrum
optical is
study
of
in its
This
illustrated
in and its local
of
an
excitation
example
crystal defects These
temexcitation
yield was
crossing flux
mW the triplet lifetime rT.
25 and
10 to 15 MHz. The
about S~
qT from
i. e. , that
of the
isolated
but reproducible
dotsps (Ref. 9)
the 78.excitation
of a sublimost
this,
PACSby
numbers:
55. Kz, 42. 50.—
70.Rj
Fig. 1, curve B,
p, 61.spectrum
71 mWcm,
fluxvalues
i.
the
e. , below
and rT =45
Thesaturation
published
qr =0.005and

Fluorescence excitation
in dilute samples
"

cm,

"

hole burning.
Nondestructive
optical detection
single absorbing
which emitted photons are
rate
centers such as molecules in a solid would provide new
As
the
must be higher than the dark count
prospects for both fundamental science and applications.
the
is
the
A molecule could serve in a microphysics experiment as
intensity
usually limited by the bottleneck
metastable triplet
a truly local probe addressable by the
l, the maximum average
'
must be large enough
is the inI
absorption
the ultilight field. Single-molecule detection
and rT the triplet lifetersystem crossing yield from
mate limit in chemical analysis, might be
interest in
time).
sensors. More fundamentally,
designing ultrasensitive
resolveexcitation
into well-shaped
most / the dots
molecule spends an averAt high
flux, the (approximately
the
demonstrated
the spectroscopy ofGHz
a single molecule, free from the usual
Recently, Moerner and Kador
GHz
fromT~, between
widths
The
the the
peaks
rangestate
peaks.
specfar wings of the absorption
first optical SMD in the Lorentzian)
averaging over many sites, would provide direct access to
in
triplet
time
age
crys- excitation flux was about 25 mW
trum of pentacene (Pc) in
the distributions of microscopic parameters. Recent ob10a para-terphenyl
to 15 / (p-TP)The
when it carries out excitationtal. Our experiments bear on the sameemission
servation of single atoms' or atomic ions in beams or
system, using sequences
and the emisi. , emission
below
ab- saturation
modulated the
fluorescence excitation instead of doubly
electromagnetic traps has revealed several phenomena,
Therefore,
cycles betweenflux 71and
(Pc-p- TP) system
sorption. The pentacene-p-terphenyl
such as quantum jumps, that are concealed on averaging
i. , photons are
sion is'' interrupted
The
by
over many individuals. This fascinating physics could be
has been extensively studied by several authors.
the three \a
generalized to big molecules in a solid.
Ol and 02 sites of this system answer ideally
Such an eA'ect can only be obemitted
in ~r~p
M~we
~~gT~
500
above requirements.
Cooling and trapping single molecules inol a solid are
at
at
a single quantum system
served
looking
we usedwhen
for
not problems, in contrast to the case of confined ions.
The optical setup was basically the one
~
\a
However, other difficulties arise from the complexity of
hole burning.
Our excitation source, a single-mode ring
random
few
because
overlapping
them),
very
resoluCR-699,
the system, e.g. , the shift of molecular resonances by the
instant
dye laser (Coherent Radiation
D
host's dynamics and the detection of the weak signal
such signals quickly blurs bunches and dark
tion 0. 5 MHz) was intensity stabilized several
by an electroagainst the host background.
optic modulator (Conoptics, Lass II). The samples were
CO Here, we use fluorescence
excitation; i.e. , we monitor the emitted intensity as a
prepared from zone-refined p-TP (250 passes). Pc (Al- '
GHz of
sensitive
drich) was used without further purification because
function of the excitation frequency. This EX
very CY
method can be used to study hole burning in samples
the low concentration needed. A Bridgman crystal/10was
0 mol/(mol Pc), from which
with
low
melt
at
10
the
from
a
optical
density.
grown
FIG. 1. Ol,
region of the fluorescence excitation spectra
Let us try to optimize ideally a host-guest system for
the GHz
concentration in
samples could be cleaved. However,0.05
pentacene
thick
crystals. Curve A, the
SMDinby different
fluorescence p-terphenyl
excitation.
crystal was not known accurately as some Pc was reelt-grown (i)crystal
showing
Gaussian
inhomogeneous
with light,
In order to
increase thetheinteraction
the
served to pre- FREQ UENC Y
EXCITATION
jected during growth. The same crystal
decross section
must be large; narrower
i.e. , the homoands. B,absorption
sublimation
flake o presenting
bands pare
and a powder load for sublimation. A dramatic
CO
\a
crease in the Pc concentration occurred on sublimation.
geneous width yq of the transition must be small. For
bstructure presumably due to cooling-induced defects. C,
FIG.
2.
of aof single
Shape ways
M~wemolecule's
peak at
~~gT~ excitation
~ '~r~p
~
was
with
two
diAerent
achieved
selectively
strong transitions, the radiative width of the zero-phonon
SMD
ol
0
M
of a verymaysmall
volume
ectrum transition
of
a
sublimation
flake.
The
different
is approxiscales.
The
bottom
spectrum
frequency
be comparable to yp and a can take
exciting a small part of the crystal. In the first one,5the
C
valuesnarrow
of the order
of O. l pmpeaks
zero-phonon molecules.
lens (focal about 12 MHz. The vertical
. For of
of a piano-convex
So-Sl
the focusLorentzian
ots are the
crystal sat at mately
individual
excitation
with FWHM
transitions
of organic
insideis in
the counts/channel.
cryostat. The exciting light
length 12 mm)
of anmolecules
etalon. in crystals, the lifescale
spectrum
, calibration
Dcould be focused onto the crystal by adjusting the contime-limited width is usually reached at liquid-helium
temperatures.
vergence of the incoming beam. The waist diameter was
I
(ii) As the overall photon detection efficiencies are
estimated to be about 5 pm. The focal point could be
2717
'
rather low (10 —10 ), each molecule must be able to
moved by slightly changing the angles of incidence on
IGHz
emit as many photons as possible before its resonance CY the lens. In the second method, the crystal was optically
This rules out many phocontacted to the end ofl a single-mode optical fiber either
frequency shifts irreversibly.
I
0
tosensitive
matrices showing
for sublimation flakes or using an index-adapting
directlyspectra
1. Ol, molecules
excitation
the fluorescence
regionand ofamorphous
50
100
0.05 GHz

of

30

~

~

~ ~

~

~

~

~

~

~

~

0

~

~

~

~

~

~

~

~

M~we

~ye

mWcm,
S~.

~

~

~

~

gO

MHz.
e.

cm,

10

molecule studied, we assume that this hole burning has a

rr "shelved"of

I

~

"

~4 "bunches.
~

So
"dark periods";

~

~as ~~~

400

e.

(or

~4

~r~p
of
~~gT~

of

1st scan

I

1

I

~

E-EXCITATIOX FREQI
M

D

~ye

~

~as ~~~

2nd scan

p5

g.

l

0

~

~

CY

~

~ ~

~

~

~

~

~

~

~

~

~

~

gO

~

~

~

~

~

~

~

~

~4

p

ia~Qg~ ~

~

p

0

0

xcitation spectra
E-EXCITATIOX
Curve A, thick '0
g.

FREQI EX

0.05
50
'0
TIME EXCITATION
a
thick

jto

~

~

sP~~ ~™~~~
2nd scan

0 05 GHz

0

I

1

FIG.

~

C

I

p

ia~Qg~ ~

0

EXCITATION
0 05
GHz

sP~~ ~™~~~

FREQUENC Y

C

100

FREQUENC Y
FIG. 4. Two successive EXCITATION
scans of the excitation spectrum

FIG. 4. Two successive scans of the excitation spectrum of a
TIME
a
hole-burning
a photophysical
Curve Physical
single moleculesinglesuggesting
crystals.
A, Society
promolecule suggesting
a photophysical hole-burning
1990 The American
melt-grown
crystal showing the Gaussian inhomogeneous
EXCITATION
UENC Y
FREQ
cess. The sudden intensity falls and surges might arise from
function of the intenautocorrelation
FIG.
3. Time-domain
intenand
falls
sudden
intensity
surges
might
function
the
Time-domain
bands. B, sublimation flake presenting narrower bands and
the flip-flops of a two-level system in the neighborhood of the
sity emitted by a single molecule in the saturated regime. The
substructure presumably
molecule's
due to cooling-induced
at flip-flops
single
Shape of a decay
C,
a scan lasted
08 s and
channel was
molecule.
The time persystem
atthe
short
times, due
shows
bunching peak
photon excitation
neighborhood
in 0.the
a two-level
inFIG.
the2. exponential
regime.
molecule
emitted
a singledefects.
sity
by
spectrum of a very small volume of a sublimation flake. The
different frequency
bottom triplet
spectrum
about I min. The vertical scale is in counts/channel.
state.is approximetastable
in the The
to shelvingscales.
s and a scan
molecule.
time per channel was
showsmolecules.
bunching
dots are the narrow
excitation peaks of individual
photon mately
exponential
Lorentzian withshort
FWHMtimes,
about 12due
MHz. The vertical
scale is in counts/channel.
D, calibration spectrum of an etalon.

jto Y
FREQ UENC
of
cess. The
autocorrelation
wer bands FIG.
and3.
The excitation peak ofat
ed defects. C,
FIG. 2. Shape of saturated
a single molecule's
The
decay
ion flake. The
different frequency scales.at The bottom spectrum is approxiThe vertical scale
about
I
min.
2718
state.
metastable triplet
to shelving in the
dual molecules.
Lorentzian with FWHM about 12 MHz. The vertical
mately
ofinhomogeneous
in different p-terphenyl
pentacene
2716

scale is in counts/channel.

2718

l

2717

arise

0.08

is in counts/channel.

30

     Visualizing

dynamics of molecular motors

Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with
1.5-nm Localization
Ahmet Yildiz et al.
Science 300, 2061 (2003);
DOI: 10.1126/science.1084398

Fig. 3. Stepping traces of three different myosin V molecules displaying 74-nm steps and histogram
(inset) of a total of 32 myosin V’s taking 231 steps. Calculation of the standard deviation of step sizes
can be found (14). Traces are for BR-labeled myosin V unless noted as Cy3 Myosin V. Lower right trace,
see Movie S1.

What about dense 
fluorophore concentra+ons? 
Can we excite nearby 
fluorophores sequen+ally? 

     Fluorescence

inhibition from stimulated emission

S

1

1.0

Absorption

Stimulated Emission

Fluorescence

Fluorescence

0.5

0.0
0

S

0

3

ISTED [GW/cm2]

6

9

STED Microscopy

S.W. Hell & J. Wichmann (1994), Opt. Lett. 19, 780.
y
x
z

x

200 nm

y

Detector

PhaseMod
50 ps

50-200 ps

Depletion
(STED)

Excitation

The stronger the STED beam the narrower the fluorescent spot!

S1
Fluorescence

Absorption

S0

Stimulated Emission

Fluorescence

1.0

0.5

0.0

0

3

6

ISTED [GW/cm2]

9

Heavy subunit of neurofilaments in neuroblastoma

Confocal

STED

G. Donnert, et al. (2006), PNAS 103, 11440.

STED microscopy
- Resolution is not limited by the wavelength of light!
- Resolution just depends on the level of fluorescence depletion.
- Resolution at the molecular scale is possible with visible light and regular lenses!
- Resolution follows a new law; a modification of Abbe’s law:

Fluorescence

1.0

0.5

0.0
0

3

Isat

6

I>>Isat

9
S.W. Hell (2003), Nature Biotech. 21, 1347.
S.W. Hell (2004), Phys. Lett. A 326, 140.
V. Westphal & S.W. Hell (2005), Phys. Rev. Lett. 94, 143903.

Sharpest focal spot
Max Planck
Society

Validation of square-root resolution law
.
V. Westphal, S.W. Hell (2005), Phys. Rev. Lett. 94, 143903.

8

The combination: STED-4Pi-Microscopy
Max Planck
Society

Monolayer

Monolayer

confocal

STED-4Pi
53 nm

M. Dyba, S. W. Hell

Fluorescently tagged microtubuli
with an axial resolution of 50-70 nm
M. Dyba, S. Jakobs, S.W. Hell (2003), Nature Biotechnol. 21, 1303.

Table 1. Fluorophores for STED microscopy.
Fluorophor

Absorption maximum [nm]

Emission maximum [nm]

STED wavelength
[nm]

Additional information

References

Abberior STAR
440 SX
fluorinated rhodamines

437[a]

515[a]

590–620

http://abberior.com

530[b]
524[c]
574[d]
655[b]

590–620
595–615
645–665
740-760

rhodamine derivatives,
uncaging at 360–440 nm

[78, 165, 166] http://abberior.com

Abberior STAR
635
Alexa Fluor 594
Alexa Fluor 488
Atto 425
Atto 532

512[b]
501[c]
552[d]
635[b]

rhodamine derivative

[76, 77] http://abberior.com

590
495
436
532

617
519
484
553

700 and 736
592
532
603



coumarine like
rhodamine like

Atto 565

563

592

650–676

Atto
Atto
Atto
Atto

590
594
633
647N

594
601
629
644

624
627
657
669

700
700
745–750
750–780

rhodamine like,
two-photon excitation
rhodamine like
rhodamine like

carborhodamine

Atto 390
B504-MA
Chromeo 488
Chromeo 494
Coumarin 102
DY-485XL
DY-495
DyLight 594
FITC
JA26
KK114
MG-2p

390
514
488
494
400
485[f]
493[f]
594[f]
485
635
650
632

479
530–540
517
628
480
560[f]
521[f]
615[f]
514
680
670
664

532
592
592
760
532
647
592
700
592
775–781
755
730–750

[54, 167] http://invitrogen.com
[168] http://invitrogen.com
[169] http://atto-tech.com
[41, 72, 169] http://atto-tech.com
[50, 73, 167, 168, 170] http://attotech.com
[73, 167] http://atto-tech.com
[167] http://atto-tech.com
[73, 171] http://atto-tech.com
[50, 67, 157] http://atto-tech.com
[75] http://atto-tech.com
[60]
[168] http://activemotif.com
[93] http://activemotif.com
[75]
[59] http://dyomics.com
[168] http://dyomics.com
[167]
[168]
[70, 172, 173]
[74, 76, 167]
[95, 96]

MR 121 SE
Nile red
NK51
Oregon Green
488
PTCA
Pyridine 4
Pyridine 2
RH 414
TMR-Star

532
552[e]
532
496

700
636[e]
553
526

793
765
647
592

458
550[f]
500[f]
532[e]
554

530–540
770[f]
740[f]
716[e]
580

592
765
765
765
650

YOYO-1
GFP
GFP switchable

491[g]
490
488

509[g]
510
511/515

568, 647
575
595

YFP
Citrine
E2Crimson
TagRFP657
NV diamond

514
516
611
611
532

527
529
646
657
600–850/685

598
592
760
750
775/740

quantum dots

440 (excitation)

580

676

coumarine like
Bodipy like

long stokes shift dye


fluorescein-based

fluorescein- isothiocyanate
xanthene like
rhodamine like
Malachite green activated by L5MG-L90S
oxazine like
phenoxazine

difluorofluorescein
Perylene like


styryl dye
tetramethylrhodamine-derived,
permeable
dimeric cyanine nucleic acid dye

Dronpa-M159T, Padron, on/off at
405 nm


tetrameric
monomeric
fluorescent nitrogen
vacancy centers
Mn-doped ZnSe

[39]
[35] http://invitrogen.com
[59] http://atto-tech.com
[168] http://invitrogen.com
[60]
[35]
[35]
[35] http://invitrogen.com
[92] http://neb.com
[105] http://invitrogen.com
[82]
[63]
[84]
[83, 174]
[85]
[86]
[38, 98]
[97]

[a] in PBS, at pH 7.4, [b] in water, pH 7, [c] at pH7, [d] at pH 7.4, [e] in methanol, [f] in ethanol, [g] dye DNA complexes at pH 7.4. Additional information can
be found in the list of dyes used in STED microscopy at http://www.mpibpc.mpg.de/groups/hell

39

     Two

color STED
Different Fluorophores can be a<ached to different proteins:
studies of correla)ons in posi)ons between proteins
(geometry=func)on)

Fig. 3. Two-channel
STED STED
imaging
byby fluorescence
Fig. 3. Two-channel
imaging
fluorescence lifetimelifetime
separation. separation.
Tubulin and laminTubulin and lamin
were immunostained with ATTO 647N and KK 114, respectively. (A) Raw intensity STED
data and (B) channels decomposed by lifetime separation (green: tubulin, red: lamin).

     Cromophores

Photocromism: Reversible photochemical reac)on where an absorp)on 
band in the visible changes drama)cally in strength or wavelength
examples: cyanide dyes, some fluorescent proteins...

The GFP from A. victoria:
Excitation peak at
λ= 395 nm and 475 nm.
Emission peak at 509 nm

The GFP gene is frequently used as a reporter of expression.
In modified forms it has been used to make biosensors, and many animals have been created that express GFP as a proof-ofconcept that a gene can be expressed throughout a given organism.
The GFP gene can be introduced into organisms and maintained in their genome through breeding, injection with a viral vector,
or cell transformation.

It is a small fusion protein!

A (bright)

1

kfl = τfl-1

kfl

S0

kSTED = σI

γ = στT

T1 1

pA =

1

GSD

1 + γI

1995/2006

S0

τT-1

I
γ = στfl

S1

S1

kexc = σI

kexc
kfl = τfl-1

S0

1 + γI

2002/2005

I

S1

kA = σI

S0

O

-

cis
O

N

O

N

S

trans
kB = τB

-1

S1
cis
N

S
O

O
R

OFF

O

N
N

S
O

1 + γI

2003/2005

Stochastic
single cycle

O
R

A
OFF

OH

trans

O

N

RESOLFT

�=1 ms
Isat =1 W/cm2

I

S1

O

1

R

O

ON

ON

pA =

S0

O

N

S

R

O

O

N

1
OFF

OH

S0

1

2

3

B

4

1 Molecule

ON
Photochemistry

pA = 1–

γ = στB

S1

S0

1

SPEM/
SSIM

1

kfl

S0

�=1 ns
Isat =1 MW/cm2

I

kT
kT =

1994/1999

S0

kT

S0

STED

pA ∞ e - γ I

kexc

T1 k
D = σI

S1

Photophysics

S1

Ensemble

S1

proposed/verified

γ = στfl

kexc

kSTED

Acronyms

B (dark)

PALM
STORM
2006

Time

42

Fig. 3. Bright (A) and dark (B) molecular states used to break the diffraction barrier. Whereas STED, GSD, and SPEM utilize

     PALM

and STORM
Stochastic optical reconstruction microscopy (STORM)
and
Photoativated localization microscopy (PALM)
or
ct
te
De

Stochas)c Ac)va)on 
by photocromism

Localiza)on by 
fluorescence excita)on

or
ct
te
De

Photobleaching

...

BB

B B
B

B B

B
B

B

B

B

1

Diffraction zone λ/2n

AA B B

A A B
B

B

B

B B
B

B

B
B

B

B B

B

B

B

B

A
A

B B
B

B
B

A
A

B B

BB

B
B
B

BB

B B
B

B

A

Stochastic
read-out

... N

2

B

...

AA B B

B B
B

Ensemble (e.g. STED)

1
B B

(dark)

Targeted
read-out
BB

B

∆r <<λ/2

BB

(bright)

... N
B

B

B
B

B

Object
2

B B

A

B B

A

B
B

B
B

...

B
B B
B

A B
B
B

B
B

BB N B
B
B
B B
B
2
B

Single molecule (e.g. PALM)
44

view
using conventional fluorescent proteins such as
     LIMON

fluorochromes.
Image/example: View of a nucleus of a bone
cancer cell: using normal fluorescence
microscopy, it is not possible to distinguish
details of its structure (left). Using 2CLM
/SPDMphymod (right) it is possible to
localize 70,000 histone molecules (red:
RFP-H2A) and 50,000 chromatin remodeling
proteins (green: GPF-Snf2H) - in a field of
2
view of 470 µm with an optical depth of 600
nm (each spot represents a single molecule,
5
total 1,2x10 ).
Spatial molecular distribution and number of
proteins allow conclusions regarding hot

Individual molecules of the same spectral emission color can be detected.
4
Counting individual molecules up to a density of 2,8 • 10 /µm², this is possible in an area of up to 5000
2
2
µm (can be extended to more ca. 125 000 µm ).
− In a wide field of view, several to many million individual molecules of a given type can be localized
using an appropriate instrumental

Super Resolution Microscopy Patent Portfolio of Prof. Christoph Cremer

45

     Inmunofluorescence

and fusion proteins.

Fusion proteins: DNA included in the DNA of other proteins, 
so that the cell generates a combina+on of both.
Different proteins can be targeted with different flourescent 
fusion proteins, for studies of coloca+on (and thus correla+on 
of func+onali+es).  
46

     Summary

- Sub-diffraction optical microscopy opens a new era in science
- The key point is to address sequentially emitters with light:
selecting some measurable property and being able to place the
rest of emitters in states that do not present that property.
- Only a very small number of possibilities have been explored!