FLUORESCENCE IMAGING
I. Fluorescence-imaging with diffraction limited spots
The resolution in optical microscopy has been hampered by the smallest spot
possible (~/2) that can be achieved by conventional methods.
y

x
Sample region
with fluorescent
labeled
molecules
Spatial distribution
of the excitation
Diffraction-limited
beam
Spot size
Fig.1. Excitation with a diffraction limited beam. The minimum area that can
be excited is determined by the ability to create a spot with the minimum size.
II. High resolution fluorescence-imaging
A key aspect is to find a way to reduce the number of fluorescently labeled
molecules that are excited simultaneously. Here we mentioned a couple of
methods that have been successfully applied.
II.1 Stimulated Emission depletion (STED)
In this approach, the effective size of the exciting beam is reduced by
quenching (reducing) the fluorescence emission of the fluorophores
located in the periphery of the excitation beam with a doughnut-shaped
beam.
Fig.2 Experimental setup (left) for creating a doughnut shape beam Right)
for quenching the fluorophore emission.1
Although the application of a laser to quench the fluorescence may appear
counterintuitive, this is indeed what happens (as will be explained below.)
As a result, the effective area of excitation is reduced. “The result is akin
to sharpening a pencil to draw finer lines. By scanning the ‘sharpened
spot over the sample, an image Is built pixel by pixel, with a resolution
currently down to 20 nm.” [Ref. F. Pinaud and M. Dahan, 2008]
Quenched
molecules
Fluorescence emission
Fig.3 At the focal point, the effective excitation beam is much
narrower than lambda.2 A key feature in STED is that, it turns
out, the effective quenching process depends non-linearly with
the intensity of the quenching beam.
SHATTERING the DIFFRACTION LIMIT of LIGHT
I. STRATEGIES
IA. Volumetric-shaping of the excitation light
Most recent superresolution methods rely on the volumetric shaping of the
excitation light (through a near-field aperture tip; periodic light gradients
generated by interference, as in 4PI.)
IB. Exploiting non-linear light-matter interactions
More specifically, using a nonlinear relationship between the excitation
and the fluorescence emission
IB.1 Non-resonant Processes: Two- and multiphoton excitations
But they suffer from two main drawbacks.
a) Fluorophores that emit in the visible require multiphoton excitation
with doubled or tripled wavelength, which results in the doubling or
tripling of the extent of the excitation spot (hence spoiling the
resolution gained by the non liner process.)
b) Multi-photon excitations are higher-order non-resonant processes.
Consequently, their absorption cross sections are many orders of
magnitude smaller than that of the linear one-photon process,
which forces the use of intense short laser pulses for efficient
excitation. The latter are phototoxic to the cell because they
accelerate radical production, thereby limiting the available
observation time before cell damage.
IB.2 Resonant processes:
Exploit spectroscopic properties of fluorophores to produce
nonlinearities of large cross sections.
Previously fluorescence has been treated as a linear process. But
Hell’s group has been looking for ways to exploit the spectroscopic
properties of fuorophores to produce non-linearities. This effort has
led to the Stimilated Emission Depletion (STED).
From reference: S. Weiss, “Shattering the diffraction limit of light,” PNAS 97,
8747 (2000)
II. STIMULATED EMISSION DEPLETION (STED):
Exploiting non-lnearities in resonant processes
In 1994 Jan Wichmann and Hell published a theoretical paper on STED,
outlining a concept to eliminate the resolution-limiting effect of diffraction
without eliminating diffraction itself.
Reference: Stefan W. Hell and Jan Wichmann, “Breaking the diffraction resolution
limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,”
OPTICS LETTERS 19, 780 (1994).
STED exploit the selective quenching :
a) Excitation process
A short pulse 200 fs and exc~ 560 nm excites the fluorophores from S0 into
high vibrational states S1 vib.
y
Spatial distribution
of the excitation
beam
x
0.2 ps
pulse
Time
S1vib
S1
Sovib
So
Fig.1 Spatial (left) and temporal (center) distribution of the excitation
beam. The diagram on the right side displays the energy levels of a
typical fluorophore. S0 and S1 are the ground and first excited singlet
states, respectively. S0vib , and S1 vib are higher vibrational levels of these
states. The excitation of the dye takes place from the relaxed state S0 to
the states S1 vib.
The intensity distribution of the excitation beam in the focal plane of the
lens is determined by diffraction effects.
2
I exc (r ) ~ J1 (r ) / r
( Joules per unit area per unit time.)
where r  ( x 2  y 2 )1/ 2 , and J1 is the first order Bessel function.
The spatial extent of I exc (r ) determines the resolution of the microscope.
The efficiency of absorption is characterized by the absorption cross
section coefficient  01 .
 01 ~ in the 10-16 to 10-17 cm2 range
If no is the population of the state S0, the temporal population change due
to stimulated absorption is given by,
dno
 no  01I exc / 
dt
b) Relaxation processes
Vibrational relaxations S1 vib  S1 occurs in ~ 1 to 5 ps.
Fluorescence by radiative transitions occurs due to transitions S1  Sovib ;
with an average fluorescence lifetime  fluor ~ 2 n ps.
This is three orders of magnitude slower than the vibrational transitions.
S1vib
S1
ps
S1vib
S1
S1vib
S1
ns
So
vib
So
So
vib
Sovib
So
So
ps
The transition from S1 to Sovib can also be induced by stimulated emission,
which is
of particular interest here. The transitions Svib
1 ! S1 and
Svib
0 ! S0 are vibrational relaxations. In the discussion of
stimulated emission we can ignore the triplet state. Detailed
reviews of dye properties are given by Lakowicz [12] and
Sch¨afer [13].
Figure 1 also displays
Iexc = 1300 MW/cm2.
I exc (r ) / 
photons per unit are per second,
quantifies the probability that an
excitation photon arrives at r.
Stimulated emission is the basis of laser action and
one of the most widely applied physical phenomena. First
reports of stimulated emission in organic fluorophores go
back to Sorokin and Lankard [5] and to Sch¨afer and coworkers
[6], who pioneered the development of the dye
laser. The operational requirements in a laser are somewhat
different than those for depletion of fluorescence. In a laser,
the role of stimulated emission is to strengthen the beam
by collecting stimulated photons, whereas in microscopy
one is primarily interested in the depletion of the excited
state by stimulated emission, irrespective of the population
of the excited state.
When analyzing a sample containing organic fluorophores, STED exploits
a) the depletion of a molecular fluorescent state through stimulated
emission, and
b) the fact that the product if two point-spread-functions (PSF) is narrower
than a single PSF.
The first aspect is to it was experimentally demonstrated in 1999.
References:
Thomas A. Klar and Stefan W. Hell, “Subdiffraction resolution in far-field
fluorescence microscopy,” OPTICS LETTERS. 24, 954, 1999.
Thomas A. Klar, Stefan Jakobs, Marcus Dyba, Alexander Egner, and Stefan
W. Hellt, „Fluorescence microscopy with diffraction resolution barrier roken
bty stimulated emission,“ PNAS 97, 8206 (2000).
Thomas A. Klar, Egbert Engel, and Stefan W. Hell,“ Breaking Abbe’s diffraction resolution limit
in fluorescence microscopy with stimulated emission depletion beams of various shapes,”
Phys. Rev. 64, 066613 (2001).
2 Fabien Pinaud and Maxime Dahan, “Zooming Into Live Cells,” Science 320, 187 (2008).
1