...
the combination means optical nanoscopy for the first time.
| Scientist
involved: |
||
Marcus
Dyba |
3
x 5 = 15 ?
The
setup:
The 4Pi-STED-microscope is the result of combining the two unrelated concepts
of STED- and 4Pi-microscopy
[2]. Here, the fluorescent sample is placed in the common focus of two
opposing lenses, but excitation and detection are performed through a
single lens. The green excitation pulse is immediately
followed by a red STED-pulse,
which enters the focal region through both lenses inducing stimulated
emission of the excited fluorescent molecules to the ground state [10].
Click on image for enlargement!The
combination: To reduce the size of the focal spot, the
PSF of the STED irradiation has to vanish at the center of the focal region,
but must be high elsewhere, as we have demonstrated in our STED-microscope.
This can be accomplished by employing a standing wave. A planar standing
wave, however, exhibits many minima in which fluorescence would still
be present. To create a single minimum, we make use of the large focusing
angle and symmetry of the 4Pi-microscope [10].
From
λ/3 to λ/23: The "diffraction barrier"
of about λ/3 realized by Ernst Abbe
in 1873 is no longer limiting the resolution in light microscopy. In our
experiments, we have shown spots of excited molecules of 33 nm width with
focused light at 760 nm. These sub-diffraction spots enable for the first
time far-field microscopy with resolution at the tens of nanometer scale
[10].
Biological
image:
The
upper figure shows a standard confocal xz-image of membrane labeled bacillus
megaterium. In comparison to the STED-4Pi-counterpart shown on the
picture below, the figure reveals the vastly improved axial resolution,
but also exhibits the side-lobe effects due to incomplete supression of
the side minima. Fortunately, the effect of the lobes can be dealt with
mathematical filtering which is shown in the lower image [10].
Click
on image for enlargement!
Hell,
S. W. (1997). "Increasing the Resolution of Far-Field Fluorescence
Microscopy by Point-Spread-Function Engineering." Topics
In Fluorescence Spectroscopy; 5: Nonlinear and
Two-Photon-Induced Fluorescence, edited by J. Lakowicz. Plenum
Press, New York: 361-426. |
|
Dyba,
M. and S. W. Hell (2002). "Focal spots of size λ/23
open up far-field fluorescence microscopy at 33 nm axial resolution."
Phys. Rev. Lett. 88: 163901. |
|