2009 APS March Meeting
Volume 54, Number 1
Monday–Friday, March 16–20, 2009;
Pittsburgh, Pennsylvania
Session H7: Cellular Imaging at the Nanometer Scale
8:00 AM–11:00 AM,
Tuesday, March 17, 2009
Room: 407
Sponsoring
Unit:
DBP
Chair: K.C. Huang, Stanford University
Abstract ID: BAPS.2009.MAR.H7.2
Abstract: H7.00002 : Far-Field Fluorescence Nanoscopy
8:36 AM–9:12 AM
Preview Abstract
Abstract
Author:
Stefan Hell
(Max Planck Institute for Biophysical Chemistry/ Dep. of NanoBiophotonics)
The resolution of a far-field optical microscopy is usually
limited to
$d=\lambda \mathord{\left/ {\vphantom {\lambda {\left( {2\,n\sin
\alpha }
\right)}}} \right. \kern-\nulldelimiterspace} {\left( {2\,n\sin
\alpha }
\right)} \quad >$ 200 nm, with $n\sin \alpha $ denoting the
numerical aperture
of the lens and $\lambda $ the wavelength of light. While the
diffraction
barrier has prompted the invention of electron, scanning probe,
and x-ray
microscopy, the 3D-imaging of the interior of (live) cells
requires the use
of focused visible light.
I will discuss new developments of optical microscopy that I
anticipate to
have a lasting impact on our understanding of living matter.
Emphasis will
be placed on physical concepts that have overcome the diffraction
barrier in
far-field fluorescence microscopy. To set the scene for future
directions, I
will show that all these concepts share a common strategy:
exploiting
selected states and transitions of the fluorescent marker to
neutralize the
limiting role of diffraction.
The first viable concept of this kind was Stimulated Emission
Depletion
(STED) microscopy where the spot diameter follows$d\approx \lambda
\mathord{\left/ {\vphantom {\lambda {\left( {2\,n\sin \alpha
\sqrt {1+I
\mathord{\left/ {\vphantom {I {I_s }}} \right.
\kern-\nulldelimiterspace}
{I_s }} } \right)}}} \right. \kern-\nulldelimiterspace} {\left(
{2\,n\sin
\alpha \sqrt {1+I \mathord{\left/ {\vphantom {I {I_s }}} \right.
\kern-\nulldelimiterspace} {I_s }} } \right)}$; $I \mathord{\left/
{\vphantom {I {I_s }}} \right. \kern-\nulldelimiterspace} {I_s
}$is a
measure of the strength with which the molecule is send from the
fluorescent
state to the dark ground state. For $I \mathord{\left/ {\vphantom
{I {I_s
}}} \right. \kern-\nulldelimiterspace} {I_s }\to \infty $ it
follows that
$d\to 0$, meaning that the resolution that can, in principle, be
molecular.
The concept underlying STED microscopy can be expanded by
employing other
transitions that shuffle the molecule between a dark and a bright
state,
such as (i) shelving the fluorophore in a dark triplet state, and
(ii)
photoswitching between a `fluorescence activated' and a
`fluorescence
deactivated' conformational state. Examples for the latter include
photochromic organic compounds, and fluorescent proteins which
undergo a
cis-trans photoisomerizations.
Photoswitching provides ultrahigh resolution at ultralow light
levels.
Switching can be performed in an ensemble or individually in
which case the
image is assembled molecule by molecule at high resolution. By
providing
molecular markers with the appropriate transitions, synthetic
organic
chemistry and protein biotechnology plays a key role in this
endeavor.
Besides being a fascinating development in physics, far-field
optical
``nanoscopy'' is highly relevant to the life sciences. In fact,
it has already
been a key to answering important questions in biology [1, 2].
Due to its
simplicity and improving performance, I expect far-field optical
nanoscopy
to enter virtually every cell biology laboratory in the near future.
\\[4pt]
[1] S. W. Hell, Far-field optical nanoscopy, \textit{Science} 316
(2007) 1153.
\\[0pt]
[2] Westphal, V., S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R.
Jahn, S. W.
Hell, Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic
Vesicle Movement, Science (2008) DOI: 10.1126/science.1154228.
To cite this abstract, use the following reference: http://meetings.aps.org/link/BAPS.2009.MAR.H7.2