Chemistry Reference
In-Depth Information
isolation of individual molecules is necessary. The background
fluorescence from
out-of-focus regions prevents optical isolation, unless a pinhole is used on the
detection path. The
fluorescence, after passing through DM, is focused by another
lens onto a pinhole, which excludes
fluorescence from regions outside the focus. By
choosing a pinhole of similar size to the focus (multiplied by the magni
cation of the
objective),
fluorescence from regions down to 0.2
-
1.0
can be isolated; or, on
surfaces,
fluorescence from areas down to
m
2
can be isolated. This excita-
tion/optical isolation format may be used either at glass
0.1
m
water interfaces, in aqueous
solutions, or in living cells. The primary drawback of this approach is that only one
point is being observed at a time. This can slow down acquisition considerably,
especially in cases where triggered chemical reactions are occurring, and the
behavior of eachmolecule during the entire chemical reaction needs to bemonitored.
Two-photon excitation of the
fluorophores is the non-linear absorption of two
photons, exciting the
uorophore fromS
0
toS
1
. Fast, powerful IR lasers are required to
excite in this way, but there are advantages to this technique. The non-linearity of the
excitation prevents excitation outside the focus; the excitation rate is proportional to
the square of the laser intensity, which rapidly drops to negligible levels outside the
focus.Drawbacksof thismethod include intense laser intensities andphotobleaching.
In order to obtain information about the sample in different regions, either to form
an image or to search for immobilized single molecules, the sample is generally
scanned using a piezo-actuated microscope stage. This changes the position of the
confocal detection volume in the sample, allowing measurements of different
regions of the sample.
-
High concentration A second limitation of the confocal geometry is the size. In order
to isolate single molecules with a 1.0-
volume, sub-nanomolar concentrations must
be used. For intramolecular studies, where the internal dynamics of a protein are
being studied, this is not a problem. For example, protein folding studies are not
adversely affected by diluting the sample down to sub-nanomolar concentrations.
Even if the effects of molecular crowding are being studied, this is not a problem,
since unlabeled proteinmay be present at much higher concentrations. However, for
studies of functional proteins that interact with other labeled components, dilution
can adversely affect the function. At the low concentrations, the labeled proteins may
not even be bound to each other.
This creates a need for a smaller detection volume than can be achieved using
standard confocal optics. Several methods have been developed, but these have not
yet become commonplace. These are important for in vitro measurements at
physiological concentrations, as well as potential cell-based and in vivo measure-
ments. For such measurements, in vivo labeling strategies will also be needed.
One simple method of achieving this is to use total internal re
ection (TIR) with
confocal detection, reducing the volume 10-fold [31]. More recently, zero mode
waveguides [32], stimulated emission depletion (STED) [33], and supercritical angle
fluorescence (SAF) [34] have all been demonstrated. Zero mode waveguides have the
smallest volumes, but require the presence of metallic coated surfaces. STED
provides very small volumes that can be focused in the far-
eld, even inside cells.
