Chemistry Reference
In-Depth Information
However, these methods are limited to low numbers of dimensions (two or three).
This precludes their use in analyzing many multi-dimensional single-molecule
observables.
9.3.5.5 Analysis of Histograms of Single Molecules
The analysis of histograms of single molecules can be very similar to the histogram
analysis just described. The axes of the histograms may even be the same. The
difference is what is counted in the histogram. Whereas the histograms above
counted time intervals or photon pairs, here we count identi
ed molecules.
For each molecule, several quantities are calculated, revealing information about
the molecule. For example, for alternating laser experiments, two important quanti-
ties may be calculated: Alternating excitation recovers distinct emission signatures
(Figure 9.3A) by calculating two
fluorescence ratios: the FRET ef
ciency E which
reports on donor
-
acceptor distance [66
-
68], and the distance-independent ratio S
which reports on the donor
-
acceptor stoichiometry:
F
A
em
D
exc
F
A
em
D
exc
F
D
em
D
exc
E
¼
=ð
þ g
Þ
S
¼
F
D
exc
=ð
F
D
exc
þ
F
A
exc
Þ
where F
D
em
D
exc
is the D-excitation-based D-emission,F
A
em
D
exc
is the D-excitation-based
A-emission, F
D
exc
is a sum of D-excitation based emissions, F
A
exc
is a sum of
A-excitation based emissions (Figure 9.2B), and
is a detection-correction factor
[68
-
70]; all emissions refer to single molecules. In addition to E and S, each burst can
be tabulated by the number of its photon counts (giving rise to a histogram of burst
size distribution), its duration (giving rise to a histogram of burst duration distribu-
tion), its polarization, and its lifetime
g
ning a six-dimensional space.
Similar quantities and multi-dimensional histograms can be calculated using
multiparameter
fluorescence detection (MFD) [43].
already de
-
9.3.5.6 Single-molecule Sorting
Stoichiometry S provides information even without close proximity between
uor-
ophores; it allows thermodynamic and kinetic analysis of interactions, identi
cation
of interaction stoichiometry, and study of local environment (as detected by changes
in
fluorophore brightness). Combination of E and S on two-dimensional histograms
(Figure 9.3A) allows virtual molecule sorting [71]; we de
ne this analysis as
Fluorescence-Aided Molecule Sorting (FAMS), and its implementation using alter-
nating-laser excitation as ALEX-FAMS. When applied on diffusingmolecules, ALEX-
FAMS is a homogeneous, mix-and-read assay, in which interacting species are
combined and optical readouts report simultaneously on their association status and
conformational status.
This sorting procedure allows the observer to determine to the number of sub-
poupulationsand theirmeandistanceaswell as thedistancedistribution. Thedistance
distribution is time averaged over the
1-ms observation time for eachmolecule, and
there is shot noise. Hence the distance distribution has limited resolution [72].
Single-molecule sorting may also be used as just that
-
a way to sort molecules into
different subpopulations for further study. Generally, the molecules switch states
