Chemistry Reference
In-Depth Information
1
A Road Map to Single Molecule Dynamics
Yoshiharu Ishii
1.1
Visualization of Single Molecules
Signals from single
fluorescent dye molecules were
first measured in non-biological
environments. Some of the earliest studies acquired signals fromsingle
uorescence
dyes embedded in a solid matrix at the temperature of liquid helium [1] and from
excitation
fluorescence signals [2]. In early studies carried out at room temperature,
single
fluorescence dye molecules spread over a
film were observed using near-
eld
scanning
fluorescence microscopy [3].
In a biological environment where
fluorescence is largely quenched by the
surrounding water molecules, single molecule imaging was
first accomplished in
1995 using total internal re
ected
fluorescence (TIRF) and epi-
fluorescence micros-
copy [4] (see Chapter 2). Single
fluorophores attached to myosin molecules were
visualized in aqueous solution in the vicinity of a glass surface while single ATP
turnover from the same myosin molecule was measured using
uorescently-labeled
ATP. TIRF microscopy has been widely used for single molecule imaging. Other
studies have measured single
fluorophores bound to biomolecules in solution while
passing through a
fixed small volume using confocal microscopy [5].
1.2
Single Molecule Position Tracking
The motion of biomolecules has been tracked by following the visualization of
immobilized single biomolecules. The motion of processive motors has been
visualized by using large particles or by attaching if
uorescent dyes to the motor.
In 1996, the smooth and processive motion of a molecular motor, kinesin attached to
a single
fluorescence dye was detected along microtubules immobilized on a glass
surface using TIRFmicroscopy [6]. Epi- and TIRFmicroscopy were used to monitor
the 2Ddiffusion of
uorescently-labeled phospholipids on phospholipidmembranes
