Chemistry Reference
In-Depth Information
2.1.2
Single Molecule Imaging and Nano-Detection
To fully comprehend molecular machines, it is necessary to understand the dynamic
properties of biomolecules themselves and their interactions with each other. Single
molecule detection (SMD) techniques have been developed to directly monitor the
dynamics of biomolecules and molecular machines. SMD techniques are based on
two key technologies: single-molecule imaging and single-molecule nanomanipula-
tion. The size of biomolecules and even their assemblies are in the order of
nanometers, so they are too small to observe by optical microscopy. To overcome
this problem, biomolecules can be
fluorescently labeled and visualized using
fluorescence microscopy. Single
fluorophores have been observed in non-aqueous
conditions [1] (see Chapter 1). In 1995, we successfully demonstrated that single
fluorophores can be seen in aqueous solution by using total internal re
ection
fluorescence microscopy (TIRFM) and conventional inverted
fluorescence micros-
copy [2]. The major problem to overcome when visualizing single
fluorophores in
aqueous solution is the huge background noise caused by numerous sources
including Raman scattering from water molecules, incident light despite
filters,
luminescence arising from the objective lens, immersion oil and dust, and the
instability of the
uorophores. In our system, the evanescent
field was formed when
the laser beam was totally re
ected by the interface between the solution and the
glass. The evanescent
field was not restricted to the diffraction limit of light, thus it
could be localized close to the glass surface which resulted in a penetration depth
(
150 nm) being several-fold shorter than the wavelength of light. Therefore, the
illumination was restricted to
fluorophores either bound to the glass surface or
located close by, thereby reducing the background light. Furthermore, by careful
selection of optical elements, the background noise could be reduced by 2000-fold
compared to that of conventional
fluorescence microscopy. By adding an oxygen
scavenger system, the instability of the
fluorophores was signi
cantly reduced. This
made it possible to clearly observe single
fluorophores in aqueous solution. Fluores-
cence measurements from single
fluorophores attached to biomolecules and ligands
have allowed the detection of, for example, the movements [3], enzymatic reac-
tions [2, 4
-
7], protein-DNA interactions [8] and cell-signal processes [9] at the single
molecule level. The single molecule imaging technique has been further extended to
detect detailed reactions of biomolecules coupled with (1) detection of position with
nanometer accuracy by computer image analysis (FIONA) [10]; (2) distance between
two
fluorophores with sub-nanometer accuracy by
fluorescence resonance transfer
(FRET) [11]; and (3) orientation by
fluorescence polarization [12] or DOPI [13] (see
Chapter 1).
The second key technology is single-molecule nanomanipulation. Biomolecules
and even single molecules can be captured on a glass needle [14
-
17] or on beads
trapped by optical tweezers [18, 19]. Optical tweezers are the tools used to trap and
manipulate particles of between 25 nm and 25mm in diameter using the force of the
laser radiation pressure [20]. The particle is trapped near the focus of the laser light
when focused by a microscope objective with a high numerical aperture. The optical
