Chemistry Reference
In-Depth Information
molecular motor propelling a cytoskeletal
filament on the surface of a microscope
slide. In order to image single
uorescent probes and tomeasure parameters that are
relevant to the functional mechanism of the target molecule, the detector needs to be
sensitive enough to respond to the limited
fluorescence emission from the probe and
the intensity of
fluorescence from non-speci
c sources and other sources of noise
must be minimized.
To keep the intensity of extraneous background below the
uorescence of the target
molecule, several approaches are used. Samples prepared for in vitro work are
assembled onto very clean microscope slides [10] in a clean environment and with
reagents
ltered to remove dust and aggregates. Once the sample is placed into a glass
microscope slide
flow cell for microscopy and sealed, the cleanliness of the micro-
scope itself is not as critical. The sample volume in which
fluorescence is detected is
minimized to reduce background
fluorescence from outside that region. A common
way of achieving a shallow excitation volume at the sample surface is termed Total
Internal Re
ection Fluorescence (TIRF) microscopy [11]. When the
uorescence
excitation, typically a laser beam, is directed at a very glancing angle toward the
interface between the aqueous medium and a glass or fused silica microscope slide,
all of the light is re
ected there and no energy is propagated into the sample chamber.
Due to the boundary conditions in Maxwells equations for electromagnetism that
require continuity of the electric
field (more properly, the electrical displacement
vector), a non-propagating, oscillating electromagnetic
field is generated at the
interface and only extends a few hundred nanometers into the aqueous medium.
This so-called evanescent wave will excite
uorescent probes that are at or near the
surface. Its limited reach into the rest of the
ow chamber, however, prevents
excitation of any non-bound sample molecules or contaminants, thus keeping the
background intensity very low. The
first report of the optical detection of single
myosin molecules used TIRF microscopy [3].
The
fluorescent detection volume is also highly restricted in confocal microscopy
[12], and confocal imaging of singlemolecules diffusing in solution has been reported
[13
-
16]. For molecules that are attached to a substrate and for those that exhibit lateral
motions (motor proteins), the evanescent wave at the slide surface is ideal. Further
characteristics of evanescent waves are described later.
Figure 3.1 shows two optical arrangements used for imaging single molecules by
TIRF microscopy. In an objective-type total internal re
ection
fluorescence micro-
scope (Figure 3.1A), a carefully collimated laser beam is projected by lens L1 and
mirror M1 onto the back focal plane of the microscope objective. L1 is located one
focal length away from the back focal plane and generates a beam waist there. The
microscope objective is designed with a high converging power. Its numerical
aperture, NA
o
¼
n
1
sin(
q
max
), where n
1
is the index of refraction of the cover slip
(typically n
g
¼
q
max
is the angle, relative to the optical axis, that the
objective lens refracts rays entering at the margin of its aperture. Microscope
objectives, designed for this purpose, with numerical apertures from 1.45 to 1.63
are produced by several manufacturers. The input light is recollimated by the
objective so that the rays of the exciting beam are parallel to each other at the sample
plane. When themirrorM1 is translated in the direction shown by the double-headed
1.515 for glass) and
