Biology Reference
In-Depth Information
Fluorescently labeled receptors tagged with Halo7 can be labeled at high efficiency
(
80%) (
Suzuki et al., 2012
). To maintain receptor functions, some linkers with
lengths between 15 and 21 amino acids should be inserted between Halo7-tag and re-
ceptors. Because the fluorophore-conjugated Halo ligand is often membrane-
permeable if the fluorophore molecules are membrane-permeable, Halo7-tag at the cy-
toplasmic site of receptors can also be labeled with membrane-permeable fluorescent
ligands. The receptors are labeled with fluorescent halo ligands according to manufac-
turer instructions (Promega), but for single-molecule imaging, cells are incubated with
50 nM Halo ligand conjugated with fluorophores for 15 min to label the extracellular
Halo7 of receptors and for 60 min to label cytoplasmic Halo7 (
Suzuki et al., 2012
).
Under these conditions, more than 80% of the Halo7-tag can be fluorescently labeled.
ACP-tag (molecular weight
>
8 kDa) is a small protein tag based on ACP from
Escherichia coli
. ACP-tag can be enzymatically conjugated with fluorophores using
the substrates derived from coenzyme A (CoA). Because CoA and fluorescent CoA
substrates are membrane-impermeable, receptors tagged with ACP at its extracellular
surface can be fluorescently labeled. More than 95% of ACP tagged at the extracellular
site of receptors can be labeled with fluorophores (
Meyer et al., 2006
). The receptors
ar
e labeled with fluorescent ACP ligand according to manufacturer instructions (New
England Biolabs), but note that for single-molecule imaging, cells are incubated with
50 nM ACP ligand conjugated with fluorophores for 15 min (
Suzuki et al., 2012
).
¼
20.2
SINGLE-MOLECULE IMAGING
Single-fluorescent-molecule tracking is performed using an objective lens-type
TIRF microscope (
Tokunaga, Imamoto, & Sakata-Sogawa, 2008; Tokunaga,
Kitamura, Saito, Iwane, & Yanagida, 1997
) constructed on an inverted microscope.
Figure 20.3
shows a schematic diagram of the TIRF microscope we built to perform
single- or dual-color imaging at the level of single molecules. We used an Olympus IX-
70 microscope as the base with a modified mirror turret to allow side entry of the ex-
citation laser beams to the microscope. A laser beam attenuated with neutral density
filters and circularly polarized by a quarter-wave plate is expanded by two lenses (L1,
f
80 mm for
594 nm excitation), focused at the back focal plane of the objective lens with an L3
lens (
f
¼
15 mm; L2,
f
¼
150 mm for 488 nm excitation or L1,
f
¼
20 mm; L2,
f
¼
350 mm), and steered onto the microscope at the edge of an objective lens
with a high numerical aperture (Plan Apo 100
¼
¼
1.49; Olympus).
A single- or dual-color dichroic mirror (Chroma Technology) is also used.
The laser beam is totally internally reflected at the coverslip-medium interface,
and an evanescent field (1/e penetration depth
; numerical aperture
100 nm) is formed on the surface
of the coverslip. The basal membrane is locally illuminated with this evanescent
field. The emission from the fluorophore is collected by the objective and passes
the single- or dual-color excitation path dichroic. For dual-color observation, the
emission is split into two imaging systems by an observation path dichroic mirror.
¼
