Biology Reference
In-Depth Information
A
Specimen
Glass
Objective
Incident light
Reflected light
B
Evanescent field
<100 nm
Specimen, low refractive index
Glass, high refractive index
Reflected light
Incident light
Range of incidence
angles greater than
the critical angle
q
Fig. 3
Total internal reflection fluorescence (TIRF) microscopy. (A) Overview including the inci-
dent and reflected laser light paths within the objective. (B) Once the incident light reaches a medium
with a lower refractive index at an angle greater than the critical angle
y
, the incident light does not
penetrate the specimen, but an electromagnetic field is created that penetrates up to ~100 nm above
the surface, called an evanescent wave. This may excite fluorophores within the range of the evanes-
cent wave.
However, the reflected light generates an electromagnetic field that penetrates
beyond the interface and into the lower refractive index medium as an evanes-
cent wave. This wave, with a wavelength similar to the excitation light beam,
decreases exponentially with the distance into the medium. The penetration
depth of the evanescent wave may be manipulated by changing the angle of
incidence beyond the critical angle, which may be calculated accurately by
knowing the angle of incidence, but will typically be limited to
100 nm or
less (
Cleemann et al., 1997; Mashanov et al.,2003
). This allows for imaging of
Ca
2
รพ
events occurring in close proximity to the plasma membrane of live cells.
However, this does require that the cell is positioned within the evanescent wave
and not above it on the glass surface, which may well be the case for the
majority of the cell if the cell is of a certain size. In our experience, it requires
an experimental e
ort to physically position large cells within the evanescent
wave and yet still maintain physiological conditions for the cell. This is required
because the evanescent wave is generated from the glass surface and not from
the boundary of the cell.
V
