Biomedical Engineering Reference
In-Depth Information
5
Applications of Optical Resonance
to Biological Sensing and Imaging: I. Spectral
Self-Interference Microscopy
M.S. Unlu, A. Yal
.
in,M.Dogan, L. Moiseev, A. Swan, B.B. Goldberg,
and C.R. Cantor
5.1 High-Resolution Fluorescence Imaging
Fluorescence microscopy is an essential tool in modern biological research.
It is a powerful method that allows noninvasive monitoring of specifically
labeled targets within living cells, and simultaneous detection of multiple tar-
gets using different labels. The spatial resolution in fluorescence microscopy is
limited because of the diffraction limit; the resolution in transverse direction
is proportional to
λ
/2
NA
=
λ
/2
n
sin
θ
(where
n
is the refractive index in the
object space, and
θ
is the half-angle of the largest cone of rays that can enter
or leave the optical system), whereas the longitudinal resolution is given by
2
λn
/
NA
2
. High-spatial resolution to detect fluorescent molecules below the
diffraction limit can be achieved in several ways, such as by increasing the
effective numerical aperture (as in 4Pi confocal microscopy) [1], introducing
spatial variation in the excitation light creating finer spatial features in the
image (as in standing wave microscopy) [2], using multiple-photon fluores-
cence absorption or emission mechanisms that lead to nonlinear effects in the
light field (as in 2-photon microscopy) [3], and by selectively quenching the
fluorescence from a focal spot to obtain a very small fluorescing volume (as
in stimulated emission depletion microscopy) [4].
5.2 Self-Interference Imaging
Fluorophores, when immobilized on surfaces, are often quenched because of
mechanisms of energy transfer, standing wave nodes in the excitation field,
and destructive interference in the emission. Fromherz and coworkers noted
that the intensity of the total fluorescence oscillates as a function of the fluo-
rophore height above a reflecting substrate [5]. Their technique, fluorescence
interference contrast microscopy (FLIC), is based on measuring the intensity
of fluorophores located within
∼λ
of vertical distance from a reflecting sur-
face. On the one hand, this proximity to the surface causes the entire emission
