Biomedical Engineering Reference
In-Depth Information
partly determines the signal-to-noise ratio of the measurements. While SHG does not involve absorp-
tion of photons and therefore are not restricted by the matching of energy of photons (i.e., wavelength)
to the energy gaps between the ground states and the excited states of the chromophores, it is enhanced
around the wavelength that induces absorption. In addition to this, basal interactions of chromophores
with photons, local electrical fields generated by the plasma membrane heavily influence the molecules
that are inserted in the plasma membrane [4]. When neurons fire action potential, for example, the
membrane potential changes from the resting state of ~−65 mV to the peak depolarized point with a
magnitude of ~+100 mV. This change in voltage is huge considering the thickness of the plasma mem-
brane being <10 nm, which gives rise to >10
7
V/m at the plasma membrane. Under these conditions,
several things are expected to occur for these chromophores inside the plasma membrane. First, hyper-
polarizability of the chromophores changes under different electrical fields, which alters the hyperscat-
tering of light by the chromophore array. This, so-called electrooptic mechanism is what is considered
to be the main mechanism by which membrane potentials are measured by SHG imaging [15]. This is an
instantaneous phenomenon and therefore there is no delay between actual membrane potential changes
and SHG signal changes, which is crucial in measuring fast membrane potential fluctuations. In con-
trast, membrane potential changes may induce slow changes in chromophores by altering their distri-
bution, especially between those on the plasma membrane and those outside the plasma membrane [4].
Obviously, changes in dye concentration at the plasma membrane affect the SHG photons generated by
the same intensity of laser, but this redistribution of chromophores takes time and is not instantaneous
and therefore, not suitable for measuring the fast membrane potential changes.
Another factor to consider is the balance between membrane affinity and solubility in the aqueous
solution. Many chromophores, especially those designed to stain plasma membranes, are hydrophobic
in nature. These chromophores can be dissolved in a nonpolar medium such as DMSO and be applied
to the samples. However, biological samples are sensitive to these media and physiological properties of
cells can be easily affected by such solvents. In this regard, chromophores should be soluble in aqueous
solution under physiological ionic strength. To suffice these two contradictory requirements, amphiphilic
chromophores have been chosen for SHG detection at the plasma membrane. These chromophores insert
themselves into the plasma membrane with their nonpolar tail group while leaving the charged head
group outside the membrane. Due to this charge group, they cannot freely pass through or flip inside the
plasma membrane and thus when applied from one side of the plasma membrane, they accumulate on a
single side of membrane leaflet in a highly ordered fashion, sufficing the requirement of SHG.
10.4 experimental Setup
10.4.1 General Description of the SHG Setup
As the SHG is a two-photon phenomenon, it requires two-photon microscopy setup, as described in
detail in Figure 10.1 and [18]. One thing that is special about SHG setup is that due to the forward
propagating nature of SHG, detectors on the forward path are also required. The best wavelength of
femtosecond laser for membrane potential measurement by SHG depends on the chromophores to be
used in the experiment. Things to consider in determining the wavelength include basal SHG signal
strength, membrane potential sensitivity, and phototoxicity. We have empirical evidence to suggest that
wavelength around 1000 nm works well in these aspects for our experiments using FM4-64 as a dye.
The laser power is then modulated by acoustooptical modulator, Pockels cell, or by other means before
entering the microscope. The cells loaded with SHG chromophores will then be illuminated by the laser
in the recording chamber. In contrast to the two-photon fluorescence (TPF) signals that are emitted
isotropically in all directions, which allow the collection of signals both in forward (transmission) and
backward (epi) directions, SHG photons can only be detected in the forward direction. While both
TPF and SHG photons are collected through the condenser nonselectively, these signals can be easily
separated by the combination of appropriate dichroic mirror and bandpass filter that allow only the
