Biomedical Engineering Reference
In-Depth Information
neuroscience [4]. Although current optical methods, which are mostly fluorescence based, remain
useful in the study of many biological systems, they are severely limited by their lack of selectivity. In
fact, to optically measure membrane potential in neurons, it would be ideal to use an optical method
that can selectively probe the plasma membrane, which is the only part of the neuron that sustains the
biologically relevant membrane potential. But in fluorescence, one obtains fluorescence photons from
the dye molecules wherever they are located, either cytoplasm, membrane, or neuropil, and this lack of
selectivity generates a significant background signal that interferes with imaging the plasma membrane
of the neuron, producing poor signal-to-noise, and imposing the need for extensive averaging. Indeed,
although fluorescent voltage-sensitive chromophores have been used to image dendrites and axons [5,6],
their lack of sensitivity, partly resulting from the lack of specificity when staining plasma versus intra-
cellular membranes, and the large background fluorescence signals, which originate from other regions
of the neuron, could severely limit their usefulness, for example, to measure dendritic spines or axonal
voltage dynamics.
10.2 SHG: theoretical Background
To circumvent this problem, one can apply the second-order nonlinear optical method of second-
harmonic generation (SHG) to optically measure the membrane potentials. We will not review the theo-
retical basis of SHG, which is covered in the chapters in Part I of this volume, but will briefly provide
some pointers to its essential features. In SHG, incident light at frequency ω generates light at 2ω on
interacting with the neuron, which selectively images membranes, and not bulk regions because of sym-
metry requirements [7]. Therefore, by cancelation of the dipole moments of the chromophores, there is
essentially no SHG signal from inside or outside the cell. Meanwhile, the plasma membrane contributes
to position the SHG chromophores in the same orientation, either by lipophilic adsorption to the mem-
brane leaflet, or by electrostatic forces. Moreover, if the SHG chromophore is delivered only to one side
of the membrane (either extra or intracellularly), the plasma membrane then acts as a symmetry-break-
ing interface, generates therefore an array of oriented chromophores with a dipole asymmetry, which
is an ideal circumstance for strong SHG. This means that when one measures SHG from neurons there
is essentially no background SHG signal and all SHG photons are generated in the plasma membrane,
exactly where the electrical field is located. This physicochemical “perfect storm” has not passed unno-
ticed by investigators: Indeed, in the last decade, following the pioneering work of Lewis and Loew [8],
a number of groups, including ourselves, have successfully applied SHG and performed high-resolution
optical measurements of membrane potential from living neurons [9-15].
10.3 SHG Voltage chromophores
Successful measurement of membrane potential by SHG depends heavily on the choice of chromophores.
So far, many chromophores have been examined for their use in SHG imaging of membrane potential
and several of them are now being used routinely [9-13,16]. However, this by no means suggests that
they are best chromophores for this purpose. In fact, there exist tremendous numbers of already avail-
able chromophores as well as chromophores being developed that have high potential to show good SHG
response to membrane potential changes. Detailed review of chemical properties of these chromophores
can be found elsewhere [17]. Here, we describe several key aspects of the SHG chromophores.
In general, chromophores used for SHG have electron donors and acceptors bridged by π-bonds
provided by double bonds or aromatic rings. Chromophores successfully applied to SHG membrane
potential measurements, including FM4-64 and ANEPP chromophores have these characteristics.
These molecules are polarized and are highly susceptible to electromagnetic fields induced by laser
illumination. When these chromophores are aligned in an ordered manner breaking the center of
symmetry, they become good SHG materials to generate SHG photons. This hyperpolarizability of
chromophores determines how many SHG photons can be recorded under basal condition and thereby
