Biomedical Engineering Reference
In-Depth Information
experimental evidence about forward propagation of voltage signals upon synaptic input onto dendritic
spines. Efforts have been made to elucidate the membrane potential changes upon synaptic stimulation
using different techniques [25,38], but SHG recordings will be the ideal tools to answer this question.
With regard to axons, changes in axonal electrical information processing have drawn much attention
[39], but the direct observation of such processes needs to follow. Here again, membrane potential imag-
ing by SHG will be a powerful tool that can be employed to meet these needs. With the improvement of
this technique, we expect that SHG imaging will become a precious tool to complement other existing
techniques in investigating the physiology of neurons and other cells.
10.6.2 Genetic Approaches
Fluorescent proteins have been modified to probe various aspects of cell physiology, including calcium
and membrane potential [40]. While these are developed for fluorescence imaging, there are reports
suggesting that GFP (green fluorescent protein) and their relative proteins generate SHG signals [41,42].
In addition, efforts have been made to generate membrane-tethered fluorescent proteins for the use of
membrane potential measurements, which also should help increase SHG signals by aligning the mol-
ecules in an ordered manner [43]. This type of genetic approach has two potential benefits: (1) it would
not require intracellular dye-loading processes and, therefore, may be able to circumvent the slow dye
diffusion issues that FM4-64 and other organic chromophores have experienced; and (2) in combi-
nation with proper promoters, it would allow labeling of genetically defined subpopulation of cells.
Furthermore, this genetic approach may pave the way to
in vivo
SHG imaging. Protein-based imag-
ing of intracellular calcium concentration has been explored for a long time and improved indicators
have been utilized to image neuronal activities
in vivo
[44,45]. Recently, a genetically targeted voltage-
sensitive fluorescent protein was successfully applied to
in vivo
imaging [46]. Clearly, these are ideal
methods to monitor neuronal behavior
in vivo
as they circumvent dye loading using invasive methods.
Unfortunately,
in vivo
imaging of SHG is difficult as most of the SHG signals go to the transmission
path and do not come back to the objective lenses. However, if the SHG signals are strong enough, back-
scattered SHG signals can be detected and provide unique information about the membrane potential
dynamics
in vivo
. Indeed, recent demonstrations of intrinsic SHG imaging of muscle and collagen
in
vivo
clearly show that
in vivo
SHG is feasible, and further suggest that it can also be applied to membrane
potential measurements [47,48].
For genetic approaches, however, time-resolution issues must be considered, as these techniques quite
often have time lag between actual voltage change and signal response. It can be predicted that if the
SHG has electrooptic response from these probes, the response can be near instantaneous and therefore
does not have any time lag. However, if changes in SHG by membrane potential fluctuation involve
realignment of the probe or conformational changes of the molecule, then these processes take certain
time and therefore it cannot be instantaneous. Although it can practically be used if these changes are
fast enough, careful characterization will be required for this type of genetic approach.
10.6.3 Hardware Devices
In addition to chromophores, modifications of the imaging devices are expected to improve the current
SHG imaging of membrane potential and further expand its applications.
First, to overcome the inherent issue of time resolution in obtaining 2D images by laser scanning
microscope, efforts have been made to utilize random access microscopy in obtaining SHG signals [49].
In this setting, the position of laser illumination is not regulated by a set of mirrors (galvano mirrors),
as most laser scanning microscopes do. Instead, these utilize acoustooptic devices to direct laser in 2D
space, or even in 3D. This technique has been successfully implemented into calcium imaging [50,51].
There still remains a problem of spatial resolution, but the temporal resolution and freedom of collection
patterns are far superior to conventional mirror-based laser scan system [49]. This technique should be
