Biology Reference
In-Depth Information
5. Optical Imaging
of SD In Vitro
Fluorescence microscopy empowers investigators to assess
functional aspects by measuring ion variations (e.g., Na
+
, K
+
, Cl
−
,
Ca
2+
, Mg
2+
, and Zn
2+
) and pH and to detect changes in membrane
potential of whole cells or mitochondria using voltage-sensitive
fl uorophores. In addition, insights regarding the metabolic state of
the tissue can be gained from monitoring the autofl uorescence of
key components of oxidative phosphorylation, like nicotinamide
adenine dinucleotide (NADH). The development of more advanced
imaging techniques, in particular multiphoton microscopy, now
permits 3D functional imaging in brain tissue, even in specifi c cell
compartments (e.g., dendrites and spines), and transgenic tech-
nologies can create organisms that produce their own fl uorescent
chimeric molecules. In contrast to fl uorescence microscopy, intrin-
sic optical signals (IOS) can be detected over a broad waveband of
light and do not require loading of foreign substances into the
tissue.
Assessment of IOS is perhaps the most commonly used imaging
approach to study SD in vitro and in vivo. IOS represent altera-
tions of optical properties of unstained tissue which can be mea-
sured as a change of either light transmittance or refl ectance.
Measurement of IOS does not necessitate any tissue manipulation
other than illumination of the preparation. The sustained signal
changes feature comparably large amplitudes and can be assessed
with a charge-coupled device (CCD) camera mounted to a micro-
scope or an array of photodiodes. Detectable IOS in brain tissue
can be induced by intense neuronal activity (e.g., induced by repet-
itive stimulation or during seizure-like activity), osmotic changes
leading to swelling or shrinkage of neurons, and SD. In contrast to
intense neuronal activation or mild hypotonicity and despite the
swelling of brain cells with subsequent shrinkage of the extracel-
lular space (
35-38
), the main optical change during SD is an
increase in light scattering (
39
) that coincides with the extracellular
DC potential shift. The decrease in light transmittance has been
shown to be Cl
−
dependent (
40
) and has been hypothesized to be
due to swelling of intracellular organelles (e.g., mitochondria)
(
41
). Other fi ndings indicate that it could be the consequence of
structural changes, located mainly in dendritic processes (
36, 42
).
The advancing wave of optical changes accompanying SD can be
roughly resolved into three phases (
43
) starting with a brief, weak
increase followed by a large decrease, succeeded by a weaker and
prolonged increase of light transmittance. During the course of
SD, recording IOS permits 2D mapping of the spreading event,
thereby allowing the calculation of the propagation velocity and
the extent of spread. If quantifi cation of the IOS is intended, a
5.1. Intrinsic Optical
Signals
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