Biology Reference
In-Depth Information
blood volume, hemoglobin concentration, cytochrome oxidation,
and light scattering (
34, 52-56
). Hemoglobin is the main tissue
absorber of light of visible wavelength and oxy- and deoxyhemo-
globin have specifi c absorption spectra which differ and are used to
calculate concentration changes in oxy- and deoxygenated hemo-
globin. This enables to follow hemodynamic changes in response
to changes in neuronal activity. The wavelength and algorithm
chosen to calculate the changes in oxy- and deoxyhemoglobin can
have profound effects on the conclusions that are drawn (
54,
57-60
). Optical spectroscopy systems use white light sources with
fi ber probes or slit spectrography to measure changes in a single
location (
58, 61, 62
). Extension to these models was introduced
when CCD cameras were used in combination with fi lter wheels to
illuminate a large region of the cortex. This allows the assessment
of temporal and spatial characteristics of changes in oxy- and deoxy-
hemoglobin in response to SD or functional activation (
34, 56,
63, 64
). Optical imaging spectroscopy also provides information
on relative changes in oxygen consumption when measurements
are combined with concurrent measures of rCBF change (
50,
62, 65
). The rapid technical improvements in the fi eld of optical
imaging and related techniques, like depth-resolved imaging (
66
),
will make them powerful tools to investigate the mechanisms and
pathology of neurovascular coupling.
The ability to image rCBF dynamically in the living brain through
a cranial window has greatly improved our understanding on rCBF
regulation. Imaging techniques described above examine rCBF
changes in larger areas of the cortex, have limited spatial resolu-
tion, and are often restricted by limited penetration depth. With
introduction of confocal microscopy into studies on brain blood
fl ow regulation, it became possible to image the cerebral vascula-
ture in three dimensions in the intact brain of living rodents and to
analyze perfusion changes at the capillary level (
67, 68
). The adop-
tion of the capillary blood fl ow measurement technique for two-
photon microscopy increased penetration depth and allowed for
direct observation of capillary-level perfusion in deeper cortical
layers (
20, 69-71
). It is currently the technique which provides the
highest time and spatial resolution of blood fl ow and has become
increasingly important for studies on neurovascular coupling, brain
ischemia, and SD (
20, 70, 72-75
).
The technique of capillary blood fl ow measurements by two-
photon imaging was introduced by Kleinfeld et al. (
69
). The cra-
nial window preparation is modifi ed accordingly. If superfusion of
aCSF is not required, the space between brain and coverslip is fi lled
with agarose (e.g., Type III-A; 9793, Sigma Aldrich) to minimize
movement. The general principle of two-photon microscopy has
been described in detail (
76, 77
) and the general techniques to
analyze rCBF in microvessels follow previously described
4.4. Two-Photon
Imaging of rCBF
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