Cranial Window Assessments in Experimental aSAH in Mice - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 489

25. Intravenous labelling of the leukocytes with 0.1 ml of 0.2%
rhodamine 6G.
26. Observation of the window using the fl uorescence stereo-
microscope and recording for later off-line analysis. Blood
vessels are visualized with blue light epi-illumination, leuko-
cyte-endothelial interaction is visualized with green light
epi-illumination.
27. Microvascular mapping prior to the observation is recom-
mended for later reassessment of the regions of interest.

3.3. Confocal
Microscopy

In contrast to conventional fl uorescence microscopy confocal
microscopy uses a laser for point illumination of the tissue of inter-
est instead of a mercury lamp. Information collection occurs
through a pinhole in front of the detector to eliminate out-
of-focus information. Due to this pinhole, an increase in spatial
resolution is at the cost of decreased signal intensity. After activa-
tion by the laser light, the targeted focus point in the tissue emits
fl uorophores, which are then detected through the same objec-
tive. The increase in spatial resolution along the light axis, espe-
cially in the sample depth, elicits the possibility of evaluation of 3D
images. Via a scanning or raster technique single point information
can be collected automatically and later transferred into a larger
image of the tissue.

3.4. Two-Photon
Microscopy

Two-photon microscopy is a fl uorescence imaging technique, which
can be a superior alternative to fl uorescence or confocal microscopy.
Still the application of fl uorescent dyes is necessary to visualize the
different structures. The collection of emitted fl uorophores from
the tissue remains the same. The main advantage of two-photon
microscopy is the excitation of the fl uorescent dyes in the tissue
with signifi cantly less energy due to the longer wavelength of the
emitted photons [approx. 700-1,050 nm (infrared) in Titan:Sapphire
Laser]. As light of longer wavelength shows less scattering effect in
tissue, it can penetrate deeper into the tissue of interest, and thus
allows for intravital imaging of up to a depth of 200-1,000

m
(Fig. 4 ). Two-photon microscopy can give access to regions and
structures of interest otherwise inaccessible for imaging analysis.
It also minimizes the fading of fl uorescent dyes.
The major disadvantage of two-photon microscopy is the high
cost expenditure, which contributes for a relatively low distribution.
Also, for very thin objects a higher optical resolution can be achieved
with single-photon microscopy (multi-photon microscope: LSM 7
MP, Carl Zeiss).

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