Biomedical Engineering Reference
In-Depth Information
astrocyte structures due to the uptake by astrocyte limiting
membranes that completely cover the pial surface
(41)
, whereas
the latter protocol loads both astrocytes and neurons
(40,46)
.For
the surface bulk loading, Fluo-4 AM (0.5-1 mM) was dissolved in
dimethylsulphoxide (DMSO) with 20% pluronic acid and mixed
in artificial cerebrospinal fluid (aCSF) containing 126 mM NaCl,
2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2,
10 mM glucose and 26 mM NaHCO3 (pH 7.4), gassed with 95%
O2and5%CO2at37
◦
C
(47)
. aCSF containing the dye (10-15
μ
l) was dropped on the pial surface, then the cranial window was
covered by a small piece of parafilm to prevent the dye from dry-
ing. After a 45-min incubation, the exposed brain was washed
for 15 min with aCSF without dye. In selected experiments, the
exposed cortex was also loaded with the astrocyte-specific fluo-
rescent indicator sulforhdamine 101 (SR101, 10
M)
(44)
for
10 min after fluo-4 AM loading (
Fig. 5.2)
. To minimize brain
pulsation, 0.9% NaCl containing 1% agarose (37
◦
C) was poured
on the cranial window and the glass coverslip was glued to the
metal plate with dental acrylic cement. A small opening between
coverglass and metal plate was left for inserting recording elec-
trode into the cortex (
Fig. 5.2)
.
Serial images from pial surface to barrel cortex layer2/3
μ
(
m deep) revealed that fluo-4 AM signals strictly co-
localized with SR101 staining (
Fig. 5.2)
. Astrocytes appear as
bright fluorescent cells with multiple processes. Astrocyte end-
feet are clearly labeled which outline the vasculatures. In contrast,
neuronal cell bodies appear as dark, round shaped areas which can
be detected at
∼
300
μ
m deep in the barrel cortex, owing to their
lack of uptake of both fluo-4 AM and SR101(
Fig. 5.2)
.
∼
90
μ
4.3. Two-Photon
Imaging
A custom-built microscope attached to Tsunami/Millinium laser
(10W, SpectraPhysics) and a scanning box (FV300, Olympus)
using Fluoview software and a 20X (0.9 NA, Olympus) objective
was used for in vivo imaging. Excitation wavelength was in the
range of 820
840 nm for both fluo-4 AM and SR101 imaging
simultaneously. Two-channel detection of emission wavelength
was achieved by using a 565 nm dichroic mirror (Chroma) and
two external photomultiplier tubes. A 525/40 bandpass filter
(Chroma) was used to detect fluo-4 AM emission light, and a
620/60 bandpass filter (Chroma) was used to detect SR101 sig-
nals (
Fig. 5.2)
. For image collection,
∼
∼
1 s was needed to record
a single frame of image at 512
512 pixels resolution. Time-lapse
images of astrocytic Ca2+ signaling were collected every 1
×
3s.
Normally, a sampling interval of 3 s was found sufficient to detect
whisker stimulation evoked astrocytic Ca
2
+
elevation, and this
low sampling rate was used to avoid the photodamage. The two-
photon laser power was carefully adjusted according to the depth
of imaging in the brain, since the photodamage induced by high
∼
