Biomedical Engineering Reference
In-Depth Information
The invention of 2PLSM by Webb's group in 1990
(39)
has
tremendously improved our understanding of neurobiological
phenomena in both normal and diseased brain. With two-photon
excitation (2PE), it is possible to image deep brain tissue in
the live animals with single cell spatial resolution
(42)
.We
have used 2PLSM because 2PE provides several key advantages
compared with single photon excitation (1PE). First, because of
the localized excitation in 2PE, all emission fluorescent photons
contribute to the useful signals. In contrast, a pinhole has to be
used to block the emission light from the unfocused plan with
1PE. Therefore, 2PE has much higher efficiency than 1PE. Sec-
ond, since only the fluophores in the focused point are activated
by 2PE, whereas the entire light pathway is activated during 1PE,
phototoxicity and photobleaching are greatly reduced in 2PE.
This is especially important for time-lapse imaging of live tissue.
Moreover, the excitation wavelength used in 2PE (740
∼
900
nm) penetrates tissue better than 1PE (350
∼
550 nm), which is
essential for in vivo imaging of live animals.
Although blind intracellular recordings
(43)
have been
widely used for loading Ca
2
+
indicators into neuronal structures
under 2PLSM, delivering Ca
2
+
indicators into single astrocyte
through recording pipette in live animals is still challenging.
However, recent studies demonstrated that astrocyte population
can be labeled by either local dye injection
(40)
or pial surface
dye application
(41)
. Together with the discovery of specific
astrocyte fluorescent indicator sulforhodamine 101
(44)
,these
pioneer studies enable, for the first time, the observation of
astrocytic Ca
2
+
activity in response to physiological stimulation
in the live animals
(9)
(
Fig. 5.2
).
We found that astrocytic Ca
2
+
signals can be clearly detected
from pial surface to
m deep in adult mouse primary soma-
tosensory cortex (barrel cortex) following surface loading with
fluorescent Ca
2
+
indicators. This penetration depth correlates
with the layer1 and part of the layer 2/3 of mouse barrel cortex.
∼
300
μ
4.1. Animal
Preparation
Adult FVB or C57BL/6 mice (
8 weeks old) were used for in
vivo imaging primarily because application of fluorescent Ca
2
+
indicators on mouse pial surface produced satisfactory astro-
cyte loading in vivo. Same surface loading procedure only works
on immature rats up to
∼
∼
3 weeks old (unpublished observa-
tions). Animals were anesthetized with intraperitoneal injection
of ketamine (0.12 mg/g) and xylazine (0.01 mg/g). Depth of
anesthesia was monitored by field potential recording and hind
limb pinch withdrawal reflexes and kept constant at stage III/3
(45)
with supplemental doses of anesthetics. Tracheotomy was
performed and the mice were intubated and artificially venti-
lated with a small animal ventilator (SAAR-830, CWE). The
