Biomedical Engineering Reference
In-Depth Information
TheroleoftheCa
2+
waves in the heart in situ is poorly understood, be-
cause Ca
2+
waves have been studied mostly in enzymatically isolated cells.
We have developed a system for in situ imaging of [Ca
2+
]
i
equipped with
a multi-pinhole-type confocal scanning device, which enabled us to visualize
real-time
x
-
y
images of Ca
2+
waves [14,15]. Using this system on Langendorff-
perfused rat hearts, with simultaneous recording of electrocardiograms, we
found that Ca
2+
waves were completely abolished by ventricular excitation,
suggesting that the waves in the whole heart play little, if any, pathophysiolo-
gical role. Nevertheless, it is possible that Ca
2+
waves play some aggravating
role in cardiac function if they occur frequently and propagate beyond in-
dividual cells on a large scale under certain Ca
2+
-overloaded conditions. In
this regard, quantitative analysis of Ca
2+
waves in the working whole heart
is essential in order to understand their functional significance.
Two-photon Ca
2+
imaging was also performed in situ with muscle cells in
a whole heart of a rat. The heart was excised under anesthesia with diethyl
ether, and perfused on a Langendorff apparatus for 5 min with a Ca
2+
-free
Tyrode solution under 100% oxygenation. Thereafter, the heart was loaded
with fluo-3/AM (100
g) dissolved into the Ca
2+
-freeTyrode(4ml)contai-
ning 1% fetal calf serum and 0.06% pluronic F-127 on a recirculating system
for 30 min. Following another 15-min perfusion with 0.5 mM-Ca
2+
Tyrode
to allow hydrolysis of acetoxymethyl esters within the cells, the heart was
used for the experiments. The motion artifact on the image was prevented
by 20 mM 2,3-butanedione monoxime (BDM), which was added to the perfu-
sate. Unless otherwise specified, Ca
2+
waves were analyzed under quiescence
via blockade of atrioventricular conduction produced by mechanical ablation
of the atrioventricular junction.
Figure 1.6 shows distributions of the Ca
2+
concentration in the right
ventricle of the heart. The total excitation power at the focal plane of the
objective lens was 150 mW. Each image was obtained sequentially with a
time resolution of about 33 ms. In Fig. 1.6, a Ca
2+
wave propagating from
the lower right to the upper left was observed.
In the experimental condition for observing [Ca
2+
]
i
, the excitation rate
of fluo-3 in each focus can be estimated to be about 6% from its two-photon
absorption cross-section, the numerical aperture (NA) of the objective lens
used and the excitation power. This excitation rate is not high enough for
this application, nor probably for many others. In addition, the field of view
in our microscope cannot be considered to be wide enough. The penetration
depth, the excitation rate and the field of view discussed above are currently
limited by the maximum output power of the Ti : Sapphire laser used. The
maximum output power of a commercially available Ti : Sapphire laser is
about 2 W and this value is still not high enough in our calculation. We
think that the development of a Ti : Sapphire laser with a higher output is
necessary for further advancement of the multi-point multi-photon excitation
microscope.
μ
