Biomedical Engineering Reference
In-Depth Information
Another very important aspect that must be considered during the confi guration
of an effective optical imaging system is the system integration: synchronization of
the stimulator, electrophysiological recording apparatus, and all other peripheral
equipment associated with real-time image capture. Generally, digital TTL signals
are used for synchronizing the operation of image acquisition and temporal control
of the onset and duration and frequency of stimulation. There is, however, a brief lag
between the trigger of the TTL signal and the initiation of image acquisition within
the computer. This time lag is inevitable and ranges from a few to dozens of milli-
seconds, depending on the frame rate. Therefore, it is necessary to design the cir-
cuitry for synchronization of the experimental components in a way that the time
delay is minimized and constant between trials. In our system, a TTL signal exported
from the imaging computer is used for triggering the stimulation and electrophysi-
ological recordings and also receives feedback from the stimulus monitor.
Fairly sophisticated software is typically provided along with any commercially
available advanced imaging device and is usually adequate for sequential image
acquisition required for Ca
2+
imaging. However, subsequent data analysis of the
optical recordings often requires the development and application of specialized
algorithms, and it is very typical for an investigator to use other more specialized
research-oriented image-processing software packages like “Image J (NIH)”
(Dreosti et al.
2009
). As mentioned above, singlemetric imaging techniques yield
signals that characterize changes in [Ca
2+
]
i
as relative changes in the observed fl uo-
rescence,
F
/
F
0
, where
F
0
is the fl uorescence intensity of the fi rst image (or the
mean value of all images during the prestimulation period). For visualization of
Ca
2+
signals using pseudo-color images, it is important to confi gure the minimum
and maximum values to capture the entire dynamic range of all images throughout
the recording. If the fl uorescence change is too small and/or signal-to-noise ratio is
too low, several sequences of images must be acquired in response to repeated stim-
uli to enable signal averaging. However, signal averaging for noise reduction is not
attainable for imaging of spontaneous or motor-related activity, because these activ-
ities occur with variable delay to the stimulus onset.
Δ
5.4
Application: Simultaneous Imaging
of Pre- and Postsynaptic Neurons
In the fi nal section of this chapter, we summarize a procedure we used for simulta-
neous Ca
2+
imaging of pre- and postsynaptic neurons. For these experiments, we
combined single-neuron staining of a postsynaptic neuron using microelectrode
injection of one Ca
2+
indicator and bulk staining of presynaptic fi bers with the AM
ester of a second Ca
2+
indicator. We simultaneously monitored the optical signals
from both indicators in different wavelengths using a dual-view optical system. This
method enabled us to measure the pre- and postsynaptic activity on specifi c den-
drites of the identifi ed interneuron. We adapted this method to the cricket cercal
sensory system (Ogawa et al.
2008
).
