Biomedical Engineering Reference
In-Depth Information
5.1
Introduction
Optical recording is a powerful technique in neuroscience research, which enables
us to simultaneously acquire data about the dendritic architecture of individual neu-
rons, the multi-neuronal synaptic circuitry involving those neurons, and the time-
resolved physiological response properties of those neurons. Rapid progress in
computer technology, along with signifi cant improvement of various types of imag-
ing devices and microscopes, has driven substantial advances in optical recording
techniques. At present, it is possible to visualize the neural activity not only in cul-
tured cells and brain slices in vitro but also in the brains of intact, behaving animals
in vivo. In neuroethological studies, optical recording methods have provided a
number of important fi ndings on the neural basis for various types of animal behav-
ior. To get defi nitive and reliable data, however, pretreatment methods such as dye
loading and sample preparation must be considered and carried out very carefully,
and the imaging system and peripheral equipment must be confi gured in a manner
that will support acquisition of data with the required spatial and temporal
resolution.
In many cases, visualization of neural activity is mediated by specifi c probes that
change their fl uorescence or absorbance depending on membrane potential, cyto-
solic calcium concentration, and/or pH. Typically the synthetic organic dye is
loaded into the targeted cells prior to the experiment, though it is now becoming
common for a genetically encoded probe protein to be expressed in transgenic ani-
mals (see Chap.
7
). It is also possible to visualize the neural activity as an intrinsic
optical signal without probe loading, but fl uorescent probes achieve much better
signal-to-noise ratio and higher temporal resolution. In this chapter, we address
Ca
2+
-imaging techniques which use organic dyes having high and stable enough
sensitivity to detect single action potentials or subthreshold synaptic activity. In
particular, we will focus on in vivo Ca
2+
-imaging techniques and describe points of
concern for setting up the measurement instruments and pretreatments in a manner
that are optimized for the intended research objectives. Details of more advanced
Ca
2+
-imaging methods are reviewed by Grienberger and Konnerth (Grienberger and
Konnerth
2012
).
5.2
Principle of Fluorescent Ca
2+
-Sensitive Dyes
Organic Ca
2+
indicators were developed in the 1980s by Tsien and his colleagues
(Tsien
1980
). All of Tsien's fl uorescent Ca
2+
indicators are based on a fl uorophore
combined with BAPTA, which retains high and stable selectivity for Ca
2+
in the
neutral to weak-alkaline pH range and has a fast time constant for Ca
2+
binding. The
Ca
2+
indicators alter their spectral properties depending on the change in cytosolic
Ca
2+
concentration ([Ca
2+
]
i
) associated with membrane depolarization. In general,
the Ca
2+
-sensitive fl uorescent dye is loaded into nerve cells using any of a variety of
