Biomedical Engineering Reference
In-Depth Information
We use a stationary stage upright microscope equipped with two
camera ports. One port has a standard high spatial resolution
CCD camera for infrared differential interference contrast video-
microscopy. The other camera port has a fast data acquisition
camera with relatively low spatial resolution (80
2.1.2. Methods
80 pixels) but
outstanding dynamic range (14 bits; NeuroCCD-SM, RedShir-
tImaging LLC, Decatur, GA). The analysis and display of data
are carried out using the NeuroPlex program (RedShirtImaging)
written in IDL (ITT Visual Information Solutions, Boulder, Col-
orado) running on a Windows XP computer. The brain slice is
placed on the stage of the microscope and the fluorescent image
of the stained cell projected by a water immersion objective via an
optical coupler (0.09-0.11 X; RedShirtImaging) onto the CCD
chip. A 250 W xenon, short-gap, arc lamp (Osram, XBO 250
W/CR ORF) powered by a low ripple power supply (Model
1700XT/A, Opti-Quip, Highland Mills, NY) is the source of
excitation light.
With the above dye, the best signals are obtained using an
excitation interference filter of 520
×
45 nm, a dichroic mirror
with the central wavelength of 570 nm, and a 610 nm barrier filter
(a Schott RG610). With the sensitivity of the intracellular voltage-
sensitive dyes used (1-6%
±
F/F per 100 mV in recording from
dendritic processes), relatively good signal-to-noise ratios can be
obtained in single-trial recordings with modest spatial averaging
(4-10 pixels) from dendritic regions that are about 300
maway
from the soma. Modest signal-averaging (4 trials) is often used to
improve the signal-to-noise ratio further.
In experiments where calcium and voltage imaging is car-
ried out from the same neuron, Ca
2
+
signals are recorded at the
frame rate of 500 Hz using an excitation interference filter of
380
μ
15 nm, a dichroic mirror with central wavelength of 400
nm and a 450 nm barrier filter. Calcium fluorescence transients
are expressed as
±
F)/F
0
, where F is the stimulated flu-
orescence and F
0
is the resting fluorescence. The value of F is cal-
culated after subtracting auto-fluorescence determined for an area
far from the loaded dendrite. Due to uneven illumination of the
field of view and due to differences in auto-fluorescence between
different parts of the slice, background fluorescence values vary
between different regions on the same slice typically by a factor of
up to 3. However, when signals from the same dendritic regions
are compared under different conditions, possible errors in cal-
culating
F/F=(F
0
−
F/F introduced by uncertainties about actual contri-
bution of auto-fluorescence to the resting light intensity can be
neglected. We did not detect any toxicity of [Ca
2
+
]
i
indicator
bis-fura-2; loading of neurons did not have detectable pharma-
cological effects (toxicity of the indicator dye in the absence of
excitation light) on the electrical behavior of neurons. During
