Biomedical Engineering Reference
In-Depth Information
density of blood vessels. The heart beat- and breathing-related
artifacts significantly increase their amplitudes when the skull and
the
dura mater
are removed. (The multi cell bolus loading tech-
nique does not require dura removal.) The stability of recordings
also depends on the diameter of the craniotomy. Thus, openings
larger than 1 mm in diameter are often accompanied by large
movement artifacts occurring at the heart beat frequency. Under
such circumstances, the vibration noise can be reduced by cover-
ing the skull opening with 2% agarose and a glass coverslip
(70)
.
Dark noise will degrade the signal-to-noise ratio at low light
levels.
4.1.3. Dark Noise/Read
Noise
Because of its electronic characteristics and because it is cooled,
the CCD camera dark noise is substantially lower than that of
the CMOS system. The CCD camera has read noise of 10e-
rms at 1 kfps, low in an absolute sense. The larger dark noise
in the CMOS camera accounts for the fact that the break in the
curve in
Fig. 3.11
is substantially to the right of the intensity
where the CCD camera becomes read-noise limited (10
2
4.1.3.1. Wide-Field
Imaging
pho-
tons/pixel/msec).
The dark noise of photomultipliers (anode dark current) critically
depends on supply voltage. At low-to-medium supply voltages,
anode dark current is relatively low, but it increases dramati-
cally at high supply voltages (usually higher than 700-800 V).
To minimize dark noise, it is therefore advisable to conduct
measurements at lowest PMT gain (e.g. lowest supply voltage)
provided that the preparation tolerates the required increase
in the intensity of excitation light. As a rule-of-thumb, the
illumination intensity has to be as high and the corresponding
PMT gain as low as possible to provide recording conditions just
below the dye bleaching threshold.
4.1.3.2. Two-Photon
Imaging
4.2. Light Sources
Three kinds of sources have been used. Tungsten filament lamps
are a stable source, but their intensity is relatively low, particu-
larly at wavelengths less than 480 nm. Arc lamps are somewhat
less stable but can provide higher intensity. Laser illumination can
provide even more intense illumination.
4.2.1. Wide-Field Imaging
It is not difficult to provide a power supply stable enough so
that the output of the bulb fluctuates by less than 1 part in
10
5
. In absorption measurements, where the fractional changes
in intensity are relatively small, only tungsten filament sources
have been used. On the other hand, fluorescence measurements
often have larger fractional changes that will better tolerate
light sources with systematic noise, and the measured intensities
4.2.2. Tungsten Filament
Lamps
