Biomedical Engineering Reference
In-Depth Information
intensity divided by the noise in the measurement and plot that
versus the number of photons reaching each pixel of the camera.
The dotted line in
Fig. 3.11
is the plot for an ideal camera. At
high light intensities, this ratio is large and thus small changes in
intensity can be detected. For example, at 10
10
photons/ms, a
fractional intensity change of 0.1% can be measured with a signal-
to-noise ratio of 100. On the other hand, at low intensities, the
ratio of intensity divided by noise is small and only large signals
can be detected. For example, at 10
4
photons/msec, the same
fractional change of 0.1% can be measured with a signal-to-noise
ratio of 1 only after averaging 100 trials.
In addition,
Fig. 3.11
compares the performance of two par-
ticular camera systems, a CMOS camera (solid lines) and a back-
illuminated cooled CCD camera (dashed lines), with the shot
noise ideal. A 128
128 pixel CMOS camera approaches the shot-
noise limitation over the range of intensities from 10
5
to 10
9
photons/ms/pixel. This is the range of intensities obtained in
absorption measurements and fluorescence measurements using
bath applied dye on in vitro slices and intact brains. On the
other hand, the cooled 80
×
80 pixel CCD camera approaches the
shot noise limit over the range of intensities from 10
2
×
10
5
photons/ms. This is the range of intensities obtained from flu-
orescence experiments on individual cells and neurons. In the
discussion that follows, we will indicate the aspects of the mea-
surements and the characteristics of the two camera systems which
cause them to deviate from the shot noise ideal. The two camera
systems we have used to illustrate in
Fig. 3.11
have excellent
dark noise and saturation characteristics; other cameras would be
dark noise limited at higher light intensities and would saturate at
lower intensities.
to 5
×
Similar considerations apply to two-photon measurements.
Because two-photon excitation will only occur with the nearly
synchronous arrival of two low-energy photons, excitation is pro-
portional to the square of light intensity and only very high inten-
sity sources achieve significant excitation. In practice, this restricts
the light source to pulsed lasers with very narrow pulses. In the
experiments illustrated in Example 2 (above), the laser inten-
sity was not limiting because the targeted cells were only some
150-250
4.1.1.2. The Optimum
Signal-to-Noise Ratio in
Two-Photon Scanning
Microscopy
Measurements
m below the surface of the preparation and losses in
focal intensity from light scattering were not large. For cells as
deep as mitral cells (
μ
m below the surface), the loss of
the excitation light due to photon dispersion within the tissue
becomes significant. In addition, in the neural tissue, the qual-
ity of in-depth imaging often suffers from (i) inhomogeneity of
the refractive indices within the tissue (e.g. blood vessels, clusters
of cell bodies acting as microlenses
(67)
), (ii) light absorption
by the hemoglobin, (iii) generation of out-of-focus fluorescence
∼
400
μ
