Biomedical Engineering Reference
In-Depth Information
wavelengths where most dyes absorb or emit light (500-900 nm).
In contrast, only specially chosen vacuum photocathode devices
(phototubes, photomultipliers, or image intensifiers) have a quan-
tum efficiency as high as
0.4. A relatively low fill factor and front
illumination reduce the quantum efficiency of CMOS cameras to
about 50%. This reduction will reduce the signal-to-noise ratio in
a shot-noise limited measurement by a factor of about 1.4. Thus,
in a low light level shot-noise limited situation, a CCD camera
will have a larger signal-to-noise ratio. Photographic film has a
much smaller quantum efficiency,
∼
<
0.01
(80)
.
These cameras have on-chip multiplication and should lead to
better signal-to-noise performance at very low light levels. How-
ever, the multiplication process adds noise (a factor of 1.4) and
some existing chips are even noisier than expected from the fac-
tor of 1.4. If an ordinary CCD has a read noise of 10e-, then
an ideal EM-CCD camera will have a better signal-to-noise ratio
only at light levels less than 100 photons/pixel/frame. The light
level achieved in the experiments illustrated in Example 1, where
there is relatively little dye in small distal processes, is
4.4.2. EM-CCD Cameras
1000
photons/pixel/msec. Thus, in this measurement and all measure-
ments with even higher light intensity, the CCD will have a better
signal-to-noise ratio than an EM-CCD.
∼
5. Future
Directions
Because the light measuring apparatus is already reasonably opti-
mized for all of the above applications, any improvement in sen-
sitivity will need to come from the development of better dyes
and/or investigating signals from additional optical properties of
the dyes.
5.1. Organic Dyes
5.2. Organic
Voltage-Sensitive
Dyes
The voltage-sensitive dyes in
Fig. 3.2
and the vast majority of
those synthesized are of the general class named polyenes
(81)
,
a group that was first used to extend the wavelength response of
photographic film. It is possible that improvements in signal size
can be obtained with new polyene dyes (see
(4, 5)
for a discussion
of maximum possible fractional changes in absorption and fluo-
rescence). On the other hand, the maximum fractional change
has not increased in recent years (L. B. Cohen, A. Grinvald,
K. Kamino, L. Loew, and B. M. Salzberg, personal com-
munication), and most improvements (e.g.
(9, 25, 26, 51)
)
have involved synthesizing analogues that work well on new
preparations.
The best of the organic dyes have fluorescence changes of
10-20%/100 mv in situations where the staining is specific to
