Biomedical Engineering Reference
In-Depth Information
while increasing laser power etc. For a thorough description of
approaches used to improve the imaging depth as well as image
quality, we refer the reader to the recent review of Helmchen and
Denk
(67)
.
A second type of noise, termed extraneous or technical noise, is
more apparent at high light intensities or at high spatial resolu-
tion. At high light intensities, the sensitivity to extraneous noise is
high because the fractional shot noise and dark noise are low. One
type of extraneous noise is caused by fluctuations in the output of
the light source (see below). Two other sources of extraneous
noise are vibrations and movement of the preparation.
4.1.2. Extraneous
(Technical) Noise
A number of precautions for reducing vibrational noise have been
described
(68, 69)
. The pneumatic isolation mounts on many
vibration isolation tables are more efficient in reducing vertical
vibrations than in reducing horizontal movements. There has
been a series of progressively more successful (and expensive)
solutions for vibration problems. A simple and inexpensive rem-
edy is air-filled, soft rubber tubes (Newport Corp, Irvine, CA).
For more severe vibration problems, Minus K Technology (Ingle-
wood, CA) sells vibration isolation tables with very low reso-
nant frequencies. The Halcyonics Micro 60 (Menlo Park, CA) is
an active (piezoelectric drivers) isolator and can defeat airborne
vibrations as well as those transmitted through the “ground”
(Brian Salzberg, personal communication). Nevertheless, it has
been difficult to reduce vibrational noise to less than 10
−
5
of
the total light. With this amount of vibrational noise, increases in
measured intensity beyond 10
10
photons/ms would not improve
the signal-to-noise ratio. For this reason and because of well size
limitations, the performance of the CMOS system reaches a ceil-
ing (
Fig. 3.11
, solid line).
4.1.2.1. Vibrational Noise
in Wide-Field
Measurements
Using a Newport Corporation Research Grade vibration isolation
table reduces the vibrational noise so that it is less than the shot
noise.
4.1.2.2. Vibrational Noise
in Two-Photon
Measurements
Preparation movement is often the limiting noise factor in in vivo
measurements. This is true in wide-field measurements because
the light intensity can be relatively high, reducing the shot noise
to a level that is smaller than the movement noise. It is true
in two-photon measurements because the spatial resolution and
contrast are high and thus the sensitivity to movement is also
high. The movement artifacts in vivo usually consist of irregular
movements of the entire animal (less of a problem in anesthetized
preparations) as well as of heart beat- and breathing-related vibra-
tions. The higher frequency vibrations are usually the result of
heart beat pulsation and are more profound in regions with a high
4.1.2.3. Preparation
Movement
