The FFT feature in NeuroPlex is very useful in determining the source of noise.
Dark Noise (NeuroPDA-II)
The place to begin is to examine the noise in the dark. This noise should not be larger than a certain size and should have a flat FFT spectrum. With the NeuroPDA system, measure the dark noise with the following settings:
1. Second stage amplifier low-pass filter: 1 kHz;
2. Second stage amplifier high-pass filter: AC2 on;
3. Use fastest acquisition rate: 1.3 usec;
4. Number of points: 1024;
5. First stage gain: high; and
6. Second stage gain: 50.
Determine the size of the dark-noise by measuring its RMS value under Output, determine its frequency content by using the FFT option under Trace. A flat spectrum with an absolute RMS value of less than 50 is good.
Extraneous Dark Noise (NeuroPDA-II)
There are a number of recognized causes of a not-flat FFT; all of them can be eliminated. A peak at 60 (50) Hz (or harmonics) indicates AC noise in the system; 120 or 180 Hz may come from the room light; peaks around 200 - 300 Hz may come from acoustic noise (e.g. from the air handling system); a peak near 1000 Hz may come from oscillation of the amplifier system.
Extraneous Light Noise
After the extraneous dark noise is minimized, a second class of noise sources, termed extraneous or technical noise, may be present after illuminating the preparation. Again, there are a number of recognized causes all of which can be eliminated.
Fluctuations in the output of the light source:
· Tungsten filament lamps. It is not difficult to provide a power supply stable enough so that the output of a tungsten filament bulb fluctuates by less than 1 part in 105. Tungsten filament sources are almost always used in absorption measurements and are also preferable in those fluorescence measurements where relatively large light intensities are obtained (i.e. a photocurrent > 10-8 amps).
· Arc Lamps. In the past, intensity fluctuations were a problem with arc lamps. Now, Opti-Quip, Inc., (www.optiquip.com, Highland Mills, NY, 10930, (914) 928-2254) provides 150 and 250 watt power supplies, lamp housings, and xenon arc lamps with noise that is in the range of 1 part in 104. We found that the 150 watt bulb yielded 2-3 times more light than a tungsten filament bulb (over the range 475-565 nm) and in turn the 250 watt bulb was 2-3 times brighter than the 150 watt bulb. The extra intensity was important for fluorescence measurements from single neurons.
Another source of extraneous light noise is vibrations in the light path. This noise tends to be in the frequency range of 1-50 Hz.
· Shutters. The opening of a shutter attached to the microscope can cause a very large vibration signal. We found that this noise damps out over a period of about 1 second. Vibrations from the shutter can be greatly reduced by mounting the shutter so that it is not touching the microscope (i.e. on another platform that is vibrationally isolated from the microscope).
· Floor Vibrations. A number of precautions for reducing the effects of this vibrational source include eliminating air-water interfaces, eliminating convection currents in the lamp housing, filtering the saline to remove particles, and mounting the apparatus on a platform made from two optical table-tops separated by (and mounted on) air-filled rubber tubes (obtained from Newport: isolator, air cushion, #5274). Embedding ganglia in 1-3% agar further reduced vibrational noise. Minus K Technology sells Biscuit bench top vibration isolation tables with very low resonant frequencies. They provide outstanding vibration isolation.
all of the extraneous noises are reduced enough, the remaining noise
will either be dark noise (very low light intensity) or shot noise
(higher intensity). In the NeuroPDA
apparatus this crossover occurs at a resting light photocurrent of
about 5 x 10 -10 amps. In the NeuroCCD system
The FastOne camera can pick up noise from other electrical devices. In one instance, moving an arc lamp power supply away from the camera and its power supply eliminated the noise.