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Description: NeuroCCD Cooled-CCD Activity Imaging System
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Description: Backpropagation of action potential in neocortical pyramidal neuron. Measured using NeuroCCD, frame interval .37ms. Provided by Srdjan Antic and Dejan Zecevic, Yale University.

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Unexpected Results

Occasionally (perhaps once a day or once a week) the camera will get in a strange mode and the images and values will be unexpected. First, try restarting NeuroPlex. If that doesn't solve the problem, try rebooting the PC and cycling the camera power. If it is convenient you might start each day with a PC reboot.

Both the voltage output of the CCD camera and the signal-to-noise ratio are dependent on three factors: the frame rate (Kfps), the camera gain setting, and the on-chip binning. Attention to the effects of these factors will maximize the resulting signal-to-noise ratio and minimize saturation.

Frame Rate

The output of the camera is proportional to the sum of the number of photo-electrons generated during the frame interval. This number will be approximately 10 times larger at a frame rate of 200 fps than at 2.7 Kfps.

The Camera Gain Setting

The camera has two gain settings, high and low. The high gain is about 4 times larger than the low gain.

Read Noise:

The dark noise (actually read noise in the cooled FastOne CCD) is affected by both frame rate and by gain approximately according to the following table.

READ NOISE (rms electrons)

 

Frame rate

High Gain

Low Gain

2.7 kHz

70

140

100 Hz - 1.0 kHz

35

90

Well Size:

The gain setting also affects the usable well size. At low gain (x1) the full well size is available. At high gain (x4) only a fraction (~ 20%) of the well size is available.

On-Chip Binning

On-chip binning during acquisition reduces the spatial resolution but also reduces the relative dark noise.

These effects result in the following

For lower light levels:

1. Use the high gain setting.
2. Binning during acquisition will increase the signal-to-noise ratio.
3. Slower frame rates will improve the signal-to-noise ratio by reducing the relative dark noise.

For higher light levels:

Saturation can be minimized by using low gain, faster frame rates, and not binning during acquisition. At higher light levels the noise is dominated by shot noise. In this situation binning after acquisition is just as effective as on-chip binning in improving the signal to noise ratio (while minimizing saturation).

 

For images with high contrast (both high and low light levels):

Careful consideration of the above trade-offs will be needed to optimize the information obtained from the measurement. Additional possibilities are to move the brightest part of the preparation out of the field of view or to differentially illuminate the preparation.

Dark Noise

Looking at single pixels we do not detect any systematic dark noise. However, we have seen systematic noise of about 150 Hz when spatially averaging (bin) many (>10) pixels together. The amplitude of this noise differs from quadrant to quadrant and from camera to camera.

 

Quadrant Differences

The FastOne camera has a four-quadrant readout. This allows a lower bandwith read amplifier for lower read noise. However, using the pseudocolor display in the Movie section, at high display gains, you will sometimes see small quadrant-to-quadrant differences in the signals. You will also see the quadrants clearly if you take one frame without subtraction.

 

Missed Frames

We notice occasional missed frames. If only one frame is missed, then the software offers the choice of interpolating or discarding the data. If two or more continuous frames are missed, then you must redo the trial. The number of missed frames depends on the CPU speed of the Dell computers which we provide. With a 400 MHz CPU approximately 1 frame per 2000 is missed; with 500 MHz, 1 frame per 10,000; with 800 MHz less than 1 frame per 25,000.

Synchronization

NeuroCCD uses a LED and software to synchronize the optical and electrical recordings. In our experience this works to within 1 data point. You can check it from time to time using the Include in Sync Test in the Acquire window.

Noise Pickup

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.

 

Smearing During the Frame Transfer

The FastOne camera has a frame transfer time of 26 microseconds. It takes 26 microseconds for the electrons of a pixel adjacent to the horizontal center line to reach the frame storage areas at the top and bottom of the active area. During this 26 microseconds, the electrons are at 40 different pixels and during their stay at each of these 40 pixels, electrons will be added in proportion to the light reaching each of these 40 pixels. These electrons will contaminate the signal. Thus, the signal on each pixel will have mainly correct electrons but a small proportion of photons will come from the wrong place in the image. This proportion will depend on the frame rate and the image contrast. At 2.7 kfps the fraction of time spent at the wrong pixels is 7.0% and at 1 Kfps it is 2.6%.

 

Problems caused by this smearing will be more likely to be noticed when the frame rate is high, the signal-to-noise ratio is large, and in dark regions of images with high contrast.

 

Cross-talk between BNC Channels

There will be crosstalk between BNC channels with signals and BNC channels with no inputs. You can get rid of this crosstalk by using a BNC Short (e.g. Pasternack Enterprises, PE6012) on the unused inputs.

Analog Output

The analog output function requires a DAP840 (but won't work with a DAP820) a-to-d card. To determine the type of DAP card you have:
a) Open the "Data Acquisition Processors" program in the Control Panel
b) Click on the "Browser" tab at the top of the form
c) Double click on the icon that looks like a PC (the second icon down) or click on the "+" to expand the tree
d) Click on the DAP icon and read the description below the browser window.

 

If you have a DAP840 you may need to create an empty text file with a name "dap840.txt" in "c:\rsi\idl52\" if there isn't one already.

 

If you don't have a DAP840and want to use the Analog Output function, contact Chun Falk at RedShirtImaging for instructions.

 

To use the Analog Output the connection inside the control box, DAC0OUT to the Analog Output BNC needs to be made. (Ground to proper ground.)

External Trigger

Because of firmware restrictions in the FastOne camera, this function cannot be implemented.