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A. Use only regulated DC light in the room. Switch off the AC light Use two 50mm Lenses attached back to back, as shown above. Turn the video-enhancement amplifier on. This will also turn on the camera.  B. Arrange for the DC light source to illuminate the "artificial cat" (shown below) instead of cortex . The idea is to do this test as close as possible to a real experiment.  C. From VDAQ load a parameter file appropriate for performing an LED experiment. Examples can be found in c:\VDAQ\2001TEST\led*.prm. or the VDAQ default parameter file. Also, you can refer to the example printed in the above section. It is important to select a "BLANK" stimulus for the interstimulus interval, that is, the parameter Interstim Stim should be set to "blank". D. (VDAQ/DOS). Select the Gray Reference Image & Gray Enhanced Image in the Calibration menu. Hit the [RET] key. You will see the output from the camera on the RGB video monitor (in gray levels) and the enhanced image will appear on the B/W monitor. Alternatively, without an RGB color monitor these images can be viewed on the console. (VDAQ/NT). Display the output of the camera using the camera setup dialog. E. Place the LED display under the microscope and focus. Position the "8" so that it appears in the center of the monitor. (At a macroscope gain of 1, objects of approximately 8 mm in length can be imaged.)  F. (VDAQ/DOS) Use the command Check/Experimental Stimuli or Check/Individual Stimuli turn the LEDs on and off. This should be apparent on the B/W monitor. The dim DC illumination from the room light should also let you see the dark outline of the LEDs (in addition to the highly reflecting surface of the LED). (VDAQ/NT)Use the Up/Down control on the Camera Setup dialog to turn on the individual LEDs.. The dim DC illumination from the room light should also let you see the dark outline of the LEDs (in addition to the highly reflecting surface of the LED). G. Control the level of the LED illumination itself with the potentiometer on the BNC front panel. To do that select the calibration menu and display the reference image either on the RGB monitor (optional) or the vga console. The LED light should almost saturate the illuminated pixels: lower the light level after you notice a few black pixels (at the center of the LED).  H. Place a light attenuator of x1000 (3OD) over the LED display (often called a neutral density filter). We also recommend that you cover the attenuator with a transparent but reflecting tape (e.g. a simple Scotch tape is adequate). In this situation the output from the LED is now attenuated by 1000-fold. (Edmund Scientific part numbers: M30,896 is a 10% transmission, M30,899 is 1% transmission; back to back they are 0.1%. Also note that a 1/1000 attenuator is part of the filter set sold by Optical Imaging.) Note: the focus of the camera should remain on the led and not on the attenuator.  I. To reproduce the biological situation and work under highest light level (providing the best signal to noise ratio), shine a background illumination on the attenuator. You want the "background" illumination to match (balance) the led intensity. Because the led intensity is reduced by 1000x by the neutral density filter, now the computer creates a tiny, imperceptible signal while turning the led digits on and off. This is best done with the light guide illuminators used for the real experiment. Without the transparent, reflecting tape, the attenuator may not reflect enough light into the lens especially if a black glass attenuator is used. Alternatively use the DC room light (if it is bright enough) and adjust the light level by closing the lens aperture. However, if you adjusted the aperture, you should readjust the led brightness (step g). Repeat step (g) to ensure that the image is below saturation. Under this condition the led pulse will modulate the background image of the attenuator by 1 part in 1000, similar to the intrinsic signal size.  J. Perform data acquisition for at least 2 conditions: One with the LED on and the other with the LED off. With the online maps, a modulation of 0.001 should be clearly seen after 1 trial without any averaging. K. Click here for led test results. 
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