Start-up Procedures

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RedShirtImaging, LLC.

Optical Coupler (10:1)

The coupler has C-mount connections on each end. The end with the small diameter lens is screwed into the FastOne camera. The other end is screwed to a C-mount port on the microscope. The coupler has two adjustments, one for focus and the second for centering.

Focus Adjustment

The focus adjustment can be used to make the camera and the microscope eyepieces parfocal.

1. Focus on an object using the eyepieces.

2. Loosen the connection between the coupler and the camera.

3. Loosen the two Allen screws on the part of the coupler closest to the camera.

4. Using the Acquire Continuously function in NeuroPlex, rotate the part of the coupler closest to the camera until the focus is sharpest. (Items 1-8 of Start-Up Procedure)

5. Tighten the two Allen screws and the connection to the camera.

Centering Adjustment

The centering adjustment can be used to make the camera and the microscope eyepieces have the same center.

1. Center an object using the eyepieces.

2. Loosen the three Allen screws on the part of the coupler distal to the focus adjustment.

3. Using the Acquire Continuously function, adjust the three Allen screws so that the object is centered. Then tighten the screws.

Control Box

This unit is the external connection to the A-to-D board (DAP820/DAP840) and thus has the capacity of sending out TTL and other analog signals as well as accepting incoming signals.

Below is an image describing the inputs/outputs of this box. An equivalent image is glued to the top of every control box shipped by RedShirtImaging.

Inputs 1-8 :

These 8 BNCs are intended for input of electrical data (Voltage, Current etc..) to be recorded in synchrony with the optical data. Connect the output from your amplifier(s) to any of those inputs. The inputs are digitized at 12 bit accuracy.

Clock Trigger:

Connect "Shutter Sync" on the camera to this BNC. This way - the camera triggers the electrical data acquisition by the AtoD board. (see diagram in NeuroCCD Installation).

Shutter:

Connect this BNC to the "Pulse Input" BNC on your Shutter Control Box and set the later to "remote control" (Names relate to the Uniblitz shutter control and might vary). The data acquisition module of NeuroPlex controls the shutter via this output.

Stim:

Connect this BNC to the "External trigger" of your stimulator. Any delay desired between the start of recording and the stimulus should then be set on your stimulator.

The following functions are available for systems that have a DAP840 (but not DAP820) A-to-D cards ^{(1) (2)}

Analog Output:

Any signal sent by the NeuroPlex data acquisition function "Analog Output", will be output through this BNC. Thus - in order to send a voltage command or a waveform - this BNC should be connected to the appropriate external input on your amplifier.

⁽¹⁾ To determine the type of DAP card you have see: NeuroCCD  Hardware

⁽²⁾ If your system was shipped prior to October 2001 you might have to make the connections inside the box for these 2 functions. See: NeuroCCD-sm Hardware

Start-up Procedure

1. Turn on computer
2. Turn on camera
3. Start the NeuroPlex software
4. >> Acquire
5. >> CCD Camera (The message "Error resetting board" at this time usually means the camera is not on.)
   It is recommended that the following focusing procedure be done under low light levels to minimize possible photo-bleaching or photo-damage to the preparation.
6. >> Acquire One Frame (normally using Subtraction (auto shutter control) and Auto (min/max)).
7. Open the shutter.
8. >> Acquire Continuously and adjust the microscope focus on an object with good contrast. (You will need to have adjusted the optical coupler first.)
9. Close the shutter and increase light level to maximum.
10. >> Acquire One Frame (normally using Subtraction (auto shutter control) and Auto (min/max) to check that the light levels are neither too low or too high)
11. >> Histogram
    If the values are < 0.5 volts, then you may be able to increase the signal-to-noise ratio if you change the acquisition settings in a direction to increase the values (i.e. use a lower frame rate and/or high gain). Repeat steps 10 and 11 then go to step 12.
    
    If the values are > 3 volts and appear piled up at a high-voltage boundary, it is likely that many pixels are saturated. (Saturated pixels will appear as flat traces when you take data.) Look for piled up values of 7-10 volts. If it appears that there are a substantial number of saturated pixels, change the acquisition settings in a direction to decrease the values (i.e. use a faster frame rate and/or low gain). Repeat steps 10 and 11. If the values still appear piled up at a boundary at 2.7 Kfps and low gain, then the only way to avoid saturation is to lower the incident light intensity. (You can use a faster frame rate than you need for your signal frequency and still not save excessive data by using Temporal Bin after Acquisition.)
12. Select the acquisition parameters in the left panel.
13. >> Take Data