Start-up Procedures

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Initial Start-up Procedures

1.		Turn on computer

2.		Turn on camera (switch on power supply)

3.		Start the NeuroPlex software (click on NeuroPlex icon)

4.		>> Acquire

5.		Set the offsets
		>> CCD Camera  The process of calculating and recording the offsets for a specific camera is done by us before shipping it. Unlike some 8 bit A/D cameras, once these offsets were set for the NeuroCCD-SM, there is no need to change them during the experiments. Moreover, using 14 bit digitization one can resolve the dark noise in most gains and rates. Thus changing the offsets is not very useful. In some rare situations, an experiment could be improved by reducing the dynamic range (increase bit resolution) of the camera. If you believe this is the case, and would like to change the offsets, please refer to the help manual.

6.		Test the response to light using the LED
		We have already tested the camera with LED for synchronization. All the test files are stored on the system computer in C:\Data\Camera_Test\ .

7.

Installing the Optical Coupler

About the Optical Coupler
The coupler has C-mount connections on each end. The end with the small diameter lens is screwed into the SciMeasure camera. The other end is screwed to a C-mount port on the microscope. The following table identifies the four optical couplers that have been supplied by RedShirtImaging. 

Name	Serial #	Magnification	Length (cm)
C-1	080799	0.10	21
C-2	092999	0.08	11
C-3	091301	0.10	11
C-4	091301R	0.095-0.115	11

All four couplers have adjustments for focus and centering. The C-3 couplers have an additional adjustment for image quality. This adjustment allows the coupler to provide a high quality image on many microscopes. (If image quality is a problem using a C-1 or C-2 coupler, please contact us.) The C-4 couplers have, in addition, adjustable magnification (0.115 to 0.095X).

The C-1 or C-3 coupler that we sent with your camera is about perfect (to our taste) on our Leitz Ortholux-II microscope. But, to make it perfect we had to increase the inside diameter of the C-mount adapter on the microscope to 21 mm with a lathe. This seems like a good thing to do generally. Now the camera sees about 10% bigger field than the eyepiece and only a small section of each corner is dark.

To reduce the field of view that the camera sees by 50%, purchase a 2X amplifier (part # 29-30-42) from Thales-Optem, Fairport, NY, (585) 223-2370.

Note for Zeiss Axioscope 2FS microscope users: The 1 X optic coupler/tube (part number 45 61 05) for mounting CCD camera (small 30 mm camera port) comes with a removable ring (field stop), which blocks a large portion of the visual field.

For coupler C-4 start by setting the magnification to 0.10.

	7.a.		Focus Adjustment
			The focus adjustment can be used to make the camera and the microscope eyepieces parfocal. Screw the coupler onto the camera so that it is tight. This connection should not change. Focus on an object using the eyepieces. Loosen the two Allen screws on the part of the coupler closest to the camera. Using the Acquire Continuously function in NeuroPlex, rotate the part of the coupler closest to the camera until the focus is sharpest. This mechanical manipulation is tricky. It can be difficult to rotate, when the coupler is attached to the camera and microscope, without twisting the cables to the camera head. This will work relatively easy if the connection to the microscope can rotate without changing the distance. Repeated testing after rotations with the coupler detached from the microscope would be optimal. Tighten the two Allen screws and the connection to the camera.

	7.b.		Centering Adjustment
			The centering adjustment is used to center the microscope image on the camera. Loosen the three Allen screws on the part of the coupler just distal to the focus adjustment. Using the Acquire Continuously function, adjust the three Allen screws so that the object is centered. Then tighten the screws. This process can be tedious and also a bit tricky.

	7.c.		Image Quality
			This adjustment (for C-3 and C-4 couplers) can be made by loosening the Allen head screw on the side of the coupler and moving the screw head while examining the image using the Acquire Continuously mode. This adjustment should allow you to form an image without "pin-cushion" types of distortion. If you still have distortion problems after making the adjustment, contact RedShirtImaging.

	7.d.		Magnification adjust for the C-4 coupler
			Loosen the two "Mag" thumb screws and slide the coupler up or down to change the size of the image on the CCD. Following this change you must repeat the Focus and Image Quality adjustments.

8.

Setting Camera Amplifier Gain:

		It is recommended that the following focusing procedures (8a and 8b) be done under low light levels to minimize possible photo-bleaching or photo-damage to the preparation.

	8.a.		Open the shutter

	8.b.		>> Acquire Continuously
			...and adjust the microscope focus on the preparation. (You will need to have adjusted the optical coupler first.)

	8.c.		Close the shutter and increase light level to maximum

	8.d.		>> 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.

	8.e.		>> 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 step 8d then go to step 9. If the values are > 8.5 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 many values between 9 and 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 lower gain). Repeat step 8d. If the values still appear piled up at a boundary at 2000 fps and a gain of 1, 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.)

9.		Select the acquisition parameters
		Most functions are controlled in the left panel; the parameters in the center panel are used for signal averaging. Additional information about the acquisition functions can be found in the on-line manual.

10.		>> Take Data

		For more tips on how to optimally use the camera settings, Camera Gain Settings  and  Optimal use of the CCD Camera