Centre Optical Instrumentation Laboratory

Basic Acquisition Settings

Configuration Dialogue

Select Configuration Tab, and then click on Laser icon

Laser Dialogue

1. Switch on the lasers needed for the acquisition
2. 405nm Use for DAPI, Hoechst and will also work for CFP
3. Argon Select the power required, 20% is usually enough for most imaging applications. This laser has several lines. Use 456nm for CFP, 488 for GFP and 514 for YFP
4. DPSS 561 Use for RFP or TRITC
5. HeNe 591 Use for Texas Red
6. HeNe 633 Use for Cy5 and far red dyes

Acquisition Settings

1. Select Acquire Tab
2. Select Acquisition
3. Select Image Size in Pixels (Nyquist Sampling)
4. Select Speed, Faster imaging less photobleaching but poor image quality
5. Set Pinhole Size For most applications Airy 1 to 3 is OK, wider pinhole
results in poor z resolution. Note if you open the pinhole you can always deconvolve the image to restore some of the lost resolution.
6. Set zoom factor taking note of Nyquist sampling.
7. Line average/Frame average. For normal scanning average 3-4 frames but note this can lead to photobleaching. If using the resonant scanner average around 16 times for best image (unless specimen is very bright)
8. There is a rotation of 90 degrees between eyepieces and the scan head, type 90 into the box and the confocal will image in the same orientation as the eyepiece view

Laser Settings

1. Make sure the laser group you are using is actually activated. Do this by clicking on visible, UV or MP. The laser won't work if there is no red dot.
2. Set laser power. 20% is generally a good starting point, balance laser power and PMT gain setting to produce the best possible image with the lowest photodamage

Spectrophotometer

1. Select the appropriate fluorophore emission curve this will act as a guide for setting the spectral detector
2. Choose a colour for your fluorophore colour
3. Make sure there is a tick in the Active box to turn on the PMT detector
4. Set the spectral detector for your choosen fluorophore. Note if there is no dark shadow under the spectral slider bar then that PMT is not active

Live Button

The Live button is used to optimize all the above parameters, the image produced cannot be saved. This is purely for set up.

Capture Image Button

Capture Image will collect a single plane image which can be saved.

Start is used to image z-stacks, time-lapse, tiled images.

Live Button

When you click on live for the first time no image will be displayed. This is because you have not applied any gain voltage to the PMT

Gain Controls

As you turn the dial labelled 1 (smart gain) an image will appear as the gain approachs 600. At this point you are ready to optimise your image

Optimizing the Image

Live Button

Roughly set up the imaging parameters as described above then click Live

On the right hand monitor click on the glow over/under icon

The fluorophore look up table (LUT) has been replaced with an LUT showing over and under exposed pixels. In order to make maximum use of the dynamic range of the detectors ie 255 levels of grey for 8 bit imaging or 4095 levels of grey for 12bit imaging laser power and gain need adjusting.

Adjust the gain laser power and offset until there are a couple of green pixels (black) and a couple of blue pixels (overexposed) the system is now set up to image using the full dynamic range

NOTE If the specimen is not particularly good this may not be possible. In which case do the best you can,as the more of the dynamic range you use the easier any image analysis will be.