Choose a filter set.
Wait until the computer boots and the screen reads "Ctrl-Alt-Del to logon"
and press the Ctrl-Alt-Delete keys to log on.The logon name is tcs_user and there is no password. Click OK or use the enter key.
The Delete key is not the same as the backspace key.
Setting up the Leica confocal software:
1. Start the Leica confocal software:
Double click on the TCSNT icon.
Wait until the hourglass turns to an arrow.2. Select the filter menu:
Click once on Acquire Images.
Choose a filter set.
If the fluorophore you are using isn't listed then choose one that is excited by the same wavelength.
Click once on the "Filters" button:
3. Configure the filter menu:
1. Select the Stain. Note: Don't worry if the fluorophore is not listed - this shows the emission spectra but does not affect acquisition.
2. Select the Clut. Start with GlowOvUn.
3. Activate the PMT's to be used.
4. Adjust the slit width and position for the emission of the fluorophores used. The slits must be at least 15nm from the laser lines used for excitation. Double click on the slit bar to get the numerical position of the slits.
Check for the appropriate dichroic:
RSP500 single label FITC, GFP
DD488/568 double labels FITC/Rhodamine or single labels with 568 excitation
RSP465 single label Lucifer Yellow, CyanFP
Substrat 2-photon imaging
TD488/568/633 use with triple labels or any 633nm excitation (Cy5, ToTo3)
Check the active laser(s) and the transmission through the AOTF (0-100%).
Laser lines not in use should be at 0.
Lasers in use can be at 100% except for the HeNe laser (633nm) which should be set at 33%. A setting of 100% means that all of the intensity is transmitted through the AOTF.
4. Configure the tool bar:
Click on the lens button and select the objective you will start scanning with from the list.
Properties of each lens can be found under "Settings" at the top of the screen and "Preferences". Note that the working distances and cover glass thicknesses in this table are in millimeters, not microns.
Mode: select XY for normal scanning.
Format: Selects the # of pixels to be collected:
128 x 64
128 x 128
256 x 256
512 x 512 - start here
1024 x 1024
2048 x 2048 - single channel only
Settings with a "2" should be used for 2 photon or live imaging. The phase will need to be adjusted.
Pinhole: do not use this button.
Use the F9 key to set the pinhole to 1.00.
A pinhole of 1.00 gives the best signal to noise ratio. A pinhole of 0.65 improves resolution.
The pinhole is not used for 2-photon imaging.
The objective lens needs to be set before the pinhole is adjusted.
When scanning, adjust the pinhole with the panel box.
Electronic zoom:
Start with a zoom of 1
