Page Comparison

Starting up

1. Remove the dust sheet from the stand and put it in the plastic storage box
2. Turn on the EL6000 light source
3. If the Shutter Open LED is off, press the Shutter button
4. Turn on the Supply Unit by flipping the green rocker switch and turning the Laser Emission key from 0 to 1
5. Wait for Windows to boot into the TCSSPEUser account
6. Wait for the hourglass cursor to turn into the pointer
7. Double-click the LAS AF icon on the desktop
8. When the splash screen appears, check the Configuration is set to Machine and the Microscope is set to DM Manual 6
9. Click Open and wait for the software to launch
10. Place your specimen on the stage and focus using a suitable objective lens

Setting up the Beam Path

1. Click on the Beam Path tab
2. Select the correct objective lens from the Objective drop-down menu
3. If you have settings saved on the machine you can load them by selecting them from the drop-down list labelled 'load/save sequential setting'
4. You can also Apply Settings from the Properties of a previously saved experiment. Right-click an image or series in the Experiments tab and select Properties
5. If you have no saved settings then add as many channels as you need for the fluorophores in your specimen by clicking the plus symbol below the Scan 1 button
6. Set up the laser for the first channel by clicking the Visible button above the laser power sliders to open the laser shutter and dragging the slider up to 10%
7. Use the drop-down menu under the PMT1 control to superimpose the emission spectrum of your dye (or a similar dye) on the representation of the visible spectrum
8. Use this as a guide to setting the emission range by dragging and resizing the slider beneath the spectrum
9. Apply a suitable colour look-up table (LUT) for the image by clicking on the coloured bar next to PMT1 and selecting a colour from the list
10. Set the PMT1 gain to 800 volts
11. Click the Live button to initiate a continuous scan of the specimen. You can click it again to Stop the scan
12. Click the Quick LUT button in the toolbar on the left of the image window to apply the Glow Over/Under LUT
13. Saturated (too bright) pixels will appear blue in the Glow Over/Under LUT; black pixels (zero value) will appear green
14. Ideally you should adjust the laser power and gain so that there are a few scattered blue pixels in the image. This might not be possible if your specimen is very dim
15. Use laser power to increase the amount of fluorescence from your specimen but beware of photo-bleaching and saturation at high powers
16. Use gain to amplify the signal from your specimen but be mindful of amplifying noise as well
17. Use the Offset slider to adjust the image so there are a few scattered green pixels in the background areas
18. Repeat the process for each channel, specifying a suitable laser, LUT and emission range for the dyes in each channel

Setting up an Acquisition (single slice)

1. Click on the Acquisition tab
2. Set the resolution of your scan by adjusting the zoom to an appropriate setting and the Format to a suitable size
   • High zooms bleach more quickly than low zooms
   • Large scan formats give high resolution with a broad field of view but are slow to scan and have more noise
   • Small scan formats are fast to scan and can have high resolution but need more zoom and so bleach more quickly
3. If you require optimum resolution you can use the Confocal Resolution Calculator to work this out
4. Set the Speed to a suitable level and specify Bi-directional scanning if necessary. 400 Hz and unidirectional are usually fine for fixed specimens
   • Increasing speed increases noise
   • The scanner will automatically jump to zoom 3 at 800Hz speed
   • You will need to adjust the phase correction slider in bi-directional mode to line up the forward and reverse scan
5. Click the Pinhole button and then click the 1 Airy button to optimise the pinhole size and hence the axial resolution
6. Use the zoom arrows to pan around the area if the features of interest are not centred
7. Set the number of frame averages required to reduce noise in the image. 4 to 8 is usually suitable. All channels will be averaged
8. Click the Capture Image button to capture an image of the currently selected channel (specified in Beam Path)
9. Click the Start button to capture images of all of the channels
10. Use the overlay button in the toolbar on the right of the image window to bring up an overlay of all the channels

Setting up an Acquisition (Z series)

1. Click on the yellow plane between the Begin and End arrows in the Z-Stack control on the left
2. Move the track wheel of the mouse away from yourself to lower the focus of the specimen
3. Click the Begin arrow (turns from black to brown) to set the start position of the Z series
4. Move the track wheel towards yourself to move the focus up
5. Click the End arrow to set the end position of the Z series
6. Use the 'Go to' button to go to the centre plane if necessary
7. Leave the number of steps on System Optimized or use the Confocal Resolution Calculator to work out the optimum step size
8. Click on Start to acquire a Z series of all channels
9. Use the scroll bar on the right of the image to scroll through the planes
10. Use the maximum intensity projection, orthogonal projection and other tools in the toolbar on the right of the image to change the view on the data

Saving data

1. Click on the Experiments tab
2. Rename the images or series in the directory by right-clicking and selecting Rename
3. Right-click the Experiment (top level item in the structure) and select Save Experiment 'Experiment' As
4. Save the experiment in your folder on the server. The data will be saved as a LIF file

Shutting down the system

If no-one is booked on the machine for three hours after you, you must shut the system down

1. Save all experiments
2. Quit the software
3. Shut down Windows from the Start menu
4. Turn the Laser Emission key to 0 and switch off the Supply Unit
5. Switch off the EL6000 light source
6. Wipe the objective lenses with lens tissue
7. Cover the microscope stand with the dust sheet