TITLE: Leica SP8 Confocal
AUTHORS: Condon ND

[DESCRIPTION]
This is a training guide for the Leica SP8 Confocal Microscope at the Insitute for Molecular Bioscience at The University of Queensland, Brisbane, Australia.

More information about the system is available here: https://imb.uq.edu.au/microscopy-confocal-1

[STEPS]
SECTION: System Start Up
1. Using the Microscope Power Switches, turn on the system in the following order
Turn on the PC by the power button on the top right of the tower.
Turn on the PC Microscope Switch, wait until the PC is powered on and you have logged in with your AD credentials
Turn on the Scanner power, wait 10 seconds
Turn on the Laser Power, wait 10 seconds
Turn the Laser Emmission key to the ON position


 

SECTION: System Start Up
2. Turn on the fluorescent Lightsource
Press the "LAMP ON" icon on the desktop of the computer
If the lamp doesnt start, ensure it is not in "Cooling Mode" by checking the Yellow LED isnt illuminated. If it is illuminated, wait 5 minutes before trying again.
The Lamp will run for 90 minutes, if you need more than that, a beep will be heard at 5minutes before it is powered off.
SECTION: System Start Up
3. Start LAS X Software
Double Click on the LAS X icon on the desktop
When the Startup screen appears ensure the following set (Configure = "Machine without climate chamber" and Microscope="DMi8"
NOTE: AFC cannot be used for STED
NOTE2: If you wish to change between modes (ie. change to resonant scanning mode) you will need to close and restart the software.

 

SECTION: Finding your sample
4. Selecting an objective
Use the LCD touch screen on the front of the microscope to choose the relevant objective. Ensure you understand which immersion media shuold be used on each objective (10x/40x Dry; no media applied, 40x Water, 93x Glycerol, 100x Oil.
Note that the immerion media is temperature dependant so choose the correct one (23/37 degrees)
NOTE2: Clean objectives before and after use with Lens tissue only (no solvents)
Press the objective Icon on the microscope LCD screen to change into objective mode
Press one of the buttons to change to that particular objective
NOTE3: If the button is flashing it is indicating a change in immersion media has occured and to check that the objective and sample has been cleaned.

 

SECTION: Finding your sample
5. Mount  your specimem
Use the appropriate stage insert (universal, slide/dish or multi-well plate). 
NOTE: Be very careful when mounting holders or samples as the stage is sensitive to pressure and can be easily damaged. Holders are held in by tiny screws, talk to facility staff if you havent been trained to change them over.
Mount your sample coverslip down or in the holder as appropriate.
Change to the XYZ menu on the LCD touch screen
Press the two front buttons (XY course & Z course) on the joystick
Use the two larger knobs on the front of the joystick to centre your sample over the objective.

 


SECTION: Finding your sample
6. SP8 Specific considerations

MotCorr Cord
Be mindful of the cord. The MotCorr is powered and tethered by a cord that can:
Get caught in stage windings
Limit the objective turret rotation
Push up on the slide such that focus cannot be achieved

Galvo Stage Holder
The galvo stage cannot bare weight, do not push down on the stage as it will damange it.
The galvo stage needs very small screws to hold the inserts in place, these can be easily lost.

Quarter wave plate slider
STED imaging requires the quarter wave plate to be engaged below the objectives, accessibly on the right hand side of the stage.


 
This quarter waveplate might dimminish the number of photons collected in other imaging modes so move it to the "OUT" position before imaging. It is fully out when you can see the writing on the insert.

 


SECTION: Finding your sample
7. Illuminating your sample 
Pres the FLUO icon on the Microscope LCD screen to change to the eyepeice mode.
Select one of the FLUO-filter cube buttons to select a filter cube that matches your sample
Press the IL-Shutter  to illuminate your sample
Alternatively you can use transmitted light modes to illuminate your sample (BF/DIC/POL)


 

SECTION: Finding your sample
8. Finding focus
Use the single rear knob on the joystick to raise the objectives to focus on your sample.
Press the Z Fine button on the joystick base and fine tune the focus on your sample.
Press the XYZ icon on the microscope LCD Panel to go to the stage mode menu.
Press the Set/Clear buttons to clear the previously stored focus position, before adding your own infocus position by pressing "Set". This allows you to quickly lower and raise the objective between samples.
Close the IL-Shutter to prevent bleaching of your sample under the Fluo menu.

 

SECTION: Setting up an Aquisition
9. Software Lightpath

 
Laser Lines. To activate a laser line, either click on the box corresponding to the relevant wavelength, or the "+" symbol for any of the WLL lines. Note if using the WLL you can click and drag the laser line to the relevant wavelength. Double-clicking on the laser will reveal specific notch filters for common wavelengths.
Objectives.  This drop down menu lists the objectives currently on the microscope and also shows the immersion media (e.g water, glycerol, oil).
Software tools. Here buttons provide shortcuts to the menu for assigning controls to each knob, the Dye Assistent and filter controls.
Emission spectrum. Active detector gating is visible, and active lasers appear as vertical lines.
Detectors (HyD and PMT). Detectors are arranged from left to right (HyD1, PMT2, HyD3, PMT4, TLD) You can toggle them on and off, and adjust their gating by clicking and dragging.
SECTION: Setting up an Aquisition
10. Turn on the Lasers
Click the laser overview button at the top right of the lightpath window
Turn on the relevant lasers for your imaging setup.
NOTE: STED lasers will only be accessible if STED has been activated at startup
NOTE: STED lasers should be turned on to 100% laser power
NOTE: WLL (White light laser) should be turned to 70% laser power.

 

The WLL does not have equal power across all wavelengths, instead the software by default corrects this for you if you "Constant Percentage" enabled.

405 Diode (UV) laser is for DAPI
440 Pulsed laser is for CFP FALCON (FLIM/FRET) imaging
442 Diode laser is suitable for CFP imaging
WLL (470-670nm) is suitable for all other fluorophores
SECTION: Setting up an Aquisition
11. Setting up Laser lines
Adding a laser line to your acquisition can be done by selecting either the diode buttons (1) or the "+" button (2) for WLL lines.
To engage the laser, the small tick box next to the wavelength indicator must be selected
To open the shutter on the relevant laser, the small red circle must be selected (3). NOTE when laser power is se above 0% and all the shutters are open, the lightpath will change from white to orange.
When using the WLL, specific notch filters (4) exist for common lines.
Move the slider up to increase laser power (5)
 

SECTION: Setting up an Aquisition
12. Main Acquisition Parameters
In the top left hand corner of the LAS X program window is the main control panel for setting up acquisition parameters.
Select the type of acquisition you wish to capture from the drop down (1). NOTE for a normal 3D acquisition select XYZ, other modes include time (t) or excitation wavelength (λ) or detection wavelength (Λ)
If you require 'complex' imaging modes select one of them using the buttons (2). These include Navigator, FALCON, Autofocus and Sequential Scan.
Ensure you final image has enough pixels for Nyquist by clicking the symbol (3) to automatically calculate it for you. Alternatively, manually chose a relevant image size from the dropdown.
Scan speed is measured in Hz in LAS X. Meaning the number displayed is the 'number of lines per second' (eg this image of 512 lines @ 400Hz would take ~1.2s to acquire.
Depending on your imaging setup you may wish to acquire bidirectionally. The toggle (4) allows you to turn this feature on. (Note using bidirectional scanning will decrease your acquisition time by half).
The field of view can be zoomed in (5) by adjusting the slider or by selecting "ON/OFF" toggle and drawing a ROI over the image area you wish to acquire.
TO reduce noise, and/or increase signal in your images you may need to perform either averaging or accumulations (6). These can be done on either the line level or frame level. Note you can only perform an average OR accumulation for each acquisition, you can for example do 2 line averages and 3 frame accumulations.
Ensure the pinhole is set to 1AU for confocal imaging.

 

SECTION: Setting up an Aquisition
13. Detector Settings & Adjustments
Generally, detector ranges should not need to change between sequences as this is a slow process. For example, for the following setup, detectors are set to a sample containing DAPI, Alexa488 and Alexa568. In this scenario all 3 dyes will be imaged at the same time, (which may result in cross-talk & bleedthrough). To overcome this, simply duplicate the sequence and turn off PMT2 & 488 laser on one sequence, and HyD1 & HyD3 and the 405 & 561nm lasers on the alternate sequence.

 

Setting up this imaging experiment
Working from left to right (in emission wavelength) and top to bottom (in detector number) turn on HyD1 and set its look up table to be blue.
On the right hand side of the detector window, set the dye to be DAPI
Adjust the detector range for HyD1 to cover most of the DAPI emmision curve shown above (e.g. 415-478nm) and set a gain of 100
Turn on PMT2 and set its look up table to be green and set its dye reference to be Alexa488
Adjust the detector range for PMT2 to cover most of the emission curve shown above (e.g. 498-551nm) and set a gain of 700.
Turn on HyD3 and set its look up table to be red and set its dye reference to be Alexa568
Adjust the detector range for HyD3 to cover most of the emission curve shown above (e.g. 571-747nm) and set a gain of 100.

Note: For this setup lasers have been set for 405nm, 488nm and 561nm. Leave 10nm either side of the laser for all detectors. NEVER place a laser line over a detector range.

Note: HyD1 & HyD3 are gated detectors. Gating the detectors will remove autofluorescence and laser reflection/scattering. When in use select the relevant laserline from the WLL in the dropdown on the right hand side.

Note: Gain may need to be adjusted for either HyD or PMT detectors. If you find you require high laser power (>30%) and HyD gain >400, or PMT gain >1000 talk to microscopy facility staff.

SECTION: Setting up an Aquisition
14. Image Window Controls

 
Note: In LAS X the toggle buttons display what you are ‘on’ now, not what you would like to do. Ie above on the right hand side the second button displays multi-channel display mode, select this to change to overlay window mode.

Note: When adjusting brightness scale select the window first to choose the active channel to change.

Note: If you need to change the LUT of your channel after acquisition right-click on the displayed LUT on the left hand side of the image display window.
SECTION: Setting up an Aquisition
15. Using Range Indicator to correctly set up your channels
In order to correctly set up you image acquisition parameters the range indicator LUT is a tool which should be used.

The LUT ranges from Green > Black > Red > Orange > White > Blue

With Green = 0, and Blue = 255 (saturated)

 
Note: To access this look-up-table select the LUT tool from the top left button in the image control panel above.
SECTION: Setting up an Aquisition
16. Setting up multiple sequences
Turn on sequential scanning mode by selecting "SEQ" at the top of the acquisition panel.
Using Seq.1 set up your lasers and detector ranges for all channels
Press the "+" button in the Sequential Scan tool window to duplicate the current settings
Turn on and off relevant detectors and lasers for each sequence
Ensure you select "Start" to acquire all sequences.
NOTE: for STED "Between Frames/Stacks" is required not lines.

 

SECTION: Setting up an Aquisition
17. Leica Dye Assistant
Similar to other venders’ solutions, Leica has a wizard for automatically configuring the light path for you, called Dye Assistant. It is not as thorough, nor as complex as other solutions so use it carefully!

 
Select the Dye Assistant icon from the Tools panel (Middle of the lightpath panel)
Add your labels using the "+" symbol. NOTE if your label isnt in the database please select a simialr dye or speak to facility staff.
Once you have selected your dyes you can indicate whether you need a HyD or PMT or either. Remember a HyD is more sensitive so is suitable for dimmer dyes.
Next select one fo the options generated by selecting Apply.

 
Remember it is important to reduce crosstalk as much as possible.

The least number of sequences is generally the fastest during your capture, but this option may result in high levels of crosstalk. If more than one sequence is required (most often the case) choosing a “Line Sequential” option will generally be faster than “Frame Sequential.” To increase signal, detectors can be set to capture more of the emission range, however these adjustments are slow, and can only be made between frames, during line sequences, detector positions cannot be changed.
SECTION: Setting up an Aquisition
18. Reusing previous settings
Load a .lif file into LAS X
Right click on the project file and select Apply Image Settings
Note: It won't apply settings such as XY stage position, Z position, however it does recall laser power, laser lines used and Z-step size.
SECTION: Setting up Multi-dimensional Acquisitions
19. Setting up Z-stacks
Ensure you have an acquisition setting chosen that includes Z dimension
Use the Z-drive (Galvonic Stage) controls on the USB controller to navigate to the top or bottom of the sample and select begin. (NOTE: not the joystick)
Move to the the top of your sample and select End

Notes:
You can quickly move to the beginning/end by selecting the arrows next to the respective buttons
System Optimised will chose Nyquist settings for your objective and wavelengths used
Begin and End are independant of top and bottom

 

SECTION: Setting up Multi-dimensional Acquisitions
20. Image Capture
Single image Captures the current XY image, with the single sequence.
Capture image captures the XY image with all the sequences and saves it to the project directory
Start captures the full experiment including channels, z-stack and time points if activated.
 

SECTION: Setting up Multi-dimensional Acquisitions
21. Time Series
Repeatedly collecting data from live samples over time will capture the sample dynamics. Selecting “t” in the image acquisition mode will enable another menu to set the time intervals.

Clicking “Minimize” will show you the estimated time to for a single image acquisition cycle. It is advised to select a time interval that is a multiple of 30 sec to give your sample time to recover from
light exposure.

The total time of the acquisition can be set below. 
Acquire until stopped can be used if you are unsure of the timing of the sample dynamics
Duration can be set to have defined end point, e.g. 6 hours
Cycle can be set if you just want to see a set number of timepoints e.g. 100 cycles.

 

It is likely that when acquiring a long timelapse, that the focus may drift, either due to the sample settling or moving. Enable autofocus which is a software-based digital autofocus. That will find the best focus within the sample and keep that Z-position as the middle of the Z-stack. Choose the best channel for the software to do the autofocus on by choosing the focus mode. Under "timelapse" you are able to choose whether this autofocus is performed every cycle or every nth-cycle, as this will introduce a time delay to perform a z-stack range to find the best focus.

 

 

SECTION: Leica Navigator
22. For multi-position acquisitions or tile scanning of your sample, enable Navigator to launch a new window that has interactive control of the stage.

 

 

SECTION: Leica Navigator
23. Multi point, tiled region and focus map
At the lower edge of the screen, you find the following buttons for creating scan regions and scan region tools.

 
Mark DO NOT USE THIS BUTTON
Selection Tool: Allows you to select one or more plotted regions (scan regions)
Single Image: Marks the current position (x,y) and adds it to the list
Polygon tool: The polygon tool can be used like a free-hand tool, to select the region for tiling
Rectangle: Rectangle tool to select a region for tiling
Elipse: Elliptical tool to select a region for tiling
Circle: Circle tool to select a region for tiling
Magic Want: Detects the boundary of the object and plots a scan region that completely encloses the object.
Focus Map Point: Sets a point for the focus map, refer also to Focus Map
Auto-focus Point: Sets a focus point for the automatic focus search if the focus search is only to be carried out at one position, see also Autofocus> Stage >First Position only. The focus search is performed at the position of the nearest tile.
Keep drawing tool: The selected plotting tool remains active so that you can plot more regions using the same drawing tool. If this function is disabled, the selection tool will automatically become active again after plotting is done.
Select all Shapes: Selection tool for all plotted scan regions.
Delete Select Shapes: Deletes all of the plotted scan regions
Hide: You can hide individual scan regions. These regions will not be considered when saving using Save Overview...
Focus Map: Opens the dialog for the definition of the focus map, refer also to Focus Map.


 
To Close the navigator window, use the "Close" button and not the "X" in the top right.
SECTION: Shutdown procedure
24. Remove your sample and wipe the objective lens with a clean lens tissue
Save your files to the local data drive and begin copying them to a backed up location
Close down LAS X (NOTE it can take a couple of minutes to perform system checks)
Shutdown the computer
Turn off the laser interlock key
Turn off the switches from right to left pausing 5 seconds between (Laser Power, Scanner Power, PC Microscope)
Leave the room clean and tidy for the next user.
SECTION: Leica Material - Light Path
25.