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Wellcome Trust Centre for Human Genetics Cellular-Imaging Microscopy Core

The Leica SP8-X SMD Confocal

Abridged INSTRUCTIONS

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Introduction The Leica SP8-X SMD confocal microscope is located in room 00/075, Lab 1. The microscope is suitable for both live cell and fixed specimens on slides, and it will run alongside the old Zeiss LMS 510 MetaHead confocal.

The new Leica SP8 has:

• An inverted DMI 6000 AFC microscope for live cell viewing • Full live cell chamber with temperature, CO2 and humidity control • SuperZ Galvo Stage and hardware Auto-Focus • 10x & 20x HC PL Fluotar air objectives, and 40x & 63x HC PL Apo oil CS2

objectives • HC PL Apo x63 Mott Corr Water CS2 Objective with software control of the

correction collar • Transmitted Light Brightfield Detector • Inverted motorised scanning XY Stage including Mark & Find and

Autostitching/Mosaic Modules • Passive anti-vibration microscope air-table • AOBS filter free scanhead • WLL2 tuneable pulsed single diode laser with 200 lines from 470 to 670nm,

including Pulse Picker for FLIM (80, 40, 20 10 MHz rep. rate) • 405nm Diode Laser for DAPI • Two Internal Spectral Detector Channels (PMT) • Two Internal Spectral Detector Channels (HyD) GaAsP • SMD Excitation: 440nm laser pulsed for FLIM • SMD Emission FLIM+FCS 2Ch HyD Fibre coupled X1 port • SMD Filter Cubes Alexa488/Alexa633 and CFP/YFP, others available • Twin premium Workstations and 30 inch Monitors • Software Modules for all Acquisition modes, 3D, FRAP/FRET/FLIP, Spectral

Dye Separation, Online Dye Separation, Live Data Mode advanced live cell imaging module, Image Processing, Measurement & Reporting

• Extra licence for PQ ST Offline Software FCS_FLIM

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General information The temp files during scanning are stored in C:\Temp\ which is generally fastest on the C: drive. If the system crashes you may be able to recover images from there.

On the Belkin USB monitor selection box under the monitors, the LED shows yellow when the Leica SP8 Master PC monitor is active and green when the PicaQuant Slave PC monitor is active. There’s a small button on the front left of the USB box to switch between PC monitors, but this shouldn’t be necessary as the mouse curser can move across both PC desktops to select that PC for keyboard input and mouse control

Shut down the PicaTech Slave PC last first and switch on the Slave PC last (the Leica SP8 Master PC goes on first). Log in with the same WTCHG account on both PCs.

In the Leica LSM software, if you set the WWL to Constant Percent, at say 10% the actual laser power will vary depending on the laser-line selected. If you set the WWL to Constant Power, the power output of the WWL will be adjusted automatically to give the same power level at every wavelength, so say 10% will be approximately the same laser power at every laser line.

Pulse Picker gives the best image quality at the highest repetition rate.

Make sure the XC-Port is in ‘mirror’ position to block off any of the SMD detectors.

When setting up a Tile Scan with the motorised stage, be careful not to accidently click on the XY stage grid pad as the stage will move rapidly to that XY position.

There are hardbound manuals for the DMI6000 microscope and the Leica SP8 software/hardware. These are also available as pdf files in the both Master and Slave PC hard drives.

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The Leica DMI 6000 Microscope Turning the right hand microscope focus control clockwise (away from you) raises the objective (up). Turning the focus control anti-clockwise (towards you) lowers the objective downwards (down).

The SmartMove remote control module on the desktop controls the motorised XY stage and the motorised z focus. The top rotary knob controls stage travel in X (across) and the bottom rotary knob controls stage travel in Y (up/down). The rear rotary knob is the z focus.

Change objectives using the LAS AF software as you can directly select the objective you want and it will allow you to go from air to oil objectives via a software ‘yes/no’ prompt.

The microscope uses standard Zeiss 518F immersion oil. If oil drips off the bottle applicator in use, you have too much oil on it. To avoid oil contamination of air objectives, never change from an oil to an air objective when focussed on the specimen under oil (you can start with the low power air objective and then move up to the hi-power oil objective).

The large HC PL Apo x63 Mott Corr Water CS2 Objective with cable control of the correction collar is for use with water immersion only – not 518F immersion oil. Make sure you don’t select it in error when using the 63x oil objective. The water immersion objective is easy to spot as the large correction collar motor sticks out of the side of the objective.

For live cell work switch on the 37oC stage incubator at least 2 hours before use, or ideally the night before. Type the words ‘Live Cell’ in your booking to tell other prior SP8 users that the 37oC incubator heater is being left on for your later live cell imaging slot.

We have a small 5% CO2 in air gas cylinder that can be used with the SP8 live cell incubator. CO2 can be used with 2.5cm Mattek petri dishes, Labtek 3x1” glass slides and multiwall plates using the appropriate stage insert.

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To transfer user hardware control to the Leica DMI 6000 microscope from the LAS software when looking down the microscope, press the diochroic filter selection button (e.g. DAPI) and then ‘shutter’. Also use the fluorescence/transmitted light toggle button on the bottom left side of the microscope.

The LCD screen on the microscope is simply a display screen (it does not have a touchscreen).

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SmartMove XYZ Stage Controller

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Switch on Procedure 1) Switch the epi-fluorescence Xe mercury lamp power box on 2) Push the DMI6000 microscope top stand/condenser backwards

The motorised stage undergoes a fast initialisation sequence that moves the stage rapidly to its end limits

3) Switch the PC Microscope rocker switch on (triple Green LED switches on the right of the desk)

4) Switch the Scanner Power rocker switch on Note that the Scanner Power must be on at least 30 seconds before you start the Leica SP8 confocal software

5) Switch the Laser Power rocker switch on 6) Turn the Laser Emission key on

The White Light Laser (WLL) box will read 70% power by default. The WLL goes to standby automatically if not used for a minute or so, but flicks back to full power when scanning

7) Switch on the black mains distribution block on the rack on the far right (Red LED rocker switch) Note: this controls power to the PicaQuant PC and both PC monitors

8) The Leica SP8 Master PC monitor will switch on and this will start up the PicaQuant Slave PC automatically

9) Press CNTRL-ALT-DEL to activate the login popup on the Leica SP8 Master PC. Log in either with your Wellcome Trust Login account (WTCHG network/internet access) or as Administrator (PC only no network/internet)

10) Move the mouse curser to the PicaQuant Slave PC and click on the screen. Press CNTRL-ALT-END to activate the login popup on the PicaQuant Slave PC. Log in with either your Wellcome Trust Login account (WTCHG network/internet access) or as Administrator (PC only no network/internet)

11) Start up the Leica ‘LAS AF’ laser scanning software and select the machine.xlhw and DMI6000 options

12) Select the objective required using the LAS AF software Ensure that the 40x and 63x oil objectives are not the ‘travel’ lock down position: turn anti-clockwise to release the objective top (ask if you need more information on this)

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Switch off procedure 1) If used, clean the oil/water immersion objective lenses with Lens Tissues

The 10x and 20x air objectives don’t need cleaning after use 2) Leave the 10x objective selected on the microscope nose-piece to

minimise any possibility of damage during start-up when the XY motorised stage initialises.

3) Drop the 10x objective to the load position (lowest) via the microscope’s focus down button or the microscope coarse focus knob

4) Exit the Leica LAS AF software 5) Shut Down the PicaQuant Slave PC first

Note: you can push the Slave PC mains on button quickly to shut down the PicaQuant Slave PC if you accidently shut down the SP8 Master PC first

6) Shut Down the SP8 Master PC Transfer any data files to the network drives first

7) Turn the Laser Emission key to off There’s no argon laser to cool down and the White Light Laser (WLL) goes to standby automatically on exiting the SP8 LSM software, so you don’t have to wait for any of the lasers to cool before switching off

8) Switch the Laser Power rocker switch off 9) Switch the Scanner Power rocker switch off 10) Switch the PC Microscope rocker switch off

Both PCs must be OFF first 11) Switch off the distribution block on the rack (Red LED button)

Note: this controls power to the PicaQuant PC and both PC monitors 12) Switch the epi-fluorescence Xe mercury lamp power box off

If you notice or encounter any problems when using the Leica SP8 microscope, please write the details in the SP8 log book provided (A5 black diary).

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________________________________________________________________ If you have any microscope/imaging problems or queries, just ask a member of the Microscopy Core staff for help. There is additional help and advice on our Core web-pages: http://www.well.ox.ac.uk/microscopy WTCHG Leica SP8-X SMD confocal microscope user guide. This version updated 11th March 2014.