Confocal 6 Guide. Switching On Components of the system should be switched on in the following order: 1. Lasers Argon laser (488nm and 514nm)

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1 Confocal 6 Guide The system is kept at 37⁰C. If you require the system at a lower temperature book at least one hour before the beginning of your session to allow the system to cool down. It is your responsibility to turn the heater off before your session and to turn it back on after your session. Make sure the air conditioning is on, as changes in room temperature can affect temperature within the unit. CO 2 is available if required. Switching On Components of the system should be switched on in the following order: 1. Lasers Argon laser (488nm and 514nm) 5. Turn key to horizontal to switch laser on. 1. Switch on mains power. Lights come on. Fan will come on 2. Make sure small switch on circuit board of the argon laser is pointing towards you (in STANDBY mode). 4. Make sure laser power dial is turned fully anticlockwise to minimum. 3. Lights are on when system is in standby 568nm laser Turn key to horizontal position. Orange laser caution light will come on.

2 2. Laser control box 2. The small green light on the top of the box will come on when the lasers are on 1. Turn on red switch and key on box Turn on the rest of the system in the following order (devices are numbered as below) 3. Synchronisation box on shelf (switch at rear) 4. Power supply to microscope (behind microscope) 5. Camera. Switch on 20min before imaging. 6. Proscan Box (filter wheel) 7. Fluorescent lamp 8. Start up computer and software Log in to computer using UOB username and password. If you are logging in for the first time, please ask one of us to set up master preferences for you. Open Volocity software Volocity login: User ID Password Configuration Press connect or confocal_6 leave this blank acquisition acquisition + FRAP (for FRAP)

3 Setting up microscope for use Identify the lens you want to use. The lenses on confocal 6 are: 10x air (yellow ring) 40x oil (light blue ring) 63x glycerol (thin red ring) 100x oil (white ring) To move a lens into position you must first lower the lens turret completely (using the Smartmove focus dial) before turning the lens into position (otherwise you can damage the lenses by scratching them on the underside of the microscope stage). You may need to unscrew a lens and screw into position yourself. Great care must be taken to avoid touching lens top and bottom and dropping the lens! When rotating the lens turret by hand, take care that the wires connected to the piezo drive do not get tangled. If there is resistance to the lenses rotating, try rotating in the opposite direction. If capturing z stacks it will be beneficial to screw the lens you are using in to the piezo focus drive (faster z movement than with the microscope focus drive). The piezo is the blue box on the lens turret. Screw spare lenses into a lens container and keep within the incubator box. Ensure one of the paper towel rings is placed around the lens as the microscope is vulnerable to any fluid running down the side of the lens (either immersion oil or medium). Before putting sample on the microscope, check lens is ready to use: Piezo Focus Drive Paper towel ring Smartmove focus device Focus dial Check lens is clean wipe with lens tissue. If dirty, dampen lens tissue with 100% ethanol, wipe lens, then wipe again with a dry part of the tissue. Check spring loaded top is fully extended. Use the correct immersion fluid glycerine or type F immersion oil. Put a small drop on the lens or if using a slide put the drop on the slide. Course focus setting Fine focus setting

4 There are 3 sample holders. Ensure the selected one is inserted correctly into the stage so that your sample will be flat push the sample holder into the spring clips on the bottom left of the microscope stage. Universal holder 35mm dish holder Multiwell plate holder If running your experiment at 37 C, you should ensure that the sample holder you want to use is placed inside the environmental chamber (ideally on the microscope stage) to warm up prior to beginning your experiment. The environmental chamber should be left at 37 C by default but it is always worth checking that it has not been switched off before your booked time. The microscope will take about 3 hours to warm up to and stabilise at 37 C from room temperature. The temperature will fluctuate after opening the doors and top of the Current environmental chamber. Allow time for the temperature of your temperature sample, immersion oil, microscope and environmental chamber to stabilize after changing sample and before capturing images otherwise your sample will drift out of focus. Push to adjust desired temperature

5 Microscope controls Note. There s no need to use any of the buttons on right hand side Left hand side Front Light goes to eyepieces Light goes to camera Shutter for fluorescence and brightfield lamps Dapi filter cube GFP filter cube RFP filter cube Analyser for viewing DIC with eyepieces

6 Setting up microscope for transmitted light images Press the TL-BF button to activate the brightfield contrast method and open the shutter (on front of microscope). Place specimen on stage Focus the specimen Adjust the light intensity with the INT buttons Close the field diaphragm with the FD button. The edge of the diaphragm should appear in the field of view, but will not if the condenser it at the wrong height or not aligned Using the condenser height adjuster, adjust the condenser until the edge of the field diaphragm appears in sharp relief (hexagon) If the field diaphragm has been badly aligned you may not see it down the eyepieces at all. In this case ask one of us to align this for you Open the field diaphragm just enough for it to disappear from the field of view Condenser height adjuster TL-BF Toggles between brightfield, phase contrast and DIC INT buttons FD buttons Focus Wheel

7 Setting up microscope for DIC Set up the condenser as described on previous page Push 2 nd from top button behind the focus wheel to switch to TL- DIC. This will be shown on the front display of the microscope. The polariser will swing into position. The metal rod can be moved left or right to adjust the polarisation of the light To view DIC down the eyepieces, you need to put an analyser into the light path. Push the analyser button on the front of the microscope. Ana should be displayed on the front. Note that when capturing DIC images a different analyser is placed into the lightpath so this one is not required Note that you should not have any plastic in the lightpath (e.g. plastic lid for an imaging dish) or you ll lose the DIC. Adjust direction of light polarisation

8 Volocity software controls Open an existing library or create a new library and select Show Video Preview from the Window menu. In the instructions which follow, please refer the number of the item back to this diagram, to locate its position on the video preview control panel Time lapse option 2. Take snapshot of image 3. Acquisition set-up 4. Start/stop acquisition protocol 5. Pause acquisition protocol 6. Freeze image 7. Light paths 8. Selected light path 9. Save light path settings 10. Laser shutter 11. Brightfield shutter 12. Camera exposure 13. Camera gain 14. Imaging mode 15. Laser selection 16. Ultraview dichroic 17. Emission filter 18. Laser power Continued on next page

9 Camera settings 20. Binning option 21. Bit depth 22. Image rotation 23. Microscope settings 24. Fluorescence lamp shutter 25. Filter cube position 26. Leica focus drive 27. Dummy lens Continued on next page

10 Ultraview settings 29. Image capture mode 30. Laser power settings (AOTF) 31. Ultraview (Piezo) focus drive

11 Optimising light path settings To view your sample on screen, push the camera icon on the front of the microscope to send light to the camera Select one of the predefined light path settings Change imaging mode to TL-BF Open the laser shutter (10) Adjust settings until you get the desired image quality: Exposure time (12) depending on desired speed of acquisition Camera sensitivity (13) for optimal image quality keep quite low (approx ). Increasing will increase the amount of noise in the image but can be useful to reduce laser power or exposure time Laser power (18) keep low to reduce photodamage. Increase to allow lower exposure times. If using the argon laser see next page. Remember to save any changes you make by clicking top right (9) Other controls to check during image optimisation: Focus position Binning (in camera controls) 1x binning gives you the full resolution of the camera. 2x binning light from 4 pixels on the camera put into single pixel in image. Quadruples the amount of light per image pixel. The software automatically changes the exposure time when using binning. Binning reduces the resolution of the image but allows you to use lower exposure times or lower laser power or gain. Bit depth (8 bit = 256 levels of intensity. 14bit = levels of intensity) Also check emission discrimination mode is selected (29) to allow separation of fluorophores with emission filters and that the correct laser (15), dichroic mirror (16) and emission filter (17) are selected.

12 Using the argon laser Always start by having the argon laser at standby power. Using higher power mode decreases the lifetime of the laser. Only switch laser to high power mode if 100% power using AOTF is not sufficient To switch to high power mode: Check dial is turned full anticlockwise (minimum power) (1) Flick switch so that it points away from you (2) Now you can increase the power by turning the dial clockwise. 12 o clock is about maximum power. When you have finished your session, always turn the laser power down to minimum (turn dial fully anticlockwise) And flick switch to standby mode (switch points towards you) Finishing a session Once switched on, the laser must be left on for a minimum of 1 hour. When finishing a session, check to see if anyone is booked on after you. 3 If someone is booked on within 3 hours: leave the laser on at minimum power with the switch in 4 the standby position. If nobody is booked on within 3 hours Turn power dial to minimum and the switch in standby position Turn key on front off (3) Leave fan running for at least 5 minutes to allow laser to cool 1 2

13 Setting up an Acquisition Protocol (multiple channels, z-stack, timecourse etc ) Double click acquisition protocol icon on Video Preview Screen (3) Select channels If you wish to acquire a multi-channel sequence select the Change channels using light paths box. Light paths are optimised using the video preview window before setting up the acquisition protocol. Add or remove channels using the + and - buttons and select the correct light path for each channel by clicking on the dropdown menus. Channel/Z tab Summary A summary of the acquisition protocol is given in this dialogue box (including time-course set up see next page) Change focus (if required) for Z-stack) Z stacks can be acquired using the Ultraview focus drive (Piezo drive) or the Leica focus drive which is slower. To define the top and bottom of a z stack, see next page. You can capture a Z-stack by defining the Z-spacing or number of images (slices). Order channels and Z Z first then channels will capture the z stack in 1 channel then repeat stack with 2 nd. Channels then z will capture all channels at each z position before moving to next z position Shutter management Select maximum sample protection, balanced sample protection, maximum speed or not managed (left open)

14 Setting up a z-stack using Ultraview focus drive (aka piezo drive) 1) Your objective lens must be screwed into the Piezo drive 3) Whilst viewing a live preview image of your sample, move the slider to lowest desired position 4) Click Set bottom 5) Move the slider to highest desired position 2) Select the focus device under the Ultraview section of the software controls 6) Click Set Top Alternative method set zero when positioned at centre of desired range then type values as required for top and bottom. Displays distance between the set bottom and set top positions Note: The piezo drive has a maximum range of 100 microns. If you need to capture a z stack greater than 100 microns thickness then you ll need to use the Leica focus drive (see next page).

15 Setting up a z-stack using Leica focus device The Leica focus drive is the same one as is controlled with the microscope focus wheel and the smartmove controller. It is slower than the piezo focus drive but does allow z-stacks of over 100 microns thickness 1) Select the focus device under the Leica microscope section of the software controls 2) If the slider is fully at the bottom or top of its range, click set zero to bring it into the middle 3) Whilst viewing a live preview image of your sample, move the slider to lowest desired position 4) Click Set bottom 5) Move the slider to highest desired position 4) Click Set Top

16 Setting up a timecourse Time tab Timelapse. Determines how often images are taken Duration Determines duration of timecourse. Can set a specific time limit or run timecourse until stop is clicked Summary A summary of the acquisition protocol is given in this dialogue box (including light path and z-stack settings see previous page)

17 Capturing Phase contrast and DIC images Phase contrast If you want to capture phase contrast images rather than brightfield, change the imaging mode (14) in the BF light path from TL BF to TL PH. Remember to save this new setting by clicking (9) BF light path selected 9 If capturing a fluorescence channel as well as phase contrast channel, you will get a quicker switch time between the channels if you have the TL PH imaging mode selected on both. 14 DIC If you want to capture DIC images, use the DIC light path and ensure the imaging mode (14) is on TL-DIC DIC light path selected The DIC light path will put an analyser in the emission filter wheel infront of the camera (17) so the analyser that is used to view DIC down the eyepieces is no longer need. Push the button (a) a to remove the microscope s analyser. Instead of Ana on the microscope display panel, you should see EM Analyser selected

18 Saving images Capture an acquisition protocol (multiple channels, timecourse, z-stack etc ) by clicking the red button. Capture a snapshot by clicking the camera icon Captured images are automatically saved to the hard drive and are viewable in the library space to the right of the image window. Multiple experiments can be acquired in the same library although if your experiments are 1GB or larger it may be advisable to create new libraries to separate your data into more manageable chunks. The default file format is.mvd2 file which has an accompanying data folder. The mvd2 file and data folder must be kept together to be readable. Images can be exported from Volocity as tifs, avis and other formats by right clicking on the image or image sequence in the library, selecting export. If you have captured a timecourse or z stack you can split the Volocity file into multiple images and then exporting these as a batch.

19 Ending the session Clean oil off lenses with lens tissue - dampen lens tissue with 100% ethanol, clean lens, then dry lens with a dry piece of lens tissue. Clean other used surfaces with 75% ethanol. Fill in log sheet shortcut on desktop of computer you need to enter the date, your UOB username and the time you started and ended your session. Save file after completion. Check Google calendar to see if system is booked after you: If someone else is booked on within next 3 hours: Turn Argon laser power down to minimum and flick switch to standby position. The 568nm laser, fluorescent lamp and other devices can be switched off if there is a small gap (>5 minutes) before next booking. Log out of computer but leave on. The default temperature of the system is 37C. Leave the heater on with the door and lid to the environmental chamber closed unless the next user has specified that they want to work at room temperature If there is no booking within the next 3 hours: Turn Argon laser power down to minimum and flick switch to standby position. Then turn key off but leave the fan running for at least 5 minutes. Turn off the 568nm laser, fluorescent lamp and other devices. Log out of computer but leave on. Leave the heater on with the door and lid to the environmental chamber closed.