Title: Leica SP5 Confocal User Manual

Title: Leica SP5 Confocal User Manual Date of first issue: 23/10/2015 Date of review: Version: Admin For assistance or to report an issue Office: CG07 or 05 Email: Igmm-imaginghelpdesk@igmm.ed.ac.uk Website: www.igmm-imaging.com Download a PDF copy of manual: \\igmm-smbhost\microscope-users\microscope user manuals\leica SP5 Facility Usage Policy 1. You must have the relevant Risk Assessment/COSHH form for the work you are undertaking before using imaging facility resources 2. Users must be trained before using facility equipment 3. Please leave the microscope clean and tidy for the next user 4. Please report any issue, even if it seems minor, to facility staff 5. Any clinical waste must be placed in the orange bins provided 1

Table of Contents System Startup... 3 System Shutdown... 4 Mounting a sample... 5 Finding the sample by eye... 5 Configuration Menu... 6 Turning the Lasers on... 6 Setting Image Bit Depth... 6 Selecting a beam path setting... 6 Scanning a live image... 8 Creating a new beam path setting... 8 Simultaneous vs Sequential scanning modes... 8 Setting up Sequential Scanning (Recommended)... 9 Image Optimisation Procedure... 10 Capturing a Multi- Wavelength 2D Image (Optical Slice)... 11 Saving Images... 11 Capturing a Multi- Wavelength 3D Image (Z Stack)... 12 Acquiring Images at different stage positions (Mark and Find)... 14 Acquiring a Time- Lapse... 14 Appendix... 15 Exporting Images... 15 FRAP... 16 FRET Acceptor Photobleaching... 25 FRET Sensitised Emission... 32 Spectral Imaging... 42 2

System Startup * See the labelled image below for location of each switch 1. Mercury-Metal Halide Fluorescence Lamp 2. Green confocal control switches found on the right side of the desk (from left to right) -PC/Microscope, Scanner Power and Laser Power Turn the Laser Emission key to the ON position 3. The PC will boot automatically, login using your windows user account. If you have been trained on the system a user account will have been created for you. 4. Launch LAS AF using the Desktop icon 5. The software initialises devices during start up, when messages appear click OK to proceed. 6. Select the Configuration tab and laser. Tick the boxes of the lasers you want to turn on. Move the Argon slide bar to 50% output 3

System Shutdown 1. Check the booking system to see if anyone is booked on the system within the next hour. If not shutdown. 2. Select Configuration>Laser and drop the Argon slider back to standby 3. Close the LAS-AF program and shut down the PC 4. Turn the Laser Emission key to the horizontal position 5. DO NOT SWITCH OFF THE LASER POWER SWITCH UNTIL THE ARGON FAN HAS STOPPED. Continue to next step while waiting. 6. Switch off Scanner Power 7. Switch off PC/Microscope (Ensure PC is off before doing this) 8. Switch off the Mercury-Metal Halide Fluorescence Lamp via the power switch 4

Mounting a sample 1. Ensure the correct stage insert is mounted (see facility staff if unsure) 2. Remember to invert your slide so that the cover glass is the closest surface to the lens 3. Select the objective lens either using the two buttons behind the right focus wheel or in the software within the Acquire tab then select the Objective text to see a drop down list of lenses 4. Use the joystick to move the stage if necessary. Change the XY speed of the stage by pressing the black buttons on the left side of the joystick base. The black buttons on the right change the speed in Z. Z X Y Finding the sample by eye 1. Choose a fluorescence filter cube via the buttons on the front of the microscope. Push the shutter button to toggle open/close shutter. I3 = FITC N2.1 = TxRd A = DAPI Filters Fluorescence shutter BF intensity (Controller on LHS of microscope Observation mode BF/FL Focus speed (course/fine) Z position 2. Once the sample is in focus you can record this Z position by holding the two outer Set Z buttons on the right side of the microscope. This sets this Z position to 5

zero. Pressing the lower Z set button drops the lens to its lowest position. This is useful when you want to change to a different cover glass on the same slide if you re using an oil immersion lens. Dropping the lens is always good practice when changing slides too. 3. To view brightfield select an empty filter cube position using the buttons on the front of the microscope. 4. Switch on the transmitted light using the TL/IL button on the left side of the microscope. Adjust the intensity up/down with the INT buttons. 5. At this point you can either change to a higher magnification lens if required or begin the scanning process 6. Lenses 60x and above always require a drop of oil between the lens and the coverglass. 40x lenses can be either a dry or oil type. The software menu used to select the lenses tells you if the lens requires oil. Configuration Menu Turning the Lasers on 1. Select Configuration tab>laser 2. Tick the boxes next to the lasers you want to use 3. If using the Argon laser (458/488/514 lines) set the percentage output to 50% in the text box next to the power slide bar then press enter Setting Image Bit Depth 1. Select Configuration tab>settings 2. From the Image Resolution drop down list select 12bit 3. The detectors have a 12bit analogue to digital converter so if you select 12bit image resolution this ensures the full dynamic range of the detector can be utilised Selecting a beam path setting 1. Under the Beam Path Settings heading select the drop down list and scroll down to the heading User Settings, here you will find the personal beam path settings you have already configured. 6

Scanning a live image 1. To see a continuously scanned live image of the sample press the Live button at the bottom of the screen. You can change any of the capture parameters with or without Live mode enabled. Creating a new beam path setting 1. Activate the laser lines required by moving the vertical slide bars to >0%, the laser line will be added to the spectral diagram 2. Activate as many detectors as required using the tick box below each detector 3. Select the emission spectrum label for each channel, choose the fluorophore/protein you re using if possible. This will add the emission spectrum of the dye to the diagram. 4. By moving the spectral sliders you can adjust the detectors detection bandwidth. Click and drag a slider to move it. Hover the cursor over the edge of a slider to allow the width to be adjusted. Try to match the width of the slider to the emission spectrum of the dye. Avoid overlapping the spectral sliders with the excitation laser lines (5nm min separation). 5. Select the Channel colour box for each detector if you d like to channel the image colour (Lookup table) 6. Press a detectors PMT text to access that detectors Gain and Offset values 7. Use the Save button to store the adjusted beam path setting. This new setting will appear below the User Settings heading at the bottom of the drop down list. Simultaneous vs Sequential scanning modes 1. The process of setting up the beam path setting above describes the setup for simultaneous scanning. This means that all active laser lines and detectors scan and acquire simultaneously. This has the advantage of speed but the disadvantage is that fluorescence cross-talk (signal from one fluorophore recorded in more than one detector) is more likely. 2. In Sequential scanning mode only one detector/laser is active at any one time, making cross-talk much less likely but at the expense of speed. 8

Setting up Sequential Scanning (Recommended) 1. From the left hand margin of the screen under Acquisition Mode select the Seq button to open the sequential mode window 2. Select the + button which will add a scan. Each scan corresponds to a single laser/detector combination. Therefore if you are imaging in 2 channels then you will need to add 2 sequential scans. 3. For a GFP-RFP setup for example you would select the newly added Scan 1 button then configure the beam path setup so that only the 488nm laser and the detector assigned to GFP are active. Use the vertical slide bars to turn all other laser lines off. Uncheck the other detectors to inactivate them. 4. Now select the Scan 2 button and repeat the procedure but this time enable the laser line and detector for RFP and switch off the first detector and 488 laser by setting to 0% power. 5. Now when you scan the lasers/detectors are sequentially turned on and off rapidly to avoid cross talk. 6. Note that there are 3 sequential scan options: Between lines- (Recommended) The laser currently scanning switches on a line by line basis. I.e. one line of the GFP channel is imaged, then one line of the RFP channel etc. Between frames-an entire image of one channel is created first, then the laser switches and acquires an image of the second channel. Between stacks-if acquiring a Z stack, then the stack of Scan 1 (GFP) is acquired first then the stack of Scan 2 is acquired (RFP). 7. Press the Save button in the sequential scan window and save to your folder on the D: Disk. Include which wavelengths the sequential scan settings relate too, to make it easy to select another time. 9

Acquisition Settings 1. Select the XY window in the left hand margin 2. Format changes the size of the image in pixels. For better xy sampling choose 1024x1024. For speed choose 512x512. 3. Speed determines how long the laser spot dwells over any particular part of the sample. The higher the frequency the lower the dwell time and thus the lower the exposure. Optimal values can range between 400-100Hz. 4. Tick the Pinhole box which will expand the Pinhole window. Press the 1 Airy button. This selects the best compromise between light throughout, resolution and noise. It is also responsible for determining the optical section thickness with higher values increasing the thickness of the optical section. 5. Use the Zoom Factor slider to increase the image magnification. This can increase resolution because the pixel size decreases (microns per pixel). 6. Line/Frame Averaging can be used to reduce the image noise level by scanning each channel a number of times and averaging the resulting signal. Only use if you have a noisy image. Image capture time is increased as is the likelihood of phoobleaching. 7. Rotation allows the whole scanned field of view to be rotated, if you need to rotate your image while scanning. Image Optimisation Procedure 1. Start with a format of 512x512 and a speed of 400Hz. Set the laser power between 5-10% for a each channel. Do not apply zoom or averaging at this stage. 2. Press the Live button hopefully you will see an image of each channel displayed on the right hand monitor. Each channel has its own window by default and you can toggle them off/on using the panel on the far right of the image monitor. The lowest button pictured here toggles the merged view on/off. 3. Check the detector gain settings for each channel by clicking on the PMT text at the top of each detector window. Gain is used to amplify the signal thus the image brightness will increase. 4. However, high gain levels will increase the noise level in the images therefore achieving optimum exposure is always a compromise between laser power, gain and scan speed. 10

5. The Offset is also found in the PMT settings menu and controls the image black level. As an example setting an offset of zero means that pixel intensity values between 1-4095 have a grey scale value and will be visible in the image. If you set the offset to -10% this means that any pixel with an intensity lower than 409.5 (4095 x 0.10) will be set to black (zero). You can use this to reduce background fluorescence but be aware that you can cancel out specific signal if not set correctly. 6. You can determine if the signal fills the dynamic range of the detectors by using the saturation indicator which is found in the toolbar to the left of the images. Press the button once and the indicator will be activated. Now any pixels which have an intensity of 4095 (max) will be coloured blue. 7. The aim is to set each channel image so that the pixel intensities are just under saturation level (4095). It s not a concern if there are a few blue pixels. Press the saturation indicator button again twice to see the channel images in their original colours. Capturing a Multi- Wavelength 2D Image (Optical Slice) 1. If you have followed the sections above you just need to decide if the image is to be captured simultaneously or sequentially. 2. Press the Capture Image button at the bottom right of the left monitor and a single scan of each channel will be acquired. Saving Images 1. Captured images will be added to the experiment. You can access the experiment images by selecting the Experiments tab in the top left corner of the left monitor. 2. Images are given a default name Image followed by an incremental number unless you uncheck the Default image basename option. 3. You can rename an image by right clicking on it then select rename. 4. Images in the experiment are stored temporarily so you must ensure you save the experiment to disk at regular intervals and at the end of the session using the Save All option at the bottom of the experiment window. 5. Images are stored as Leica LIF format by default which can be opened with FIJI s Bio-Formats plugin. 11

Capturing a Multi- Wavelength 3D Image (Z Stack) 1. Adjust scan parameters as for 2D image capture. 2. When optimising the image intensity for a z stack, move through focus while live scanning to find the focal plane with the highest signal intensity. Turn on the pixel saturation indicator to aid in this process. 3. Ensure the Z stack panel is visible. View the live image and move in one direction through focus using the joystick Z control until you find the top/bottom of your sample. Click on the Begin arrow on the diagram to record that position. Then move to the opposite end of the sample and click the End arrow. Stop scanning. 4. You can change the number of steps or step size by typing in the relevant boxes. It is recommended that you use the System Optimised option. This will calculated the step size and number of steps automatically according to the Nyquist-Shannon sampling theorem. 5. Click Start button to acquire the Z stack. 6. Z stacks appear in the experiment folder with the name series by default. 12

Capturing a Tile Scan Image (Image Stitching) 1. Enable the tile scan option in the Acquisition mode window. The tile scan window will appear. 2. Here you can see an image of the stages travel range. The cross hairs point to a small square box which represents your current XY position. Click on the + magnifying glass to zoom in on the box. 3. Clicking the mouse anywhere within the scaled region will cause the stage to move to that position, USE WITH CAUTION. Only click small distances from the current position (indicated by cross hair). 4. The Scan Field box allows you to define how many images should be captured in XY. For example type 2x2 for a square grid of 4 tiles. 5. Click on the rubbish bin button to remove the scan field setting. 6. You can customise the region to be tile scanned further by using the Mark Position button. For example to define your own xy grid, begin live scanning and move to the top left corner of the region you want to scan by using the xy joystick. Your current position is updated within the tile scan window. Click Mark Position, then move to the bottom right corner of the region you want to scan, click Mark Position again. 7. The software will calculate how many xy tiles are required to scan the area you have defined. Once finished the tiles will be stitched together. 13

Acquiring Images at different stage positions (Mark and Find) 1. Enable the Mark and Find option in the Acquisition Mode window. The mark and find window will appear. 2. Here you can see an image of the stages travel range. The cross hairs point to a small square box which represents your current XY position. Click on the + magnifying glass to zoom in on the box. 3. Clicking the mouse anywhere within the scaled region will cause the stage to move to that position, USE WITH CAUTION. Only click small distances from the current position (indicated by cross hair). 4. To mark a field of view to be captured click the Mark Position button. 5. Continue to add further positions to be scanned by using the joystick to move around. The positions are added to a dropdown list, selecting a position from the list will move the stage to that position. 6. The Show All Positions button will highlight within the stage area the fields of view you have marked for scanning. 7. You can delete a single position using the Clear Position button or delete all using the rubbish bin button. 8. If acquiring a Z stack at each position you can either use the same stack dimensions as defined for one position at all positions or define individual Z stacks per position. This is set using the Same Stack For All tick box. Acquiring a Time- Lapse 1. Select a beam path setting and configure each wavelength channel as required. Follow the sections above to add a tile scan, mark and find etc. 2. In the Acquisition Mode window choose the relevant time experiment from the dropdown list xt, xyt, xyzt etc. 3. This will open the Time window. Define the total duration of the time lapse and the frequency of captures in the Interval box. 4. The minimise tick box will set the shortest possible delay based on your scan speed etc. 5. The Stacks option allows you to choose how many time points you collect instead of having to input a duration + interval. 14

Appendix Adding a scale bar to an image 1. On an open image select the Scale icon from the set of tools left of the displayed image 2. Hold the left mouse button and drag across the image to draw a scale bar 3. To edit the length or position of the bar select the Cursor icon then click on one end of the scale bar to adjust its length or in the middle of the scale bar to move it within the image 4. To delete the scale bar select the bar using the cursor tool then select the Delete button 5. To save a copy of the image with the scale bar, right click on the image then select Snapshot all Exporting Images 1. To export an experiment or single image, right click on it in the Experiments view then select Export>As TIFF. 2. Select the destination folder for the exported images. 3. The Overlay Channels option will save a single file (RGB colour) that contains all of the wavelength channels captured. This is not the raw data and cannot be used for quantitative intensity analysis. 4. If the Overlay Channels option is not ticked then a Raw data check box appears, select this option. The greyscale 16bit images will be saved as individual TIFF files. 15

FRAP 16

FRET Acceptor Photobleaching 25

FRET Sensitised Emission 32

Spectral Imaging 42