MM-Leica User guide Feb 2013
Contents
1. OPERATING INSTRUCTIONS LEICA MM pmaoo0 epi fl microscope You must not operate this equipment without prior training from a BALM facility staff member To arrange training and for help please contact Dr Ann Wheeler ext 2406 a p wheeler qmul ac uk Dr Katy Cogger ext 2407 k f cogger qmul ac uk Isma Ali ext 2407 i ali qmul ac uk Standard Operating Procedure Basic instructions How to turn the equipment on 1 Switch on the metal halide lamp 2 Switch on the microscope the switch is on the back RHS of the microscope 3 Switch on the computer and log in How to turn the equipment off 1 Switch off computer 2 Switch off Microscope 3 Switch off metal halide lamp Rules of use This microscope should be treated with respect and care at all times This Microscope can only be used by Masters by Research or PhD students Postdocs and members of staff The microscope lenses must be cleaned after every usage and the equipment treated carefully at all times If you have any problems at all with the microscope no matter how trivial they may seem please see a technician immediately REMEMBER You have 5GB of disk space on this microscope Check before you start if you have room for your experiment If not delete your old data 1 Open Metamorph software basic or standard user NB The software takes a while to load Objectives available 4x 10x 20x 40x air 2 Place slide on microscope stage and choose ob2. UT rod on LHS to direct the light to the camera 82 Multi Dimensional Acquisition D Illumination FL GFP FITC 7 9 Click Show Live icon _Wavelengths ame E W1 FL GFP FI Digitizer live image will appear on screen Display Scene nny Exposure po a ms you may need to adjust the focus ee Auto Expose No Auto Expose O00 Alignment Cropping X fo Xe fo Set Alignment 10 Adjust Gain and Exposure for each wavelength E Previous Next wjf 3 e 3 lel el firr l BB BB Acquire Cose 2 Multi Dimensional Acquisition Main _ llumination FL DAPI Saving Wavelengths Gain o W1 FL DAPI Wi FL GFRYFITI Wi FL AFSES E Exposure Display To change the assigned wavelengths select an illumination Digitizer from the dropdown menu igitizer FL GFF FITC Phase 10 20 Phase 40x Phase 60s Summary Auto Expose If you need to use Auto Scale click on the icon on the LHS of the Live Image Window and select it from the dropdown menu v Best Fit Range 8 Bit Range 0 255 10 Bit Range 0 1023 12 Bit Range 0 4095 14 Bit Range 0 16383 16 Bit Range 0 65535 Within the parameters of the gain and exposure that have been set you can alter the image displayed by sliding the Scale Within Active Region triangles on the side bar Scale Image NB If the uppermost tail of the histogram is not viable then some areas of your image will be overexpose
3. d Multi Dimensional Acquisition Illumination FL GFP FITC x e e Wavelengths Gain fi 4 11 Click Acquire W1 FL GFP FI Digitizer omz z a separate picture will be i S Exposure 70 ms Auto Expose No Auto Expose Alignment Cropping fo x fo Set Alignment taken for each channel Previous Next we ain ft H e B E E iarr z BB O Acquire Cose Color Combine 12 Creating a Merged Image ae click Colour Combine F Color Combine in toolbar on RHS gt Source Type AOO NS ie Components C Stack C sigeinsee select 24 bit EO air Gea in Colour Combine window ee N _ GREEN component No Images assign the appropriate images Bi emacs Foiea for each colour component Destination DF Color Combine M Hue rendering Enable hue rendering N N Color Eombne Cancel Save combined image to your folder in tiff format When you have finished transfer all your data to the Z network drive PLEASE TIDY UP Clean lenses throw away used tissue lens tissue dispose of old slides in the yellow sharps bin
4. jective lens 40x 63x oil Move by hand not automated NB please LOWER THE STAGE before changing between objectives to avoid crashing lens onto slide 3 Check rod on LHS is pushed IN to direct the light to the eyepiece 4 Select a channel from the tool bar on C BF jnl the RHS and find your sample _ DAPLjnI Use these buttons to change between the channels 5 Select Multi Dimensional Acquisition Fi CY5 AF647 jnl ai Open and close shutter using the a new window will appear N amp shutter state button E v ti Dimensional Acquisition Review Multi Dimensional Data a ee Color Combine 2 Close All coy 6 Set the parameters of your experiment using the step by step tabs in the multi dimensional acquisition window 7 Once you ve set up your acquisition setting you can save them using the Save State button and reload them next session using the Load State button Sjen H fein A Me MO Me FLFR EE TBH Acquite Close 82 Multi Dimensional Acquisition Save your data on the F Drive In the Saving tab click Select Directory and PEET Multi Dimensions Experiment choose your folder on the F Drive Type in a Base Name for your experiment a Bea 040456 o Check the Incremental base name box is ticked this will automatically number your images under the base name you selected 4 Multi Dimensional Acquisition E Choose the number of wavelengths you need 8 Pull O
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