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Leica Epi user guide fluorescence July 2013
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1. base name you selected 10 Choose the number of wavelengths you need 11 Select which filters you will use for each 82 Multi Dimensional Acquisition wavelength from the dropdown menu Main Number of Wavelengths f Saving i Wavelengths Allow separate binning for each wavelength A F Multi Dimensional Acquisition Display Mair Summary T Illumination Wavelengths Gain w1 FL DAPI Gee We FL GFPYFITI ae Wa FLAFSGG E EXPOS tthe 20 Display Summary Auto Expose Alignment Cropping 3 p V0 SetAli R 2 Multi Dimensional Acquisition 12 Click Show Live icon to see you image Te B c Illumination FL GFP FITC 7 on the computer screen ae A on eee live image will appear on screen WI FLGEES Digiizer Exposure 70 3 ms Auto Expose No Auto Expose z 000 Alignment Cropping fo Y fo Set Alignment you may need to adjust the focus 13 Adjust Gain and Exposure for each wavelength acquired To move between the wavelengths click on the tabs in the 4 Frevious D gt Newt Multidimensional acquisition menu a Gain is camera sensitivity Exposure is length of time the light falls onto the camera detector Both can be changed to optimise your image acquisition lt n hes Within the parameters of the gain and exposure that have been set you can alter the image ae oe displayed by sliding the triangles on the side ba spears Tiie Barge D HEN Iii Aa
2. OPERATING INSTRUCTIONSI z LEICA Epi DM5000 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 Facility Manager Dr Ann Wheeler ext 2406 a p wheeler qmul ac uk Microscopy Technologist Isma Ali ext 2407 i ali qmul ac uk Standard Operating Procedure Fluorescence How to turn the equipment on 1 Switch on the Mercury Halide Bulb 2 Switch on the Leica controller box 3 Switch on the computer and log in 4 Ensure the lever on the right hand side of the microscope is pulled out How to turn the equipment off ie Switch off the computer 2 Switch off the Leica controller box 3 Switch off the Mercurv Halide Bulb Rules of use This microscope should be treated with respect and care at all times This Microscope can only be used by Masters 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 NB The software takes a while 1 Open MetaMorph software Basic or Standard user to load Objectives available 2 Pl
3. ace slide on microscope stage and choose objective lens l 4x 10x 20x 40x air 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 ral Set Acquisition Channel 4 Focus on your sample by eye C BF jn Use the buttons on the toolbar on the right hand side to move between DAPLnI filters for DAPI Green Red and Far red fluorescence by eye gt E GFP jni For Bright field please see Appendix 1 E Fern I vs ar647 jn 5 The shutter will control the light passing through the microscope D Sd S A To open or close it press Set current shutter state WE Multi Dimensional Acquisition jE Review Multi Dimension ata 6 Once you have found your sample by eye and focussed on it Color Combine a you can take an image of it To do this you need to send the KA Chek AN aa light to the camera and activate the camera i Pull out the rod on the LHS of the microscope to send the light to the camera ii Open Multi dimensional Acquisition to activate the camera You need to decide if you want to make quantitative measurement of the fluorescent Staining intensity in your image or to take an image which will document your sample open in Office applications e g PowerPoint One you know what type of data you want set the camera to acquires 7 Select Se
4. e DAE NB If you can t see the top tail of the histogram is not then some areas of your image will be overexposed 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 If you would rather set your own scalings recommended Click on the icon and deactivate it Ensure in the Display menu Custom is selected so scalings aren t applied when you take your image 14 Click Acquire a separate picture will be taken for each channel rin Pein Ho ejer Saye mf mf ml RTE E BB eee com gt i f 15 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 a aka ajara m A m mj m TEA 16 Creating a Merged Image N Click Colour Combine gt Zh otrontn in toolbar on RHS Select 24 bit in Colour Combine window Assign the appropriate images for each colour component Click Colour Combine Save combined image to your folder in tiff format Appendix 1 1 For Bright field you can use the multi dimensional acquisition menu or the Acquire menu File Edit Regions Stack Devices Display Process Log Mi Illum BF x Acquire ia m hi Z Stop Focusing F2 2 Focus your image and pull OUT nee rod on LHS to direct the light to the Image pF Acquired camera Set Save Shve w Sequence Display Ac
5. quire Corect Annotate Special 3 In Acquire menu click Show live sechure Time Image Scaling live image will appear on screen 10 f Gms ov tow G High 67 you may need to adjust the focus ba Binniha Autoscale 4 Adjust Exposure time and click Gamera Area Scale within the active region i l gt Full Chi Acquire to take the image e jaa Goa Center Quad es Active Reson n reer Live Bin 1 a Temp n a Setting Modified test X Display Saturation Markers Reset Display Close Less lt lt Setting Load Save Save As 5 Save images to the D drive 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
6. t acquisition channel from the tool bar on the RH gt cal Set Acquisition Channel Choose 8 bit for images which will open in office etc Choose 12 bit for images which can be used for quantification DAPI jn Set Acquisition Channel kik g Hot TT hrome DFC 490 8 bit Monochrome DFC 490 36 bit Color DFC 490 12 bit Monochrome amp Set Current Shutter State a Multi Dimensional Acquisition 2 Review Multi Dimensional Data Color Combi olor combine KZ Close All yng Multi Dimensional Acquisition 8 Set the parameters of your experiment using 1 Saing I Timelapse ummary the cP by BEEP tabs in the multi dimensional NN a ee Summa acquisition window a ae eee c V Aun Display v ep eae Load State Summary Next es Bin fi E Bin fi E E Ej 1 FL AF568 Rhodan 7 Z Acquire Close 82 Multi Dimensional Acquisition Description Images automatically saved with base file 9 Save your data on the D Drive on P e en Multi Dimensions Experiment R Inthe Saving tab click Select Directory and Wavelenaths W1 BF fe Display Summary Select Directory Z Ann colour problems Increment base name if file exists 040456 choose your folder on the D Drive Type in a Base Name for your experiment Check the Incremental base name box is ticked this will automatically number your images under the
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