510 confocal instructions Feb 2013
Contents
1. In the Ocular tab choose an objective and a filter Fluorescence Shutter On Shutter Off Configuration Avagn 5 Click Online oe and open the shutter Ocular Nj 6 Find your sample PianApoc hromat EJN Oil Ph3 wl BH None amp Lens ix Use this button to change the objectives Objectives available 10x 20x air 40x 63x 100x oil Automated do not move by hand NB please LOWER THE STAGE before changing between objectives to avoid crashing lens onto slide Use this button to change between the fluorescent filters 7 In the Acquisition tab select a Configuration and accept the prompt asking if you wish to switch on the lasers 8 In the Laser menu Switch the Argon laser from standby to on 9 In the laser properties for the Argon laser Set the tube current to 6 1A by adjusting the output NB Ignore the warning about exceeding 50 output 10 Click Auto Exposure to generate an initial image a ZEN 2008 ibe Acwasiion Merten Macro i vi wow BB SS Acquisition Configuration DAPI FITC Ehodamine fal foo fo New Auto Exposure Continuous Snap Z Stack Time Senes Bleaching Tile Scan Positions Regions Setup Manager 12 Optimise the system by clicking Merged Blue Underexposed pixels Red Overexposed pixels File Acquisition Maintain Macro Tools View Configuration CFP YFP FRET DAPI FITC Rhodamine DAPIFITC ICYS GFP RFP FRET CFP YFP FRET Laser Laser2. Lines nm Power Laser Diode 405 405 On E Argon 458 477 488 514 nw HeNe1 543 On HeNe2 633 off w Laser Properties Maximum Power 30 0 mW Wavelength 458 477 488 514 nm Status Ready Tube Current Output 11 Click Live to see the live image and select the SPLIT view to see the individual channels View Dimensions Display Player View Dimensions Adjust the Gain and Digital Offset so that the signal is within the dynamic range of the detectors see below Channels Showall WA FITC 13 In the Channels menu click Show all pee and Select all Select all Wiese v 14 Set the pinhole to 1AU 405 458 477 488 514 543 633 488 nm i 15 Alter the Digital offset so background is black not blue 16 Alter the Gain Master so there are no red pixels Z 405 458 477 488 514 543 633 405 nm 17 Repeat for each channel y s Acquisition Configuration DAPI GREEN RED 18 Stop the Live image aquisition Show manual tools a ED E m New Auto Exposure Continuous Snap Z Stack Time Seres Tile Scan Positions Regions Dimensions Display Player View Dimensions Zoom Click the Merged button again to return to the original colours Merged Ch2 T1 Ch1 T2 You can Crop your image and change the position size or rotation of the cropped area by moving the crop box Start scanning again and your cropped image will appear a Acquisition Mode _ Objective Plan Ap
3. OPERATING INSTRUCTIONS 510 Meta inverted confocal 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 Isma Ali ext 2407 i ali qmul ac uk Standard Operating Procedure How to turn the equipment on 1 Switch on the metal halide lamp 2 Switch on the Zeiss remote control 3 Switch on the computer and log in How to turn the equipment off Switch off lasers and wait to cool Switch off Zeiss remote control once lasers have cooled Switch off computer 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 ZEN software Login ZEN 2008 SP1 1 2 Cl ick Sta rt System Laser Scanning Microscope LSM 510 Boot Status Start System Image Processing 3 Place slide on microscope stage File Acquisition Maintain Macro Toos View 4
4. ochromat 20x D 8 M27 19 In the Acquisition Mode menu Scan Mode Frame Frame Size X 1024 set the image capture parameters Speed o e Recommended acquisition ee e Frame Size 1024 x 1024 pee Number Bit Depth 32 Bit Speed 7 Averaging number 4 Aicega Image Size 424 7 pm x 424 7 pm Bit depth 8 bit Pixel Size 0 42 ym 1 Reset All CEEE Tie Acqusiion Mariam Meo Tos w E 20 Click Snap m y i Acquistion Configuration DAPIFITC Rhodamine re x T Mew Aulo Exposure Continuous Snap Workspace Zoom pace Configuration KATY 21 Save your image on the E drive Open Images displayed in the top right of the workspace 2N ZEN 2008 File Edit Acquisition Maintain Macro Tools Window Help To export your images in TIF format go to File and Export Z Cutaneous Patricia 1 1 07 05 2 P10 05_0UV_Phall568_DAPI_10 Ism Export Format Tagged Image File Raw data single plane Export as Raw data single plane for overlay image Channels RGBimage Palette image Red channel Ch T3 Green channel ey Blue channel Chi Compress Select file name and sawe Tagged Image File ll Export as Full resolution image window for contents of the image window will vary depending on which tab is open Full resolution image window single plane When you have finished transfer all your data to the Z network drive PLEASE TIDY UP Clean lenses throw away
5. used tissue lens tissue dispose of old slides in the yellow sharps bin
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