Zeiss LSM 800, Standard Operation Protocol

Page 1: ...Imaging and Flow Cytometry Core CPOS LSM 800 SOP A 1 ver 2 1 Updated 11 13 2020 LSM 800 Confocal Microscope Standard Operation Protocol...

Page 2: ...tion D 6 E Locate specimen focus find cells region of interest E 7 F Confocal imaging set up F 8 G Saving an Image file G 11 H Removing sample from microscope stage H 12 I Z stack set up I 13 J Airysc...

Page 3: ...corefac downloads ZEN_BLUE2 3SOP_20200415 pdf It is recommended that user study both guides side by side B Microscope Lens list Position 1 2 3 4 5 6 Magnification 10x 20x 40 63 EMPTY 5x Numerical Ape...

Page 4: ...n the program icon on desktop Or click on Start All Programs Carl Zeiss Microscopy ZEN ZEN blue edition entry blue icon 7 Click on ZEN system button 8 Check that you have took out sample frame adjuste...

Page 5: ...oscope stage Wires should be on the right hand side and gas tubing should be on your left hand side vi Slide incubator to the left while gently pushing downwards vii Insert live cell sample viii Add a...

Page 6: ...terms will be used throughout this document to locate different buttons Reference to this part from time to time when you read the rest of this SOP i Menu bar ii Tool bar iii Left tool area Locate mod...

Page 7: ...ple finding and bright field observation 6 Move the area of the slide you wish to observe above objective lens with X Y manipulator on air table 7 Focus onto your specimen Notice Z position value show...

Page 8: ...lable when z stack is activated Will change track after finishing whole z stack 5 Select any one track for set up 6 Table s rows indicate detector choice Row 1 PMT1 Row 2 Airyscan detector when idle w...

Page 9: ...image do not exceed 250 units 16 bit image do not exceed 65500 units Important information Preventing damage to PMT units Avoid over saturating the detector 19 Turn on Live 20 Adjust laser power You c...

Page 10: ...directional insert image for quality bi directional insert img for speed 30 Switch between bits per pixel N B different degrees of intensities a pixel can represent 8 bits for presentation representat...

Page 11: ...Hard drive D User 4 Create a folder with your PI s name and Lab wording suffix Applicable if no such folder exists and you are the first one in your lab using LSM 800 5 Navigate into the folder you h...

Page 12: ...scope stage 1 On control panel press load position button Objective lens should be moved to lowest position allowing clearance for you to operate with the stage area 2 Move slider on sample carrier ap...

Page 13: ...N Live i4 5 hands on Fine Focus knob Please note current z position value greater numeric value higher focus position or 6 cursor over preview window Press Control key on keyboard Scroll with mouse wh...

Page 14: ...s option if 3D rendering volumetric calculation is needed 14 For a more graphical representation of the z sections you may view them in optimize Sectioning and Step 15 Z stack setup finished 16 Select...

Page 15: ...nting pre mature sample bleaching 9 bit Either 8 or 16 but follow carefully in step J19 when adjusting intensity parameter 10 Scan Speed maximum 11 Zoom 1 3x Please refer to steps F26 to F32 for graph...

Page 16: ...to step J19 until all channel has been optimized 23 Check ALL channels 24 Turn on Continuous scan mode J24 25 Go to Airyscan tab in centre preview area 26 Check Detector under Airyscan tab Wait for t...

Page 17: ...size of tile region 7 Stake User define two opposite corner markers with x y motorized control and button The software will define the total number of tiles based on your input region Keep on pressin...

Page 18: ...escence channel for navigation 15 Use xy motorized control to navigate your specimen 16 When you have a region of interest in view go to Acquisition tab 17 Turn ON Live view set Focus until image is c...

Page 19: ...our settings 3 Go to tile window and click on Advanced setup 4 The software should 1 automatically turn on Live 2 replace half of the preview area with a navigation map and 3 added controls for tile s...

Page 20: ...itable for your high magnification scans Red F O V mark on advanced tile area should resize to fit new F O V physical dimension 13 for navigating large area an air lens is preferred over immersion oil...

Page 21: ...on E g 6 x 12 tiles required then add minimum of 2 x 4 support points 23 click Distribute 24 If some of the support points are not in the area of your specimen you may drag the yellow marks insert ico...

Page 22: ...step verify position heights or tile region heights if support point is enabled 34 Click on verify Positions If tile setup is used click Verify Tile Regions button instead in this step 35 double clic...

Page 23: ...Select Verification Helper Method 41 When DF or AF previously Set Z and move to next will be changed to Run DF AF and Set Z After initial run then user may press Use DF AF to Verify the Remaining Plea...

Page 24: ...le insert image 46 In window named Time Series set up Duration for of cycles seconds minutes hours 47 and 48 set up Interval for of seconds minutes hours 49 Interval time must be estimated time requir...

Page 25: ...st of lens tissue compared to the potential of damaging an expensive objective 1 Fold a piece of lens tissue into a long strip insert diagram 2 Drawn the folded lens paper across objective lens surfac...

Page 26: ...er bath with M fold paper towel 7 Take out incubator unit from microscope stage GENTLY Do not use excessive force to avoid hitting condenser with incubator 8 Use a kimwipe moistened with 70 Ethanol av...

Page 27: ...17 Turn laser emission key from I back to 0 18 Turn off number 4 and 3 power supply units 19 Check that LED beside number 7 is no longer blinking indicating computer has finished shut down 20 Proceed...