OPERATING INSTRUCTIONS
Contents
1. Quit Image Sequence BMP PNG PGM FITS LUT Selection XY Coordinates Page Setup Print Measurements Text JPEG Quality 0 100 90 Frame Rate 7 fps OK Cancel ey Live cell imaging switching on the system 32 a Solent Scientific 1 Switch on the temperature controller NB System takes about 1hr to warm up Please leave the controller set to 35 C the temperature at the sample will be 37 C wA E eA _ 2 Put sample on stage 3 Position water chambers around sample 4 Position CO chamber over sample ensuring it is sitting flat and there are no gaps 10 5 Check tubing is connected to the ddH20 bottle NB Only one bottle should be connected Solent Scientific 6 Turn on CO controller ie atts Fe h 7 Check there is no pressure left in the system by turning the side valve on the gas cylinder in the minus direction ie Anti clockwise The pressure on the bottom gauge should read zero If you do not check this the tubing can EXPLODE when you open the CO air valve 11 8 Open the CO air valve on the top of the gas cylinder 9 Adjust the side valve clockwise to increase the pressure in the system to 1 bar bottom gauge 10 Check you can see bubbles in the water bottle 11 Set up Metamorph 12 Live cell imaging switching off the system Solent Scientific eo Tomberat2. BLUE component DAPI 2 Current Plane Destination mai Color Combine Hue rendering E Enable hue rendering Coefficients CY Review Multi Dimensional Data lo tal Alternatively you can make a multi coloured Select Base File C Users BALM Tech1 Do TIMESERIES ND Open Sequential stack movie using Review Multi Dimensional Wavelengths 3 a 3 a be a a a a m Kk Ke V DAPI Data my GFP A 7 RFP Select the wavelengths you want to combine then in the Display tab tick the Colour composite box Time ae 40 0 1 gt gt and select the correct sources for each E Bile Montage 2 G2 E App wavelength n ee Z Color Composte Source R RFP sss G GFP B DA v lt None gt Scale 16 Bit Images DAPI V Auto Scale GFP oo Reset Image Displays Run Joumal Loop Selections Xs Displa Go back to the Selection tab Display and click Load images as before Remember to save your movie stack usually in Tif format To change your metamorph movie stack into a universally compatible format open the Tif file in Image J Then go to File Save As AVI Make sure you choose Uncompressed And then decide the Quality and Frame Rate ___ New d Open Ctri O Open Next Ctrl Shift O Open Samples b Open Recent gt Import gt Close Ctri W Save Tiff Gif Revert Jpeg B Text Image ZIP Raw Data
3. as bei Le a i 1 Turn off the temperature controller 2 Close the top valve on the gas cylinder 3 Turn the side valve in the minus direction anti clockwise to release the pressure in the system 4 Wait for the gauge to return to zero and the bubbles in the water bottle to stop 5 Turn off the CO controller 13
4. experiment If not delete your old data Make sure you empty the recycle bin afterwards Z stacks C7 Multi Dimensional Acquisition 1 2 3 4 5 6 7 8 9 Main In Multi Dimensional Acquisition select ud sss Multiple Wavelengths and Z series Wavelengths F Multiple Stage Positions API Multiple Wavelengths W2 GFP Z Series Save as normal W3 RFP F Stream files are saved as a single file for each Z Series F Run Journals wavelength Display Summary C7 Multi Dimensional Acquisition llumination GFP Set gain and exposure for each wavelength for Gain 1 a the brightest point in the Z axis live Digtizer Exposure 30 Make sure Z series with the wavelength is ee 3000 ticked for each channel a No Auto Focus Corf Z seres with the wavelength Alignment Cropping X 0 0 amp In the Z series tab live C7 Multi Dimensional Acquisition Either Acquire wavelength set at each Z Main va en Or Acquire Z series for one wavelength at a Saving Curent Position 245 425 um Increment 1 Wavelengths time a Sate Settings for acquisition series Loop order Acquire wavelength set at each Z 0 Acquire Z seres for one wavelength at a time Focus on top bottom of image and click Set top to current Keep shutter open between steps E Range Around Current Set Top To Current Set Bottom To Current Focus in opposite direction and click Set botto
5. OPERATING INSTRUCTIONS TIMELAPSE inverted epi fluorescence microscope Advanced 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 Advanced user guide How to turn the equipment on 1 If you want to do fluorescence switch on the metal halide lamp a 2 Switch on the extension plug b 3 NEW Switch on the Camera using the button on the camera It looks like a computer on button 4 Switch on the computer and log in 5 If you want to do bright field switch on the bright field controller box d How to turn the equipment off Switch off the bright field controller box then the computer then extension plug b and finally the metal halide lamp a 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 20GB of disk space on this microscope Check before you start if you have room for your
6. advanced stage position wavelength table acquisition parameters ad Previous P Net aga Bin 1 G BR Bin H N O A amar BB Bh pcan aose 7 Click Acquire _ Timelapse 1 2 3 4 5 6 7 PECECETETEECECETETN 7 Multi Dimensional Acquisition In Multi Dimensional Acquisition select feet g Saving Timelapse Timelapse and Multiple Wavelengths if you lt lt lt gt Timelapse Multiple Stage Positions are using more than one channel o Wavelengths Multiple Wavelengths W1 DAPI C Z Series Save as normal Wa GFP Stream files are saved as a single file for each foe wavelength at each time point ame ale Display Summary 7 Multi Dimensional Acquisition SI Set gain and exposure of each wavelength live oa me m Gain 1 Digitizer 20 MHz h Make sure Auto Focus is set for Every N Exposure 20 Acquisition and N is set to 6 Auto Expose Never 3000 splay i Every Time Point Summary Auto Focus Namert Coppa X0 e Y0 E ben aea In the Timelapse tab Main a Set the Number of time points and Time Sa Number of time points 13 2 Interval Durston g Duration tells you how long the experiment will take W1 DAPI Time Interval 5 We GFP Estimated minimum interval 0 55 sec Ws RFP Display Summary TY Multi Dimensional Acquisition Main Saving In the Summary tab you can see how much Time
7. dering Remember to save the colour combined stack Pl Enable hue rendering Coefficients _ Multiple stage positions CFT Multi Dimensional Acquisition Main 1 In Multi Dimensional Acquisition select Multiple samy Pi tmekene Stage Positions and Multiple Wavelengths V Multiple Stage Positions Multiple Wavelengths Wavelengths W1 DAPI Z Series 2 Save as normal W2 GFP F Stream files are saved as a separate file for each W3 RFP F Run Journals wavelength at each position Display Summary i d Multi Dimensional Acquisition 3 Set gain and exposure for each wavelength at Illumination GFP one of the positions live E Digitizer 20 MHz a Exposure a0 ms CY Multi Dimensional Acquisition Fol ate j a Position Label 4 In the Stage tab live ae aen Remove any stage positions already stored Stage 314794 P 7 P Wavelengths A from previous experiments by highlighting U i ZER E them and selecting Delete a Offset Z for travel to this position 5 Using the joystick move the stage to a position ummary ae Ziravel oist 0 Position 314698 17376 236 4 M Position2 315438 17376 236 4 Postion 315438 18324 236 4 of interest focus type in a label for the position then click Add current position to list Position4 314794 18072 236 4 G 6 Move to the next position of interest and add it OA a O to the list and so on F Use
8. ional Data window tick the wavelengths you want to use Right click in the top left corner to select all time points NB If you do not want to include all time points you can unselect them here by right clicking in the box for each image Click Load Images A separate stack movie is created for each wavelength use the bar at the top to navigate through the different time points You can change the colour of the stack by clicking on the look up tables button and selecting a colour CY Review Multi Dimensional Data l Select Base File CAUser t BALM Tech1 Do TIMESERIES ND Wavelengths W DAPI W GFP W RFF Lala se Bs T VO T Te i Ta UR ek eax kK Kk E E Enable Montage 2 Hj G Apply Selections IX s Display Load Image s Select Best Focus E gi L ih gt Clear Reset Image Displays EE RFP 2 50 13 13 os Jie is is lt I 4 14 F DAPI 2 33 13 13 rae C D gt fim WW Monochrome Pseudocolor Red Green Blue Yellow Magenta Cyan Set By Wavelength Chroma Cy5 Cy C3 Gold Reset Contrast Configure LUT EY Color Combine o amp eae Source Type Components Stack Single Image To combine different coloured stacks use the Result Depth Colour Combine tool Display Colour combine Sbt 24bt 48 bit See processing Z stack data for more info eor gt E
9. lapse E Wavelengths storage space is required to collect you data oa set Make sure you have enough space on the W2 GFP W3 RFP E Drive and empty the recycle bin a Summary Click Acquire 5 Collection Every time point Exposure 50 ms Gain 2 Digitizer 20 MHz Auto Focus Off Wavelength 3 lllumination RFP Collection Every time point Exposure 100 ms Gain 1 Digitizer 30 MHz Auto Focus Off Image Saving Requirements 219 total images requiring 604 71 MB Description Processing timelapse data faa az Display Process Log Measure Journal Apps To make a metamorph timelapse movie go to Apps and Review Multi Dimensional Data Rewiew Multi Dimensional Data Multi Dimensional Data Set Utilities Measure Colocalization Measure Grid Or use the Review Multi Dimensional Data shortcut x yf Review Multi Dimensional Data FRET Correlation Plot In the Review Multi Dimensional Data window 4 click Select Base File PY Multi Dimensional Data Set Utilities o E eee In the Multi dimensional data set utilities window click Select Directory and find where you saved your data CA User BALM TechT Documents Katy Data Sets Description Timesenes nd a Make sure you tick the dataset you want to use g z Time 13 Time Points Then click View Wavelengths DAPI GFP RFP Data Log Not Open Append Sets In the Review Multi dimens
10. m to current Choose step size or number of steps L Number of Steps 7 see recommended step size for guidance com ended Step Size 0 9 um Click Acquire EB Acquire Processing Z stack data Y MetaMorph m a lt a File Edit Regions Stack Acquire Devices Nol Adjust Digital Contrast DE Contrast Shortcuts b GEL Scale Image To create a multi coloured stack select Display and Colour Combine Graphics b or use the Colour combine shortcut Intensity Profile Overlay Images Color Combine Color Separate Color Align Split View rd Deinterlace ar Color Combine Sy Show Hide Image at Full Screen Set Image Zoom Stretch and Mirror i7 Color Combine fo 5A Source Type Components Stack Single Image Set the bit depth to 24 bit Result Depth and select the stacks that correspond to each mnt Mbt 4Bbi RED component Zstacks2_w3RFP f component Zstacks2_w2GFP BLUE component None Destination stacks w3RFP Hue rendering fstacks2_w1lDAPI Enable hue rend stacks weGFP I w None Selected CY Color Combine EI aE Source Type Components Stack Single Image Result Depth Make sure All Planes of the stack are selected by clicking on the stack icon GREEN component 2st All Planes BLUE component Zstacks2_w1DAPI Current Plane Click Colour Combine Hue ren
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