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mDSLM User Manual 01

1. all the time but the reading is only visible in the window for the electronics box and in the log file 4 Transmission light Use 760 nm position Donotuse a filter Intensity can only be controlled by changing the exposure time laser power does not work for transmission 6 position filter wheel Semrock filters not necessarily available in their catalogue but the Stelzer group has a contact person whose details we should get One position has to stay empty the others contain bandpass or longpass filters Filter wheel can be turned manually when it is off but never try that when it is on Ludl Electronics filter wheel controller with green on off button With time filters will become loose and start to rattle and need to be tightened up again The filter wheel must not touch the microscope to avoid vibration If the filter wheel does touch the microscope casing you will get vibration visible by the generation of sinus waves on screen and the filter wheel position will have to be adjusted The air table needs to be well aligned and floating 5 Camera Andor Clara cooled CCD camera With higher magnification objectives camera vibration becomes noticeable To avoid this use the software option that allows turning camera cooling off during acquisition Auto on off There are two holes at the back of the microscope casing which contain screws The one closest to the camera adjusts the camera angle the other o
2. to get beads in focus and adjust y height as well as y center can adjust Y height and center using beads Use the z stage control to move the light sheet to the edge of the agarose block that is closest to the detection objective Set the correct z spacing Pixel size in x and y x 4 objective magnification 6 45 um x 4 10 2 58 um z spacing 6 45 um x 4 20 1 29 6 45 um x 4 40 0 645 6 45 um x 4 63 0 402 Set Start plane then set 300 planes by moving the z stage control until you get ca 300 planes By clicking on Set End Plane the number of planes is updated Set timelapse long total duration 1h interval 30 s Untick Save next to Start Acquisition tick z stack and time lapse and click Start Acquisition Use the detection Pifoc to focus beads strongest signal no rings 11 Use the illumination Pifoc to move the focussed beads into the middle of the sheet Change the laser line and align again How to centre the stage This needs to be done when a new chamber is fitted to the microscope On delivery the stage is centred 12 Remove the chamber and unscrew the basin put the basin aside Unscrew the head of the stage Fit the head of the stage to the chamber in the least wobbly position Make sure that the specimen holder keeps its correct position Put the chamber back in with the head of the stage attached The pin attached to the head of the stage will now not be centred to fit t
3. Pifoc control If not already correct move the light sheet centre along its axis to the centre of the camera image using the scanner offset control top panel and adjust the light sheet height to span the whole image or a smaller ROI Remove the alignment mirror using the reverse procedure of fitting it Alignment is finished Specimen can be inserted Camera view of the sample left top bottom right 9 Alignment procedure for several laser lines using the mirror Use the mirror only if you are not successful using beads for alignment The 488 nm laser line is now too strong to be used with the mirror The mirror is used to correct chromatic aberration between different laser lines Do the alignment below using the mirror before the bead alignment if you want to image several channels Click on Preview Insert the mirror with y 13 000 and z 1 500 turn to 45 using tweezers adjust y to right height with tweezers if not inserted into the specimen holder all the way Then move to z 0 Adjust x with the x stage controls until the laser beam is visible laser line of your choice integration time 50 ms no filter adjust dynamic range bits in between With the 40x objective only one of the two reflections will be visible Move the x stage until you see the higher beam and concentrate on that If the beam is not perfectly horizontal it does not matter and is caused by the mirror not be
4. e the specimen holder to the front right end z 1 500 um and x 1 500 um of the chamber using the z and x stage control Insert the alignment mirror using the provided forceps so that its surface normal its reflective surface is the one that intersects the centre of the cylindrical rod it is glued to roughly aligns with the detection axis mirror surface faces the detection objective Adjust the insertion height so that the mirror will when back in the centre position intersect the illumination beam centre line of the illumination objective Move the stage back to z 0 using the z stage control Turn on the Preview mode with acquisition parameters set to Laser line of your choice Power 0 Integration time 50ms No filter Now rotate the stage carrying the specimen holder and the alignment mirror by 45 using the stage control so that the illumination light is intersected and directed towards the detection objective roughly parallel with the detection axis if necessary you can also use the x or z stage controls to bring the mirror into that position If not already visible increase the power until you see a dim light sheet or light soot on the camera image 10x objective Use the x stage controls to bring the light sheet into the centre of the camera image Set the step size for the control to 1 and using the and stage controls find the angular acceptance limits range of the detection lens and rotate t
5. he base Using the software move the stage in x and z and later also in y to centre it to the pin Adjust the pin in such a way that it does not touch the sides of the hole The stage connects with the table at y 10 000 Move to 10 000 and tighten screw To access the microscope controller open http 192 168 1 2 in Internet Explorer Remote file browser button on the left Open folder Tara Settings Stage Settings Use Copy and Paste buttons to create a copy of the stage settings Double click on the file to save it on the desktop and give it the same name as before Open file in Excel if installed Home position is the important one old value movement new value for the x and y axes 7800 1100 8900 Save the file and use download upload button to move the file to the folder Could have used the Edit button for all this instead Restart realtime computer by pressing reset button on realtime controller after exiting the acquisition software Console button shows what the realtime computer is doing after restarting Internet Explorer Restart acquisition software Unscrew head of stage again and re attach it to the table to check if it is centred Move the stage up to 10 000 and tighten the screw Specimen Multi Stage Position Specimen 1 Define new specimen Sync channels sync basic setup this will copy stack size and position as well as spacing or just fill in spacing In specimen 1 and then in specimen 2 move to correct posi
6. he mirror to the centre of this range Higher magnification objectives If the above is not possible i e light sheet moves out of the field of view use detection Pifoc and stage controls to align the rotation stage When moving the Pifoc the light sheet moves out of focus but also changes position if the mirror angle is not at 45 Use the stage control to rotate the stage by 1 either direction is fine and check by moving the Pifoc over the full range whether the shift has decreased or increased If it has increased change the direction of the step otherwise continue in the same direction with another step Successive steps minimising the position shift of the light sheet bring the mirror angle closer and closer to the 45 position The mirror now intersects the illumination and the detection axes by 451 Use the detection Pifoc control to bring the mirror surface not the light sheet into focus which when achieved is apparent by well resolved always present dust particles on the surface of the mirror If this is not possible i e the Pifoc range is not large enough use the scanner offset control together with the z stage control use small step sizes e g 1Oum to bring the surface of the mirror within the range of the Pifoc while keeping the light sheet position centred on the camera range Minimize the thickness of the light sheet visible on the mirror i e in the centre of the camera image using the illumination
7. ing perfectly vertical Do not infer that the camera angle is wrong Lower laser intensity to O Use y height to reduce lines to dots y height changes the height of the light sheet the higher the light sheet the shorter the time per point which decreases the laser intensity and increases the length of the lines on screen Use y center to change left right positioning of the dots to the centre of the screen Y center moves the whole light sheet up or down Z position moves the lines up or down Do not use Now use y height to increase the length of the lines on screen so that the ends are outside the field of view and the bright end is not visible anymore Concentrate on the upper line When moving the detection Pifoc a long way the light sheet may move in x If this happens the mirror is not at precisely 45 Change in 1 steps and move the detection Pifoc until the light sheet stops moving in x Use the detection Pifoc to focus on dirt on the upper light sheet Activate the cross hairs and move the upper light sheet to the centre using the x stage control Use the illumination Pifoc to find the position where the light sheet is slimmest Now adjust the second channel by choosing the relevant laser line no filter and laser power 0 The upper light sheet of the first channel has to be in the centre of the cross hairs see above lf the laser power in the second channel is too st
8. ion tick Time Lapse tick Save Start Acquisition Alternatively use Next Specimen for all the different angles Rotation works only if the needle for compensation the capillary and the agarose are absolutely vertical and if there is no wobble One should mount samples in micropipettes for the rotation 14 Structured Illumination This generates 3 images per plane phases are moved by 120 Structured illumination Scanning Properties Laser Power Modulation Current SI Frequency modulates laser on off depends on the sample and on how deep you are inside the sample 30 60 are good numbers 15 Preparation of Tetraspeck beads for mDSLM objective lens final conc beads mL bead stock 150 uL 10x NA 0 3 3 15E 07 5 19 ul 1 um beads 20x NA 0 5 3 45E 08 56 85 uL 1 um beads 40x NA 0 75 6 29E 09 40 95 uL 200 nm beads 63x NA 1 0 2 77E 10 180 uL 200 nm beads Set the heatblock to 70 C Prepare 2 low melting agarose in 2x the buffer you are going to image your samples in Vortex your bead stock thoroughly and make up your beads in H2O dest to a total volume of 75 uL For the 63x lens the beads need to be dried down in a speed vac before use Warm the diluted beads up in the heat block Add 75 uL of the low melting agarose and vortex thoroughly Put back into the heat block and draw the liquid up into the capillary using the Teflon plug Hold capillary against ice until the agarose is set TetraSpeckTM Fluorescent Micr
9. mDSLM Manual Start up procedure 1 Switch on computer 2 Change objectives before switching on microscope 1 2 3 If you are a using a different chamber from the previous user the stage must be realigned by a member of staff before you continue 11 4 Screw chamber in fill with appropriate medium switch microscope on and start acquisition software 6 7 Check for leaks 5 If you are using more than one laser line you need to align the different laser lines 3 4 9 If you are using one laser line move on to the next item 6 Align the appropriate beads 10 7 Calibrate your rotation compensation tool if you want record multiple angles of your specimen 13 8 Insert your sample Shut down procedure 1 Set to P 0 set x stage control to x 0 set z stage control to z 0 set y stage control to y 0 Remove sample Exit acquisition software 2 Remove medium and unscrew chamber Clean chamber and tools to prevent rust Switch microscope off before removing any objective Unscrew detection objective and clean it 5 Shut the computer down at the end of the day Rw 1 llumination objectives and detection objectives 2 5x NA 0 06 illumination objective generates more homogeneous light for 10x NA 0 3 and 20x NA 0 5 detection objectives If you use the 5x illumination objective for the 20x detection objective the light sheet will be slimmer but only over a short distance in the middle and onl
10. ne adjusts the tube length How to check the camera angle on assembly Special metal tool inserted into probe slot at 45 angle just like the mirror Use transmission light 760 nm 150 ms exposure no filter Focus on the right pointed edge of the tool using the z control Draw an ROI around the edge of the blade or use the cross hairs lf the blade does not look horizontal on screen loosen screw 1 3 mm in hole at back of casing closest to the camera which fixes the camera in place Slowly rotate the camera until the blade in the field of view is perfectly horizontal and fasten screw When you move the blade in y in 100 um steps it must not move in x The stage movers The cables that connect the stage movers x y z must not hang down and pull Ensure that the heavy connectors are fixed to the table Galvo mirror 1 screw for alignment no alignment should be necessary If there is a problem contact Alexander Atzberger alexander atzberger physikalischebiologie de DIA illumination Transmission light with power switch set to maximum and therefore fixed intensity can only be controlled by changing the exposure time and changing the camera bits The chamber Finger gloves are available from pharmacies Latex without powder size M Coverslips 300 um thickness 18 mm diameter glued in with nail varnish from the outside Grease for the chamber Glisseal N Swiss grease for laborato
11. osphere Standards Life Technologies T7280 0 2 um diameter T7282 1 0 um diameter Samples are mounted in low melting agarose at 0 8 1 2 Cool the agarose down pipet it into the capillary and add the specimen Arabidopsis seedlings Grow seedlings vertically on 1 pytagel Scoop seedling with pytagel into capillary leaving the shoot exposed Dip bottom end of the capillary in 1 low melting agarose to lift up the root until it is exposed as well Add carbon pin to support root Perfusion system Ismatec ISM831C Capillaries Hilgenberg ask Alex
12. ries suitable for vacuum without silicone 6 How to change objectives Only change objectives when the microscope is turned off Remove water from chamber set x stage control to x 0 set z stage control to z 0 set y stage control to y 0 Unscrew the chamber Change objectives Check that the finger glove is in the right position and not ruptured It is better to change the finger glove when changing objectives You need a special tool to unscrew the rings that hold it in place Fit a new glove over the inner ring and cut off the finger leaving ca 1 5 cm Screw the finger glove in so that the fat end points to the inside of the chamber Push the cut off end in as well Screw the chamber back in fill with water and check for leaks Check again after 15 min Turn microscope back on and activate Preview in the acquisition software Set the y stage control to y 13 000 set the z stage control to z 1 500 Fit the sample capillary Set the z stage control to z 0 7 How to start the microscope and the acquisition software There is a red start button on the console for the microscope that also controls the realtime computer The acquisition software computer is separate When the microscope crashes press Reset button on the realtime computer mDSLM LightMicroscopy Group EMBL Heidelberg Acquisition software Computer sign in your Biochemistry login password your Biochemistry password embl password mds m sof
13. rong even when using the full dynamic range 14 bit increase y height temporarily to the highest setting Enable both channels and use light sheet position offset to perfectly superimpose both least amount of wobble match both intensities as well as possible It is always best to adjust the outer to the position of the 561 nm laser line If you want to adjust three channels set the light sheet position offset of the middle wavelength 561 nm to 0 Move the top line of the light sheet of the 561 nm laser into the centre of the crosshairs using the x stage control Switch channel and move the second laser to the same position using light sheet position offset followed by the third 10 mDSLM alignment using beads Perform a bead alignment every time you change objectives If you are not changing objectives perform the alignment once a day Use Tetraspec beads in 1 low melting agarose um beads for the 10x and 20x objectives and 200 nm beads for the 40x and 63x objectives The default stage settings before adding the beads are y 0 x 0 z 0 0 Click on preview to activate stage settings and laser Move the y stage control to y 13 000 then move the z stage control to z 1 500 Add the beads and move the z stage control to z 0 Use any laser line laser power gt 0 integration time 50 ms appropriate emission filter For coarse adjustments go to Settings under Scan Contoller Use detection Pifoc
14. tion set start plane set end plane start at surface and end further into the sample moving in the other direction is possible Set time lapse this is valid for both channels Click Save and decide where to save Set Position Here button without function Start acquisition When finished open file in Explorer Open Fiji Highlight file in Explorer and drop into Fiji Stacks Manipulation Concatenate When you have your concatenated stack Stacks Hyperstack Stack to Hyperstack Forz projection over time Image Stacks z project 13 Rotation There are two tools for rotation compensation a metal needle for the 10x and 20x objectives and a glass needle for the 20x and 40x objective To make the glass needle break a bit off a Femto tip and glue it on to the holder for thin capillaries Fit the needle into the specimen holder 760 nm 150 ms exposure filter empty and centre it using the crosshairs Focus on the needle and then fit an ROI to the tip of the needle Advanced in top menu Manual Calibration follow instructions and when finished press Finish Remove the needle and insert your sample focus on it and set up a stack that starts way outside the specimen and ends way outside the specimen Rotation start angle 0 end angle 315 angular step 45 steps 8 alternatively start angle 0 end angle 330 angular step 30 Possibly set camera gain Tick Z stack tick Rotat
15. tware Lsfm Contr 4 9 1 Recorded Data Set up network path Copy only copies data from computer to network without deleting Save in folder on computer File name saves on computer File structure Multiplane 1 Tiff per z stack and channel and time point Hierarchical 1 folder for timepoint containing 1 folder per specimen Flat 1 folder and every file is in there 1 file for each plane and each channel Format Tiff 16 regular can contain up to 4 GB Big Tiff can hold bigger files for sSCMOS cameras only Scan controller settings can be saved and loaded do not touch Gamma and Angle The minimum exposure time is 50 ms because the scanner moves too slowly for less We will get software upgrades and will be notified by email beforehand 8 mDSLM alignment procedure using the mirror The chamber must be filled with water to above detection lens level Click enter after typing each number to set scan and Pifoc parameters to Lightsheet height 1000 Illumination Pifoc 50 Detection Pifoc 50 x centre and y centre 0 Move stage up using the y stage control button x z and angle must be in their default position i e at zero until it couples fully to the specimen holder at ca y 10 000 um and then lift the specimen holder by ca 1 500 um again using the y stage control to a point where it is positioned well above its parking position and where it can now be freely moved in the horizontal x z plane Now mov
16. y slim objects can be imaged with this combination 5x NA 0 16 illumination objective generates more homogeneous light for 40x NA 0 75 and 63x not currently available detection objectives Only change objectives when the microscope is switched off and the chamber has been taken off Every time you change the detection objective change the finger glove or at least check it has not acquired any holes How to fit the laser cable on assembly The cable has two discernable ends The microscope end is just pushed in To fit the end at the laser unit press button to push end in but do not move the adjustable screws which have been adjusted and fixed in the factory 2 Objective Pifocs The Pifocs vibrate when no objective is mounted but will be still when the objective is on Always switch the Pifocs off before changing objectives In order to do that switch the whole microscope off because the Pifoc power switch is hidden 3 Laser lines 488 nm diode laser 561 nm DPSS solid state laser AOM 638 nm diode laser The lasers are not quite stable during the first hour of operation Switch on the electronics with the lasers and wait 20 min before starting an experiment Even at 0 power the lasers emit a bit of light They can be unclicked and so turned off in the software at random but it is best to leave the solid state laser on otherwise you have to wait 10 min before imaging A photodiode measures laser power

mDSLM User Manual 01

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