Confocal Microscope usage manual ZEISS LSM 510 Meta

Transcription

1 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : 2.0 Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Page 1 sur 14

2 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : 2.0 Detailed Summary 1. Pwering up the cnfcal micrscpe Zeiss LSM 510 Meta Page 3 2. Starting the LSM 510 Sftware Page 3 3. Pwering up the lasers Page 4 4. Getting ready fr viewing thrugh eye pieces Page 5 5. Setup f the scanning cnfiguratin Chsing filters fr flurphres in sample Page 6 6. Scanning and detectin tuning Page 8 7. Acquisitin image ptimisatin Page 9 8. Saving images Page Acquire a Z-stack Page Clsing yur sessin / Shutting dwn the system Page 13 Page 2 sur 14

3 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : Pwering up the cnfcal micrscpe Zeiss LSM 510 Meta Switch n the mercury arc lamp (HBO) befre turning n "Remte Cntrl" switch. If cnventinal flurescence is nt required there is n need t turn n the HBO Lamp. Let the lamp strike (abut secnds) and ensure light is nt flickering. If HBO hurs are greater than 200 hurs please infrm facility staff and put nte in lg bk. The cmputer shuld be already n. If necessary switch it n. 2 - Starting the LSM 510 Sftware Lgn with user name, passwrd and dmain name set t «CIF» Duble click n the "LSM 510 META" icn. Chse "Scan New Images". Then "Start Expert Mde". The sftware is nw started. Yu have nw t cnfigure the system fr yur wn acquisitins. Page 3 sur 14

4 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : Pwering up the lasers The «expert mde» windw appears - Chse "Acquire". Press the "Laser" buttn Start nly the laser(s) required fr the flurphres used in yur sample. If the Argn laser 458, 488, 514 nm (blue excitatin fr FITC, GFP, YFP, Bdipy, CY2, etc.) is t be started : Select "Laser unit Argn/2 " First g t standby and wait until the text says "n line" Then click n "n" Adjust the Output level (%) between 40% and 50% Running higher Output (%) is generally nt necessary Check that the cuurent des nt g beynd 8.1 Amps If the HeNe1 laser 543 nm (green excitatin fr Rhdamine, Texas Red, CY3, Alexa 543/568, etc.) is t be started: Select "Laser unit HeNe1 543 nm" click n On If the HeNe2 laser 633 nm (red excitatin fr CY5, Alexa 633, etc.) is t be started: Select "Laser unit HeNe2 633 nm" click n On If the Did laser 405 nm (DAPI, Hechst, etc.) is t be started: Select "Laser unit Dide 405 nm" click n On Page 4 sur 14

5 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : Getting ready fr viewing thrugh eye pieces Press the "Micr" buttn and then the "Vis" buttn Select the viewing mde : FITC, Rhdamine, DAPI, r BF (Bright Field) (N.B. If these buttns are blank please cntact the CIF staff) Objectives may als be changed. Fcus n sample and check flurescence If yur sample has been viewed with an il immersin bjective, d nt frget t clean the slice BEFORE switching t a dry bjective! Page 5 sur 14

6 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : Setup f the scanning cnfiguratin Chsing filters fr flurphres in sample Press the "Cnfig" buttn (see picture belw) Fr standard PMT detectin select the "Channel Mde" buttn Fr single fulrescence channel detectin r simultaneus duble channel detectin chse the "Single Track" buttn At this pint yu can chse an existing cnfiguratin, r create yur wn ne. T use an existing cnfiguratin : Press the «Cnfig» buttn Then chse the apprpriate cnfiguratin in the pull-dwn menu Page 6 sur 14

7 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : 2.0 T create yur wn cnfiguratin : Select the different bject yu want t put in the light path (filters, dichrïc mirrrs, see labels n the screensht) Check that yur cnfiguratin makes sense with the «Spectra» buttn Fr a multi-channel detectin : Select the "Multi Track" buttn (NB: gd chice fr clcalisatin trials t avid crsstalk artefacts) Create ONE cnfiguratin er channel and give them an apprpriate name in the bx designed fr this purpse : Page 7 sur 14

8 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : 2.0 Yu can chse t apply a «single track» cnfiguratin t each track by clicking the buttn «Stre/Apply Single Track», r use an existing multitrack cnfiguratin by clicking the «Cnfig» buttn. NB : Many ther scanning ptins are available such as : Spectral detectin (called Meta by Zeiss) Sequencial detectin with the Meta channel and PMT channels 6 - Scanning and detectin tuning Switch t the LSM mde by clicking n the «LSM» buttn Open the Scan Mde windw by clicking n the «Scan» buttn Press the "Mde" buttn Tune the fllwing parameters : Select "Frame" (frm spt - line - frame) Chse the image size (size in pixels, e.g : 512x512 u 1024x1024), begin with 512x512 "Scan speed" : leave the default value as it is this time "Pixel depth" (8 bit) : leave this value t 8 bit yu d nt plan t d quantitative analysis. Mde: chse "Line", r "Frame" fr nise reductin in the picture Methd (Mean) : Leave the default value. Number (tipically 1, 2 r 4, depending n nise amunt and signal intensity in the picture) Page 8 sur 14

9 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : 2.0 Select the "Channels" buttns Tune the fllwing parameters : Select a Pinhle f 1 (fr nw) Set the 488 nm excitatin laser slider t ~ 0.05% - 5% de transmissin (typically 2%) Set the 543 nm u 633 nm laser sliders t 30-40% Set the 405 nm laser slider t 1-5% 7 - Acquisitin Image ptimisatin Use the "Find" buttn t autmatically find gain and ffset values fr the detectr fr each channel individually. These settings will then have t be ptimized. Page 9 sur 14

10 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : 2.0 Press the "Palette" buttn in the image windw Select "Range Indicatr" - red pixels = saturated - blue pixels = zer value Press "Cnt." fr cntinuus scanning Chse a "Channels" buttn (that s Ch2 r Ch3) Set the "Amplifier Offset" slider t a value where blue pixels becme rare ("black clipping"). Set the "Detectr Gain" slider t a value where red pixels becme rare t ("saturatin"). Click n the "Single" buttn fr a full reslutin image with meaning Page 10 sur 14

11 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : Saving images Click n the «save» buttn n the image windw If yu haven t created any database yet, create ne by clicking n the «MDB» buttn The MDB MUST be created in yur persnnal directry (\\Cifserver\CIF-USERS\yurlgin\) Page 11 sur 14

12 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : 2.0 Then save yur image by filling the fields and chsing yur database, then click «OK» Yur images are then stred and visible in the database pannel : Page 12 sur 14

13 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : Acquire a Z-stack After having chsen a field f interest and fine tuned yur image, chse Z-Stack then «Mark First/Last» : En mde Z Stack le butn «Single» devient le butn «Start». G t «Fast XY» mde and mve the fcus in ne directin until there is n signal anymre, and clcik n «Mark First». Mve the fcus in the reverse directin until there is n signal anymre and click n «Mark Last». Select the number f slices yu want t d in this interval and click n the «Start» buttn. NB : D nt frget t click n Z Stack ne mre time when yu have finished t leave the Z Stack mde Clsing yur sessin / Shutting dwn the system If anther user is ging t use the cnfcal in the next hur, (See RESERVATIONS) the lasers can be left ON If this is nt the case, the switch all lasers t OFF, and the HBO lamp t. Page 13 sur 14

14 CIF Manuals Titre : Cnfcal Micrscpe usage manual ZEISS LSM 510 Meta Date: 03/05/2005 Authr : Yannick KREMPP Versin : 2.0 Leave the Remte Cntrl n ON (sur 1) The cling fans will still wrk during 5 minutes and then shut dwn autmatically (difference will be audible) Clean the il immersin bjectives with 2 ctn-swabs (Q-Tips) briefly dipped in ether by sweeping first the glass lens then the metal barrel Clean il and fluids frm micrscpe stage Quit the Zeiss sftware Transfer yur data (images) n e.g. \\CIF-SERVER\CIF-USERS r burn them n a CD Shut dwn the cmputer Replace the dust cver n the micrscpe Put all dispsable paper prducts, cans, wrappers int the trash can Once the fans has becme quiet switch ff the Remte Cntrl The last user is respnsible fr ensuring that the system was crrectly shut dwn. If yu have cancelled yur sessin the same day then : Call the CIF t ensure the system has been shut dwn Or cme ver t the facility t ensure the system is shut dwn yurself. Page 14 sur 14