How to use >Zen2010<

Transcription

1 Hw t use >Zen2010< Inhaltsverzeichnis General Operatin Instructins fr Cnfcal Micrscpe LSM 510 Meta (Zeiss)...2 Start Sftware >Zen2010<...2 Intrductin t Image Cntainer and Wrkflw...3 Hw t use >Zen2010< fr Acquisitin...5 Setting up a New Image Dcument... 5 Start Laser Lines... 6 Direct Observatin: Ocular View... 7 Image Acquisitin: Setting up Single r Multiple Track Cnfiguratin... 9 Image Acquisitin: Cnfiguratin Light Path and Laser Line Image Acquisitin: Setting up Scanning Parameters Image Acquisitin: Image Optimizatin Scanning a Z stack String and Exprting Image Data Clsing Sessin / Shutting Dwn System

2 General Operatin Instructins fr Cnfcal Micrscpe LSM 510 Meta (Zeiss) Turn n Main Switch, Remte cntrl fr cmputer and the cmputer (please fllw the cnsecutive numbering, 1,2,3.ff) Switch n the mercury arc lamp (HBO) 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. Start Sftware >Zen2010< Lg in with Strg+Alt+Entf: user name, passwrd and dmain name set t «LIC» The ZEN sftware is started by duble clicking the ZEN icn. The lgin panel (Fig. a) appears n tp f the ZEN Main Applicatin windw. The sftware can be perated in three different mdes. Nrmally chse Start System Figure 1 Chsing Start System initializes the whle micrscpe system and activates the entire sftware package (image recrding and analysis). The Image Prcessing mde ignres the hardware and activates nly data handling and image prcessing functinality t analyze stred images The Offline/Dem mde reads the active hardware database and simulates the respective hardware. This is nly available by clicking the little arrw left f the text "Bt Status" (Fig. b). 2

3 Intrductin t Image Cntainer and Wrkflw Zen2010 main applicatin windw after start up with Fig. 2) empty image cntainer and Fig. 3) with several images laded Figure 2 On the Left Tl Area (Fig. 2/D) the user finds the tls fr image acquisitin, image prcessing and system maintenance, easily accessible via 4 Main tabs (Fig. 3/1). Ocular: All functins needed t cntrl the micrscpe Acquisitin tls (Fig. 3/3 and 4): t acquire images. Arranged frm tp t bttm they fllw the lgic f the experimental wrkflw. Center Screen Area: area fr viewing and interacting with images, centered in the middle f the Main Applicatin windw. File management and data handling tls are fund in the Right Tl Area (see Fig. 2 and Fig. 3). Figure 3 3

4 Shw all mde, interactive: In the basic mde: Shw all is deactivated and the tls shw nly the mst relevant functins, cvering apprximately 80 % f the users applicatin. Fr each tl, the user can activate Shw all t display and use additinal functinality (Fig. 4) Figure 4 Mre features f ZEN include: The user can add mre clumns t the Left Tl Area r detach individual tls t psitin them anywhere n the mnitr. T add a clumn, drag a tl grup by the title bar (e.g. "Online Acquisitin") t the right and a new tl clumn autmatically pens. T detach a tl, click n the little icn n the very right end f the blue tl header bar r drag it by this blue header bar t where yu want it. Wrkspace Zm: allws adjustment f the ZEN windw size and fnts Wrkspace Layut: Save setting up fr a specific experiment. These cnfiguratins can als be shared between users. Figure 5 4

5 Hw t use >Zen2010< fr Acquisitin Setting up a New Image Dcument Create new image dcument (div. ptins): Click Snap r Click New The new dcument is immediately presented in the Open Images Area. Remember, an unsaved 2D image in the active image tab will be ver-written by a new scan. Multi-dimensinal scans r saved images will never be verwritten and a new scan will then autmatically create a new image dcument. Acquired data is nt autmatically saved t disc There is n "image database" any mre like in the earlier Zeiss LSM sftware versins. Figure 6 5

6 Start Laser Lines Chse the "Laser" menu Start nly the laser(s) required fr the flurphres used in yur sample. All lasers need 30 min. t run stable!! Figure 7 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 "ready" Then click n "ON" Adjust the Output level (%) between 40% Running higher Output (%) is generally nt necessary Check that the current des nt g beynd 6.1 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 633nm" Click n "ON" If the Enterprise (UV) is started: first turn n cling water by the Switch at the right side n the wall, then click Standby, set utput intensity t 50% If 2-Phtn Laser is t be started: turn the key in psitin "ON", start it frm the Sftware: "ON" 6

7 Direct Observatin: Ocular View Click Ocular (8): fr direct bservatin; press the Online buttn fr yur actins t take effect immediately. Then pen the Ocular tl (9) t cnfigure the cmpnents f yur micrscpe like filters, shutters r bjectives, lasers are blcked. Figure 8 Online: mde t set the hardware in psitin fr using the micrscpe Offline: will clse the light shutters n the micrscpe Figure 9 Selecting an bjective (9+10) Open the graphical pp-up menu by clicking n the Objective symbl and select the bjective lens fr yur experiment. The chsen bjective lens will autmatically mve int the beam path. Fcusing the micrscpe fr transmitted light (9+10) Open the graphical pp-up menu by clicking n the Transmitted Light icn. Click n the On buttn. Set the intensity f the Hal. lamp using the slider 7

8 Setting the micrscpe fr reflected light (9+10) Click n the Reflected Light icn t pen the XCite Cntrls and turn it n. Click n the Reflected Light shutter t pen the shutter f the X-Cite lamp. Click n the Reflectr buttn and select the desired filter set by clicking n it. Figure 10 Stre micrscpe settings (11) Save: Micrscpe settings can be stred as cnfiguratins by typing a cnfiguratin name in the pull-dwn selectr and pressing the Save buttn Assign: These cnfiguratins can be assigned t buttns that are easier t press Figure 11 8

9 Image Acquisitin: Setting up Single r Multiple Track Cnfiguratin Click Acquisitin: mve t the LSM system Figure 12 Open Imaging Setup and the Light Path tl in the Setup Manager tl grup t access the hardware cntrl windw t set-up the beam path.. Tw cnfiguratin ptins (select Channel Mde, if necessary): Simultaneus scanning f single, duble and triple labeling: Advantage: faster image acquisitin Disadvantage: ptential crss-talk between channels Figure 13 Sequential scanning f duble and triple labeling; line-by-line r frame-byframe: Advantage: Only ne detectr and ne laser are switched n at any ne time. This reduces crsstalk. Disadvantage: slwer image acquisitin Figure 14 Add and Remve Track Switch track every 9

10 Line: Tracks are switched during scanning line-by-line. The fllwing settings can be changed between tracks: Laser line, laser intensity and channels Frame: Tracks are switched during scanning frame-by-frame. The fllwing settings can be changed between tracks: Laser line and intensity, all filters and beam splitters, the channels incl. settings fr gain and ffset and the pinhle psitin and diameter. Frame Fast: The scanning prcedure can be made faster. Only the laser line intensity and the Amplifier Offset are switched, but n ther hardware cmpnents. Image Acquisitin: Cnfiguratin Light Path and Laser Line Setting up the Light Path (Fig.15 here: Cnfiguratin fr single track, simultaneus scanning f tw laser lines) Click n the LSM tab The Light Path tl displays the in the Imaging setup windw selected track cnfiguratin which is used fr the scan prcedure. Select apprpriate emissin filters and dichric beam splitters (HFT, NFT,BP, LP and s n) and activate channels Click Laser: t select the laser lines and set attenuatin value (%) Figure 15 Activatin / deactivatin f the excitatin wavelengths (check bx) and setting f excitatin intensities (slider). If necessary pen the Laser Cntrl tl (see abve). 10

11 Selectin f the main dichric beam splitter (HFT) r secndary dichric beam splitter (NFT) psitin thrugh selectin frm the relevant list bx. Selectin f an emissin filter thrugh selectin frm the relevant list bx. Activatin / deactivatin (via check bx) f the selected channel (Ch 1-4, mnitr dide ChM, META detectrs ChS1-8, transmissin ChD) fr the scanning prcedure and assigning a clr t the channel. Stre Track cnfiguratin Fr string a new cnfiguratin enter a desired name in the first line f the Cnfiguratins list bx and click Stre Buttn (Disc) Yu can als lad and delete an existing cnfiguratin. Figure 16 Image Acquisitin: Setting up Scanning Parameters Select the Acquisitin Mde tl frm the Left Tl Area (Fig. 17) Frame Size : Select the Frame Size as predefined number f pixels r enter yur wn values (e.g. 512x512) in the Acquisitin Mde tl. Optimal buttn. Click fr calculatin f apprpriate number f pixels depending n bjective N.A. and λ. Scan Speed: Use the slider t adjust the scan speed. (A higher speed with averaging results in the best signal-t-nise rati. Scan speed 8 usually prduces gd results. Use speed 6 r 7 fr superir images.) Bit Depth: Select the dynamic range 8 r 12 Bit (per pixel) in the pull-dwn (8 Bit will give 256 gray levels; 12 Bit will give 4096 gray levels. Publicatin quality images shuld be acquired using 12 Bit data depth. 12 Bit is als recmmended when ding quantitative measurements r when imaging lw flurescence intensities) 11

12 Average. Averaging imprves the image by increasing the signal-t-nise rati. Averaging scans can be carried ut line-by-line r frame-by-frame. Frame averaging helps t reduce pht-bleaching, but des nt give quite as smth f an image Figure 17 Adjusting the pinhle size (Fig. 18) Select Channel Tl Set the Pinhle size t 1 AU (Airy unit) fr best cmprmise between depth discriminatin and detectin efficiency. Pinhle adjustment changes the Optical Slice thickness. When cllecting multi-channel images, adjust the pinhles s, that each channel has the same Optical Slice thickness. This is imprtant fr clcalizatin studies. Image Acquisitin: Image Optimizatin Once yu have set up yur parameter as defined in the abve sectin, yu can acquire a frame image f yur specimen. 12

13 Click Live, Cntinuus r Snap buttns t start the scanning prcedure t acquire an image Aut Expsure, als starts scanning, used fr autmatic pre-adjustment f gain Click n the Stp buttn t stp the current scan prcedure if necessary. Figure 18 Scanned images are shwn in separate windws. Figure 19 Chse Range Indicatr In the View Dimensins View Optin Cntrl Blck, click inside the clr field in the buttn under the channel buttn (Fig. 19). Clicking n the right hand side f the buttn leads t a list f clrs. 13

14 Figure 20 The scanned image appears in a false-clr presentatin (Fig.20) Red = Saturatin (maximum), image is t bright Blue = zer (minimum): image is nt bright enugh Adjusting the laser intensity, gain and ffset Select Channel Tl Figure 21 Set the Pinhle t 1 AU (Airy Unit) (Fig. 21) Reduce r increase the Gain (Master) until the red pixels nly just disappear. When the image is still saturated, reduce AOTF transmissin in the Laser cntrl sectin f the Channels tl using the slider t reduce the intensity f the laser light t the specimen Increase the Digital Offset until all blue pixels disappear, and then make it slightly psitive Scanning a Z stack Activate in the main tls area. If a multi-dimensinal acquisitin tl is nt selected, the respective tl and its set parameters are nt included in the multidimensinal image acquisitin. If n multidimensinal tl is activated, the special buttn is grayed ut and nly single images can be scanned. Open the Z-Stack tl in the Left Tl Area 14

15 Figure 22 Select Mde First/Last n the tp f the Z-Stack tl.. Click n the buttn in the Actin Buttn area. A cntinuus XY-scan f the set fcus psitin will be perfrmed Use the fcus drive f the micrscpe t fcus n the upper psitin f the specimen area where the Z-Stack is t start. Click n the Set First buttn t set the upper psitin f the Z-Stack. Then fcus n the lwer specimen area where the recrding f the Z Stack is t end. Click n the Set Last buttn t set this lwer psitin. Click Optimal, set number f slices t match the ptimal Z-interval fr the given stack size, bjective lens, and the pinhle diameter. Click n the Experiment buttn t start the recrding String and Exprting Image Data T save yur acquired r prcessed images, click n the Save r Save As buttn in File menu r buttn in the main tlbar r in the main tlbar 15

16 Figure 23 The WINDOWS Save As windw appears. Enter a file name and chse the apprpriate image frmat. Nte: the LSM 5 frmat is the native Carl Zeiss LSM image data frmat and cntains all available extra infrmatin and hardware settings f yur experiment. Click n the Save buttn. If yu clse an image which has nt been saved, a pp-up windw will ask yu if yu want t save it. Chsing yes will lead yu t the WINDOWS Save As windw. Figure 24 Exprt: T exprt image display data, a single ptical sectin in raw data frmat r the cntents f the image display windw including analysis and verlays, chse Exprt frm the File menu. In the Exprt windw yu can select frm a number f ptins and prceed t the WINDOWS Save As windw t save the exprted data t disk. Clsing Sessin / Shutting Dwn System End Sessin (if smene will use this wrkstatin afterwards): 16

17 Examine, which lasers are used by fllw up in the Web Calendar, this laser line can be left ON, turn OFF the ther lasers via the sftware. Clean bjectives Turn fcus dwnward, set smallest bjective Exit Sftware, Lg OFF Fill ut Lg Sheet Remve all yur waste (samples, cver slides, ) Prtect syringes fr immersin media (H2O and/r il) always immediately with prtective cap, since therwise cntaminatin r danger f injury exists! Turn Off (nly last user) The last user is respnsible fr ensuring that the system was crrectly shut dwn. TURN OFF all lasers via sftware, wait at least 5 min t allw laser mdule t cl dwn In case f UV Laser: turn ff cling water after 5 min (therwise cndensate frms in the device!!) Exit Sftware, LOG OFF Shut dwn the cmputer TURN OFF the Main Switch TURN OFF the HBO lamp Fill ut Lg Sheet Leave the place in such a way, that we d nt have t clean up!! 17