Zeiss Apotome Fluoro 5 Quickstart Guide For ZEN v2.3. Guide V1.00, updated January 19th, 2017.

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1 Zeiss Aptme Flur 5 Quickstart Guide Fr ZEN v2.3. Guide V1.00, updated January 19th, Overview The Zeiss Aptme is an upright cmpund micrscpe with multiple autmated functins, capable f use as a transmitted light DIC micrscpe (10-60x), a basic widefield epiflurescence micrscpe, and an enhanced reslutin epiflurescence instrument (via the Aptme attachment). The system can capture tiled images, images at predefined pints, and z-stacks suitable fr decnvlutin. N.B. Be mindful f what yu are ding at all times. The micrscpe is delicate and very expensive, and must be treated as such.

2 Slide Cleaning and Preparatin Make sure yur slide is scrupulusly clean. All dust, ld il and munting medium shuld be remved with a little 80% ethanl slutin. If yu can use a munting medium that sets hard, please d s. D nt use glitter nail plish t seal slides as it scatters excitatin light; we recmmend IMBil munting media, available t rder thrugh the PPMS bking system. Be sure t allw yur slides and sealants at least eight hurs t set, prir t imaging. Startup Check the micrscpe t be sure it is clean. Yu are respnsible fr the cnditin f the micrscpe at the clse f yur sessin. If yu find it dirty, make sure micrscpy staff knw abut it; that is, r `phne us. Check the eyepiece diptre settings. If yu dn t need t make an adjustment fr yur visin, set bth t zer. Set the interpupillary distance fr cmfrtable bincular viewing. Lg in t the cmputer. If yu will be using flurescence, click the Lamp On icn n the Windws desktp. Nte, if the lamp wn t turn n, check the cntrl bx t the right f the scpe. If the cling light is n, yu ll need t wait fr it t turn ff befre yu can start the lamp. Press the pwer buttn n the bttm left hand side f the micrscpe stand and wait fr the LCD n the micrscpe t finish lading. Start the acquisitin sftware, ZEN, by duble-clicking the ZEN desktp icn. Please be patient: ZEN is a large prgram and takes a while t lad and start. Duble-clicking numerus times will nt make this prcess g any faster and may, in fact, prevent it starting. Nte that yu may als start ZEN by right-clicking n the ZEN icn and selecting Open frm the resulting menu. When ZEN starts, click Zen Pr and wait fr system t finish initialising. This t will take time. Be patient. If the system will nt bt prperly r yu get a stream f errrs, d a full restart. Clse ZEN. Wait at least sixty (60) secnds. Shut dwn the cmputer. Wait at least sixty (60) secnds, but ideally tw (2) minutes r mre, t allw the cmpnents in the micrscpe t lse any residual charge and reset themselves. Start the system again as described abve. The vast majrity f errrs are reslved this way, s just be patient and take yur time. The wrst thing yu can d is rush, as the hardware and sftware have many things t d and rushing thrugh start up will almst guarantee a fault. If a full restart des nt wrk, cntact Darren Paul r James Springfield. Objectives always inspect and clean il immersin bjectives bth befre and after a sessin. Always duble check the LCD tuchscreen, ZEN sftware r the bjective itself t cnfirm which immersin media shuld be used n each bjective; Dry r Oil. Flur 5 has bth Dry and Oil Objectives. Nte Well: Dry (these never have media applied): 5x, 10x, 20x. Oil: 40x, 63x, 100x D nt apply t much il t the bjectives; ne drp will suffice. Be sure t clean the il ff the bjective between samples. Apply fresh il fr each sample.

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4 Basic Navigatin. ZEN has fur basic interface screens, accessed via tabs tward the tp left f the display. Lcate is used when finding the bject f interest thrugh the eyepieces r via the camera system. Acquisitin prvides fr set up f imaging prvisins, and image capture. These are mst used during rutine imaging, and will be cvered in the user tutrial. The ther tw screens, Prcessing and Analysis, prvide varius methds f pst capture image mdificatin and characterizatin, respectively. Cntact micrscpy staff fr instructin in these functins. Lcate This is used t fcus n the sample thrugh the eyepieces. Ensure yu remve any remaining il frm the bjective r sample befre changing bjectives. Select the 10x bjective by clicking n the bjective icn (1) in the Micrscpe menu n the right hand side f the screen. Fllw the prmpts if asked t change the immersin il (2). Place yur slide n the stage. Nte: the edges f the slide must be slid under the silver retentin clips. Mve the eyepiece/camera slider n the right side f the micrscpe head int the eyepiece psitin. Click n the Lcate tab (3). Select the illuminatin methd r dye yu wish t image, by pressing ne f the favurite buttns (4) that mst clsely matches yur wn dyes emissin clur. Ensure that the brightfield lamp r flurescence lamp is turned n and the shutter is pen. Set the lamp t an apprpriate pwer level (abut 12 t 50%) using the knb n the micrscpe stand r n the lamp unit. Set the eyepieces fr yur cmfrt. Using the jystick, lcate a regin f interest in yur sample. Carefully bring yur sample int fcus. Establish Köhler illuminatin: Fr brightfield; Clse the Field Stp and bring the image f the Field Stp int fcus using the Cndenser Fcus Wheel (fund belw the stage but abve the Specimen Fcus Wheel). Centre the image f the Field Stp in the field f view using the sub-stage cndenser centering screws. Open the Field Stp until it is just beynd the field f view. Set the Aperture Stp t a value f three-quarters f the numerical aperture f the bjective (0.75 A N, fr the mathematically minded). Fr flurescence; There is n Field Stp in the flurescence light path n this micrscpe due t the Aptme mdule taking it s place, s n adjustment is necessary. Adjust the Aperture Stp until the image just becmes dimmer. Switch t whichever bjective yu wish t use, and refine yur Köhler settings. Mve the eyepiece/camera slider n the micrscpe t the camera psitin. Click the Live buttn (5), t see the live camera image n the cmputer display. Click the Find Fcus and Set Expsure (6) buttns t adjust the fcus and expsure f the image, r adjust the expsure manually using the Expsure Time slider (number?). Optinally, increase the image reslutin by changing the binning t 1x1 (7), then adjusting the expsure again.

5 If yu are cllecting single clur images, yu may nw simply click the Snap buttn t capture an image (8), r use the drpdwn tab t capture a quick tile f yur sample (9). Click Save (10).

6 Acquisitin This is used t cllect multiclur, z-stack r ther cmplex datasets. Multiclur Images Open the Acquisitin Tab (1). Select Widefield frm the drp-dwn list (2). Select the tickbxes fr the Channels yu wish t image (3). Mve the eyepiece/camera slider n the micrscpe ut t the camera psitin. Click the Live buttn, t see the live camera image n the display (4). Click the Find Fcus and Set Expsure buttns (5) t adjust the fcus and expsure fr all f the Channels f the image, r manually click n each Channel t highlight it, then adjust the expsure manually using the Expsure Time slider (needs a number). By default, the camera captures a 2x2 binned image (6). Yu may increase the image reslutin if desired by changing the binning t 1x1, then adjust the expsure again. Click Snap (7) t capture a multiclur image, r use the drpdwn tab t capture a quick multiclur tile f yur sample. Then click Save (needs a number). Z-stacks Perfrm the steps in utlined Multiclur images, abve. Click n the Z-stack tickbx (1). Select Full Z-Stack per Channel frm the drpdwn list (2). Un-tick all but ne f the Channel tickbxes (3). Nw setup the Z-stack range via ne f tw methds. First/Last gd fr quick scans and irregular samples: Using Live view, fcus up thrugh the sample until the tp is reached. Click Set First (4). Again using Live view, fcus dwn thrugh the sample until the bttm is reached. Click Set Last (5). Select the interval (distance) between z-sectins: the easiest ptin is t click Optimal (6). The sftware calculates the ptical slice thickness and then sets the interval between slices t be less than this; ensuring that yu wn t miss anything between slices. Center gd fr regular cell mnlayers and tissue samples, as well as fr minimizing the number f sectins imaged: Bring the sample int fcus. Press the Center buttn (7). Select interval (distance) between z-sectins; the easiest ptin is t click Optimal (8). Enter an arbitrary number f Slices (start with abut 20) (9) r, alternatively, set the Range. Select all f the Channels yu wish t image. Click the Start Experiment buttn (10) (belw the Live/Cntinuus/Snap buttns) t begin acquisitin. Save yur data.

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8 2D Aptme Carefully and gently slide the Aptme mdule int the side f the micrscpe bdy. The micrscpe will beep when it is in place. Select Aptme frm the drpdwn list (1). Select the tickbxes fr Channels yu wish t image. Mve the eyepiece/camera slider n the micrscpe ut t the camera psitin. Click the Live buttn, t see the live camera image n the display. Click the Find Fcus and Set Expsure buttns t adjust the fcus and expsure fr all f the Channels f the image, r manually click n each Channel t highlight it, then adjust the expsure manually using the Expsure Time slider. Increase the image reslutin if desired by changing the binning t 1x1, then adjust the expsure again. Click Snap. Click Create Image (2) t prcess the image file int a single, reslutin enhanced image. Save yur data. 3D Aptme Carefully slide in the Aptme mdule, select Aptme frm the drpdwn list, and select desired Channels, as described abve. Adjust expsure, reslutin (if desired), and setup a z-stack as previusly discussed. Click Start Experiment (needs a number). Click Create Image, as abve. Decnvlve the image (needs a number), if desired. Save yur data.

9 Advanced Tiling Click thetiles checkbx, situated belw the z-stack checkbx mentined previusly. A Tiles parameters panel will pen. In the Tile Regins sectin ( ), define the desired tile by number f images, size, r by marked pints. Use the Psitins sectin ( ) t define XYZ pints arund which yu can capture multiclur, z-stack and tiled datasets, if required Alternatively, use Advanced Setup ( ) t invke an interactive display which shws the live image captured in relatin t the sample carrier. Nte: it is pssible t capture a preview scan at lwer magnificatin, and then identify and mark regins t tile at the desired magnificatin.

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11 Other Optins AutSave ( ) enables a user t autmatically save the raw.czi files t a specified lcatin after capture. Users shuld always save t the D:\Data\ directry and create a flder in their name. AutExprt perfrms similarly t AutSave, but cnverts the raw.czi file int the desired image frmat such as TIFF.

12 Image Inf ( ), accessed frm the tabs n the left f the image windw, displays all f the settings used t capture the image, including pixel size and emissin wavelength.

13 Saving and Transferring Files and Nte well; unless yu have specifically set up autmated saving (as described abve) images are nt saved as yur sessin prceeds. Yu must save yur images manually, preferably, as yu g. There are three different places frm which t save: The buttns belw the image thumbnails in the Images and Dcuments panel. The diskette icn abve the menu tabs. Frm the applicatin menu system, under File > Save. Set up a lcal flder in My Cmputer / Data (Drive D:). Save all acquired data here, initially. D nt save t the Windws desktp, r t My Dcuments, etc. Never perfrm an initial save ver the netwrk: ZEN isn t built fr it and may crash. Unsaved images have an exclamatin mark in a yellw triangle appearing next t their thumbnails in the Images and Dcuments menu. When finished imaging, ensure yu transfer yur images t yur netwrk flder. Nte: data may be cleared frm the cmputer as frequently as nce a week. Nte: Re-using previus settings (as is pssible with ZEN and the cnfcal micrscpes), des nt wrk with this sftware and hardware cmbinatin. Set up yur image captures at the start f each sessin frm scratch. Shut Dwn Cleaning up the micrscpe Carefully clean the il immersin bjectives, if used, using the lens tissues prvided, in the manner demnstrated by the micrscpy staff. D nt use Kimwipes. If a dry (air interface) jective is il cntaminated d nt attempt t clean it; ntify micrscpy staff. Wipe up any spills yu may have made. Nte well; Yu, the last user, are respnsible fr the cnditin f the micrscpe and ancillary equipment. If the equipment was fund dirty, ntify micrscpy staff. Wipe up any shards f glass yu might have prduced: nbdy likes being jabbed by little slivers f glass in a bilgy lab. Make sure all f yur images are saved. Exit the ZEN sftware. Transfer yur files frm the lcal cmputer t the netwrk strage. Shut dwn the cmputer: Wait sixty secnds after quitting ZEN. G t the Windws Start menu and select Lg Off frm the Shut Dwn ptins; d nt use the shut dwn buttn in the sftware r n the keybard t shut the cmputer dwn.