Nikon Deconvolution Microscope Quickstart Guide For NIS v Guide V1.02, updated August 19, 2016.

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1 Nikn Decnvlutin Micrscpe Quickstart Guide Fr NIS v Guide V1.02, updated August 19, Startup Turn n the PC. Lg nt the PC, wait until Windws has finished lading (~60 sec). The chamber is set t 37 C, measured via a thermcuple within a small dish n the micrscpe stage. The thermcuple must be immersed in water. Add water as necessary. If yu require CO 2 Incubatin, Duble Click the CO 2 On icn n the desktp. Nte that it may take ne-half hur r mre fr the CO 2 level t stabilize. Duble Click the NIS icn n the desktp t turn n the micrscpe and lad the capture sftware. This will als cnfigure yur data file paths. Basic Navigatin Nte; there are three basic prgram screens (and mdes). Tabs fr these are presented in the lwer left crner f the mnitr display. The initial prgram screen is the ND Acquire screen. This is best used fr imaging single r a few selected pints in ne r a few slides r dishes. The Multiwell screen brings up JOBS mde, which can run cmplex experiment macrs, and is best used fr repetitive, multiple pint captures with multiwell plates, etc. The third screen is Measure, which prvides access t a range f measurement and image analysis ptin during and pst acquisitin. Basic Visualizatin and Setup (Cmmn t bth ND Acquire & JOBS mdes) Select the bjective yu wish t use frm the Macr Panel: the apprpriate DIC r phase rings will mve int place alng with the bjective (1). If yu are using an il immersin bjective, carefully clean the lens with a lens tissue. Place a drp f il n the bjective. Install the apprpriate sample hlder adaptr i.e.: 3 place slide, 4 place petrie dish, etc. and munt yur sample. Set the XY stage speed t Carse n the jystick by twisting the jystick knb and centre yur sample ver the bjective. Select Eyepiece Mde (2) (n the Macr Panel). Select the Optical Cnfiguratin yu wish t use frm the OC Panel (3), and ensure it is highlighted green. Chse either: Quad Band with Bleedthrugh quad-band dichric and quad-band emissin filter. Quad Band w/ Bleedthrugh quad-band dichric and individual band-pass emissin filters. Triple w/ Bleedthrugh triple-band dichric and individual band-pass emissin filters.

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3 Dual w/ Bleedthrugh dual-band dichric and individual band-pass emissin filters. Other single dichric and band-pass emissin filters. Clur Brightfield false clur imaging f brightfield images. Permits assembly f realistic full clur verlays. If yu wish t perfrm brightfield imaging, slide the plariser unit situated abve the micrscpe cndenser t the pen psitin. The brightfield lamp is turned n via the push buttn n the small black bx lcated t the left f the micrscpe. Adjust brightness there als, via the rtary knb. Bring the bjective up using the micrscpe carse fcus knb until the il tuches the cverslip (if yu are using immersin il). Fcus n yur sample using the eyepieces, r via the Fcus Light n the frnt f the micrscpe. Nte: the LCD panel n the micrscpe displays the Z psitin. Crrect fcus is typically between 4000 & 6500μm. Set the XY stage speed t Fine r Ex Fine n the jystick, and centre an bject f interest. Select Live Image (r Snap Image if yu want t minimise bleaching) frm the Macr Panel. Nte: Selecting the Stp buttn will terminate the live image update and sample illuminatin (4). Select the image size yu wuld like: ROI OFF (2048x2048), ROI 1024x1024 r ROI 512x512 (5). We recmmend yu adjust camera expsures manually via the drp dwn list n the Flash4.0 Settings Panel (6). Hwever, expsures can be set autmatically using the Aut-Expsure buttn. The size and psitin f the autmatic expsure calculatin area can be set by selecting the Prbe buttn n the right hand side f the image windw). Turn n the Pixel Saturatin Indicatr (7) and the aut Lk Up Table (8) in the tlbar abve yur image. If necessary, yu can adjust the LED brightness (default = 75%) by clicking n the OC Panel title bar and then selecting Spectra Pad (9). Hwever, if yu change the lamp LED brightness, yu will ntice when returning t the OC Panel that the currently selected OC bears a red exclamatin mark. This is a warning that a setting has been mdified. T prceed, save the change by clicking n the small arrw next t the red exclamatin mark. Select the Muse XY buttn (10) in the tlbar abve yur image, then click n yur image, hld dwn the left muse buttn and drag the muse t mve the mtrised XY stage. Rll the muse wheel t adjust the fcus (ttal travel = 200μm). Yu can centre an bject f interest by duble clicking the left muse buttn with the cursr n the bject f interest. Fcus n an bject f interest. Either: Adjust the fcus autmatically by pressing the AutFcus buttn (#) (yu can mdify the aut-fcus settings under the Devices > Aut Fcus Setup menu),

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5 OR Adjust the fcus manually by scrlling the muse wheel, the micrscpe r jystick fcus knb. Once the sample is in fcus, adjust the expsure settings again either manually r using the Aut-Expse buttn, ensuring that the captured image is nt ver-expsed. NB. Saturated pixels will NOT decnvlve, s it is much better t have a weak image than a bright ne. Repeat fr all ther desired Optical Cnfiguratins (flurphres, channels, etc.). Expsure times are aut-saved when they are mdified. Nte: at this stage it is pssible t perfrm a simple ne clur capture, if desired, by clicking Snap Image (11) n the Macr Panel. The methd f setting up a mre cmplex experiment n the ND Acquire and Multiwell (JOBS) screens diverges at this pint. Perfect Fcus (PFS) The Perfect Fcus System is a hardware based autfcus, that utilises an IR LED t fcus n the sample side f the cverslip. The user may then tell the system hw far away (ffset) the sample is frm the cverslip. PFS requires a refractive index change t detect the cverslip, such as a liquid sample munting media, and may nt wrk with fixed slides. T set up: Fcus n yur sample. Press the PFS buttn n the frnt f the micrscpe. If viewing a live image, the sample may well appear t defcus. Hwever, if the small recessed green FOCUS LED next t the PFS buttn lights up, PFS is wrking. Adjust the PFS fcus cntrller r muse wheel (nt the micrscpe) t refcus yur specimen (determine the ffset). When cnfiguring a multipint ND acquisitin, it is essential that PFS is turned n befre yu recrd each pint. If nt, PFS will nt functin during the experiment. If imaging mre than ne 35mm dish in an experiment, ensure that Keep PFS On During Stage Mve in the XY Overview tab is NOT ticked, r PFS may fail when the metal stage insert mves ver the bjective. Nte: if the micrscpe triple beeps, r yu dn t see the small FOCUS LED light up, try adjusting the micrscpe fcus up and dwn until yu get PFS t wrk. Yu can make the micrscpe remember the PFS fcus psitin fr each bjective by pressing the Memry Buttn nce PFS fcus lck is achieved, hwever this will be lst when the micrscpe is pwered ff.

6 ND Acquire screen, XYZ Overview Panel The XYZ Overview Panel (12) cntains a number f sub-panels, prviding verall sample visualizatin, and fr the cnfiguratin f sme capture parameters. The sub-panels are accessed via the named tabs (listed belw). ND Acquisitin Overview is the default tab (13). Within this, ne may navigate the sample and mark pints f interest. Pressing the Add Pint buttn (14) r the keybard spacebar will recrd the XYZ crdinates f the current viewing psitin. Many such pints f interest may be s defined. Once at least ne pint is recrded, pressing the Adjust t Pints buttn (15) will zm in the verview display zm t encmpass and display them. The muse wheel r magnifier buttns may be used t zm in manually. Duble clicking anywhere within the preview windw will mve the stage t that pint, r if Mve t Nearest pint buttn (16) is active the stage will mve t the clsest pint t yur click. Activating live Image will update the main image windw. Clicking Snap Image will als shw a preview image in the XYZ Overview windw. Right click t bring up a menu fr advanced features such as Scan Large Image, Clear Preview Image and Use pints fr Fcus Surface etc. Use pints fr Fcus Surface will use the Z-Height f each pint t map ut the fcal surface f yur sample, allwing the sftware t interplate and predict the fcal height f ther areas n yur slide. A Heat Map f the fcal surface is displayed by pressing the Shw Fcus Surface buttn (17). The fcus surface can be used when scanning t keep the sample in fcus ver a slide r multiwell plate by ticking Use Fcus Surface in the Advanced sectin f the XY tab in the ND Acquisitin cntrl (discussed belw). The Fcus Surface and PFS Surface Tabs The Fcal Surface and PFS Surface Tab prvide additinal visualizatin f a fcus surface derived frm marked pints and via the Perfect Fcus autmatic fcus system, respectively. Nte that the fcal surface can be mapped independently f the ND Overview pints list if desired. Duble clicking n the preview will t mve the stage t that pint. Fcus, and, then click Add Pint r press the space bar t recrd the Z heights. The Dcument Overview Tab This is used t visit XYZ pints in previusly acquired datasets. Further details f the capabilities f the Fcus Surface, PFS Fcus Surface and Dcuments Overview Tabs may be btained frm the micrscpy staff.

7 ND Acquire screen, ND Acquisitin Panel (18) Enter a name fr the experiment in the Experiment text bx (19), if desired. Nte, this is nt the image(s) filename but rather, a general descriptin f yur experiment. Checking the Save t File checkbx (20) will autsave the data as an.nd2 file. Autsaving may slw rapid capture sequences, but is safer. The Path textbx shuld be autmatically ppulated with D:\Data\yur user name\nd\. Enter a filename in the Filename textbx. Nte; a checkbx permits files t be saved as a.tif series (rather than.nd2), if desired (21). The sftware will nt display images saved as.tif files as they are captured. Select the rder in which capture functins will be executed via the Order f Experiment drp dwn bx (22). The last ptin displayed will be the fastest. The Timing buttn indicates the apprximate ttal experiment time (23). The Time, XY, Large Image, λ (lambda), and Z functin tabs may be checked t assign these functins t the capture (24). Clicking a functin tab allws cnfiguratin f the functin ptins. The Time tab (25) Click the checkbx in the Phase clumn (26) n the first line f the list display t add a time sequence. Set the Interval and Duratin r the number f Lps (cycles) t be aquired. Nte: yu must never have n delay set in the Interval clumn. Nte als: a number f Phases may be set with differing parameters, t run sequentially. Nte: Under the Advanced Menu (27), Keep Object in View t have the sftware attempt t keep a live cell in the field f view f the camera. The XY tab (28) Displayed is the list f pints defined n the XYZ Overview Panel, abve. Clicking n a pint in the list will mve the micrscpe stage t that pint. Pints may be added t the list by pressing the Add New buttn (29), r pressing the keybard space bar. Ticking Include Z (30) will set acquired images t be captured at their recrded fcus psitins. T update a pint shuld yu adjust the fcus r XY psitin, press the hrizntal arrws pinting t the value t be changed (31). XY psitin and Z psitin may be updated individually. Nte: Include Z must be ticked. Right clicking n the pints list allws update f the Z psitin fr all pints.

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9 Pint lists may be Saved and Laded (32), fr example, fr regular experiments using multiwell plates, etc. Clicking the Clse active Shutter during Stage Mvement check bx (33) will clse the shutter between pints. This will reduce phtbleaching, but again, it will als slw dwn image acquisitin. The Advanced Menu (34) cntains ptins t perfrm an Autfcus befre/after imaging, and t use the Fcus Surface derived in the XYZ Overview windw. T image multiple slides r dishes within ne experiment the system MUST be cnfigured t lwer the bjective between samples. This is essential t prevent damage t the bjective. Please cntact micrscpy staff fr assistance in cnfiguring the necessary macrs. Nte: T use Perfect Fcus (see later sectin) with pints, yu MUST ensure it is turned n befre the pints are recrded. The Large Image tab (35) This is equivalent t tiling n the Zeiss cnfcals. Set the size f yur image ( Scan Area ) (36) as a number f frames r as an area (mm 2 ). Select Stitching if required, and set the image verlap t apprximately 15% f the image size (37). Select All Channels if yu wish the sftware t stitch each channel individually, r a single channel if stitching may be perfrmed n a master reference channel (faster). Clicking the Clse active Shutter during Stage Mvement check bx (38) will clse the shutter between images. This will reduce phtbleaching, but will als slw dwn image acquisitin. The λ (Lambda) tab (39) Click the checkbx under Optical Cnf t activate a line (40), then use the textbx drpdwn t chse and add yur first Optical Cnfiguratin t the list (41). Nte that the expsure values applied t this cnfiguratin will be thse determined during basic visualizatin and setup, abve. Click the checkbx n the next line t add an additinal ptical cnfiguratin t the capture. The sftware will autmatically select the next OC in the tp t bttm rder displayed in the OC Panel. Repeat as necessary. The T Ps and Z Ps clumns and drpdwn bxes (42) (visible if Time and Z tabs are checked) allw fine tuning f channel capture with respect t these additinal parameters. Nte: if capturing a reference DIC image, set the Z Ps t Hme, rather than All. Nte als: yu have the ptin t clse the shutter between Optical Cnfiguratins t reduce phtbleaching (check Clse active Shutter during Filter Change ) (43), but this will very

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11 slightly slw dwn the acquisitin. Yu may als need t place delays befre and after the acquisitin if yu find yu are missing images, r the channel clurs are reversed, etc. These are cnfigured under the Advanced drpdwn (44). The Z-Stack tab (45) The buttns tward the tp left f this panel select the Z-series set up methd (46). Optins are; Tp Bttm (same as First-Last n the cnfcals). Symmetric (equal number f slices abve and belw a center psitin). Asymmetric (unequal number f slices abve and belw a center psitin). Set an apprpriate Step Size and Range (47). T use the faster piez z drive, select NIDAQ Piez Z, then press the Piez buttn (48) t centre n the current Z psitin and maximise travel range (maximum = 200μm, ie: +/- 100μm frm centre). If yu require a Z-stack larger than 200μm yu must use the Ti-Z Drive ptin. Nte: Relative (49) MUST be checked fr Multipint XY capture with Z stacks. This is nly available with the Symmetric and Asymmetric set up ptins. The sftware will ask t cnvert t a relative stack shuld ne attempt Multipint XY with an abslute, Tp Bttm defined stack. Nte als; if Tp-Bttm set up is selected, the exact start r end pint can be lcked in by right clicking n the relevant end buttn and selecting Keep Exact (Bttm r Tp) Psitin, as necessary. Once yu have setup yur experiment, press the Run Nw buttn (50) t acquire yur data. NB. If yur utput data file size is t large, g t Image/Cnvert/Change depth t cnvert it t 8 bit, then save it again.

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13 The Multiwell (JOBS) Screen Setting up a Multiwell Plate Select the 10x bjective, then wind it dwn s yu dn t hit it when munting yur plate. Multiwell plates insert directly int the micrscpe stage. Ensure that well A1 is psitined in the tp-left crner, the plate is sitting flat, and is pushed int the lwer right hand crner. Select the Add Well Plate buttn (1) at the tp left f the Well Plate and Slide Navigatin Panel, and chse the apprpriate well plate frm the pp up bx & list. Click OK. NB. Yu MUST NOT attempt t mve t, view, r image the uter rws and clumns f the plate as the bjective will hit the stage and cause damage. Prepare yur plate accrdingly. Duble click n well B2. D nt click n well A1. Select Eyepiece Mde (2) and either DIC r a relevant flurphre (3) frm the OC Panel, and fcus n yur sample. Nte: Selecting the Stp buttn (4) will terminate the live image update and sample illuminatin. Select Live Image (5) frm the Macr Panel. Activate the crsshair verlay n the live image via the Shw Graticule buttn, lcated n the right side f the image tlbar (6). Adjust yur expsure times manually r using Aut Expsure (n the Flash4.0 Settings Panel) (7). Select Re-Align Well Plate frm the HCA / JOBS Menu at the tp f the screen (8). The Re-Align Well Plate dialg will pen (9). Chse the plate yu are using frm the plate list, and click Next. Select well B2, and the methd f alignment (Centre, RHS r 3 pints) (10). Fllw the prmpts, using the jystick t mve t the relevant pints, and click Add (11) fr each pint. Click Finish (12). The Stage is nw cnfigured and aligned fr yur well plate. Setting up a Jb The Jbs Explrer Panel (13) at the bttm right f the mnitr is the hub f the Multiwell Screen. It is frm here that cnfigured jbs are usually run r rerun, by right clicking n the jb name and selecting Run (14), r by highlighting the jb and clicking n the green arrw in the jb list s left margin (15). In bth cases this pens the Jb Wizard (described belw), which must

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15 be clicked thrugh t run the jb. Here als, ne may return t the default view f results by clicking n the Results buttn (16), after explring ther ptins. Select yur desired bjective and fcus. G t the Jbs Explrer Panel, and ensure D:\Data\yur user name\jbs\ is set as the path (17). Create and name a new Prject, if desired (18). Jbs are defined by clicking n Add New Jbs frm Template r Add New Jb buttns (19 & 20) in the Jbs Explrer Panel s buttn bar. These bring up the Jb Template Panel and the mre detailed, Jb Definitin Panel, respectively. As an example, click n the Add New Jbs frm Template buttn. The Jb Template Panel (21) pens with basic ptins fr live and fixed plates. Selecting Live Cells and Imprt (fr example) then establishes a basic capture framewrk, and shws a Live jb in the Jbs Explrer jbs list (22). The Jbs Wizard ppup (23) is pened. The Jbs Wizard allws cnfiguratin f the experiment and capture parameters. The wizard interface is divided int tw clumns. The left clumn has basic parameter chices, and the right a series f subpanels with detailed ptins fr each f these parameters (24). Wrk dwn the left clumn, checking the ptins r filling in the infrmatin required n the right. Fr example: The Experiment Optins entry allws chice f capturing a single image r Z-stack, the use f autfcus via the built in Z-drive r Perfect Fcus, and autmated ntificatins. Selecting these ptins adds the relevent entry t the left clumn, with ptins then t be cnfigured n the right. Descriptin & Ntificatins prvides ptins fr an experiment descriptin, and the cnfiguratin f ntificatin parameters (if these were previusly activated). Acquisitin Parameters allws cnfiguratin f λ (Lambda) and (if applicable) Z-stack settings in similar manner t the equivalent ND Acquisitin Panel tabs. Sample Definitin Plate allws selectin f the well plate used and the specific wells t be imaged. Remember, the uter rws and clumns f the plate MUST NOT be specified. Sample Definitin Well allws set up f imaging within the wells, as user defined pints, randmised pints, r sampling patterns. Large image settings may als be cnfigured. If a shading (r flatfield) crrectin image has been defined, it may be applied here. Timelapse Definitin allws definitin f time series by number f lps (cycles), r interval between captures (as described in the ND Acquisitin Time tab). Nte: it is best t set a small delay between cycles, via the Wait Between ptin under Speed f Acquisitin (25). Data Analysis presents Cell Cunt Analysis, General Analysis, and Intensity Analysis ptins. Cnfigure by selecting an analysis type, clicking the Define buttn (26) and chsing desired ptins n the pp up settings panel. Analyses may be cnfigured fr each OC. Settings may be saved fr future reuse via the Save Recipe buttn (27). Upn reaching the end f the Jbs Wizard, the ptins t Test Run and Run the jb will becme available (28). Test Run executes a single cycle f the jb; useful fr verificatin. A cmpleted jb may be rerun frm the Jbs Explrer Panel, as described abve.

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17 Editing Jbs. Jbs may be edited pst deplyment. Right clicking n the jb in the Jbs Explrer list and selecting Edit This Jb will pen the Jb Definitin Panel (29). This allws cmprehensive cnfiguratin f the jb cntent, rder and timing. Fr example: The imaging surface f prer quality (ie cheap) multiwell plates may vary in height r thickness by mre than 50 micrmeters. Fr such plates it may be necessary t turn ff the PFS when mving between wells, and then turn it back n t image. T cnfigure, drag the PFS On and Fcus task (30) frm the left clumn t just abve the Run Macr1 task (31) in the jb, and drag the PFS Off task (32) t just after Run Macr 2 (33). Depending n the experiment, it may be fund that images fail t acquire crrectly unless a small delay is added befre and after image capture, and at the end f the timelapse lp. This can be set by dragging the Macr task (34) frm the left clumn, int the JOB, just befre the Capture task (35). Duble click n the Macr task t expand it, and enter either Wait (0.25), r press the Fx buttn (36) and search fr the Wait cmmand, edit the delay and press kay. Cnsult the NIS Help Menu and cntact Micrscpy Supprt Staff fr assistance with editing jbs. Sme Advanced Features Many f the advanced features f NIS are available bth under the Applicatins menu (1), and via icns displayed abve the image windw. See, fr example: Averaging Pushes image nise int the backgrund by averaging pixel intensity ver a number f images. EDF Extended Depth f Field creates a single, fcussed image frm a previusly acquired z-stack. The Z stack may be aligned first, if necessary, via the Align Sequence ptin f the EDF buttn drpdwn bx, r the Applicatins menu. The cmpleted EDF may be viewed and exprted in 3D. Realtime EDF Autmatically acquire, align, HDR and prcess a z-stack t prduce an EDF image.

18 HDR Acquire a High Dynamic Range image frm a stack f different camera expsure images. This can be useful fr samples that cntain bth very bright and very dim structures. FRET Use the FRET applicatin t acquire and analyse a FRET sample. MultiWell Plate Imaging (as perfrmed under ND Acquire). It is pssible t image multi-well plates under the ND Acquire Screen. Hwever, this is best suited t a relatively simple (ie a few wells r pints), ne ff experiment. Fr mre cmplex r repeat experiments set up under the Multiwell (JOBS) Screen. T set up under ND Aquire Screen: Click the dwn arrw at the right end f the XYZ Overview Panel s header bar (2), and select Well Plate and Slide Navigatin. This will replace the panel. Select the Add Well Plate buttn (3) at the tp left, and chse the apprpriate well plate frm the pp up bx. Click OK. A map f the chsen well plate and f an individual well will be displayed n the Well Plate and Slide Navigatin Panel (4). NB. Yu MUST NOT attempt t mve t, view, r image the uter rws and clumns f the plate as the bjective will hit the stage and cause damage. Prepare yur plate accrdingly. Clicking n a well in the plate map will mve the stage t the centre f that well. If desired, clicking n psitins within the enlarged map f the single well will mve t thse psitins. Fcus n the sample nce mre and set up the Perfect Fcus System if required, as described previusly. Pints (ie wells r intrawell psitins) may be marked via the XY tab f the ND Acquisitin Panel, by activating the next available check bx in the Pint Name clumn (5). This will recrd the current X, Y & Z psitins as well as the PFS ffset. Check the ptin Include Z (6). Click Keep PFS On During Stage Mve (7), n the Well Plate and Slide Navigatin Panel. Set up the rest f the experiment as usual. Z intensity Crrectin This can assist in the prductin f a unifrmly bright z-stack. Set up is as fllws:

19 Right click n the ND Acquisitin Panel header and select the Acquisitin Cntrls submenu, then select Z Intensity Crrectin (8). Nte that in additin t switching t the Z intensity crrectin cnfiguratin panel, this puts the link t the panel int the header bar s main menu. Clicking n the header thence prvides easy switching between the ND Acquisitin and Z Intensity Crrectin Panels. Under ND Multipint, select Relative r Abslute crrectin as apprpriate (9). Relative ffsets the expsure scaling t cmpensate fr ptential differences in z stack starting psitin acrss the sample, whereas Abslute applies the cmpensatin nly t thse heights at which it was defined. The pint list in this panel displays Z heights within a stack. T ppulate the list either: Fcus and click the Add Pint buttn (10) t add a Z value t the list. Adjust the expsure (Aut Expsure is fastest) and click the hrizntal arrw pinting t the expsure t update the stred value. Repeat t cver the height range f the Z stack. Alternately, with a Z stack already defined under the Z-Stack tab f the ND Acquisitin Panel, click Frm ND (11) t ppulate the list with the stack s tp, centre and bttm psitins. Check Mve Z t Selected Pint (12) and click n each pint t mve t each pint. Optimise and update expsure values as abve. Repeat fr each channel. The Run Z Crr (13) lcated at the bttm f the ND Acquisitin Panel immediately executes the experiment with the cnfigured crrectin values. D nt click the Run Nw buttn, as this will nt crrectly apply the crrectin t multiple channels. Nte: If the crrectins have nt been established, pressing Run Z Crr will generate an errr. The ptin T ND (14) pushes Z pints defined here t the ND Acquisitin Z-Stacks Tab, setting the stack tp and bttm psitins. The Use n Live ptin (15) applies the defined crrectin t live imaging. Shading (Flatfield) Crrectin Shading crrectin applies a preacquired image f the pattern f illuminatin thrugh the cmpnents f a given ptical cnfiguratin t images acquired with that cnfiguratin, in rder t crrect illuminatin artifacts. T set up: Click the tab at the bttm f the image screen t bring up the Opened Images Panel (16), then click n the header bar and select Shading Crrectins. Chse whether yu wuld like t apply a single crrectin t all f yur OC s, r apply individual crrectins t each OC (best).

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21 Clicking the Capture Crrectin Image buttn will begin acquisitin f yur crrectin image (17). Chse Subtractive r Multiplicative as the Crrectin ptin (18). Subtractive is apprpriate fr crrectin images taken n a clear area f the sample plate r slide, while multiplicative is used fr images derived frm a unifrmly flurescent crrectin slide. Ensure the Spline Interplatin, Smthness and Keep Mean Image Intensity ptins (19) are turned ff. Fllw the prmpts. Click the Shading Crrectin buttn (20) t apply t live and subsequently captured images. T apply t a pre-existing image pen the image and click the Apply Shading Crrectin t Current Image buttn (21). Nte: Right clicking n the shading crrectin image thumbnail als allws applicatin, viewing and deletin f the shading image. Nte als; clicking n the Opened Images Panel header bar als prvides ptins fr viewing an Intensity Prfile, and specifying the Aut Capture Flder. Shutdwn Prcedure Clean the il immersin bjectives, if used. Use the lens tissue prvided, nly, in the manner demnstrated by the micrscpy staff. If a dry (air interface) bjective is il cntaminated d nt attempt t clean it; ntify micrscpy staff. Clean up any il, sample r medium dripped n the micrscpe, within the chamber, r n the micrscpe and cmputer tables. Kimwipes are prvided fr that purpse. NB. Yu, the last user, are respnsible fr the cnditin f the micrscpe and ancillary equipment. If the equipment was fund dirty, ntify micrscpy staff. If sample r sample medium has been spilled ver any prtin f the micrscpe please wipe it up, and then ntify micrscpy staff. Clse the NIS sftware and wait at least 60 secnds befre lgging ut r shutting dwn Windws. Cpy the files yu created t yur netwrk drive. Lg ut r shutdwn Windws. This will shutdwn the micrscpe and ther hardware. Remve yur samples, lab cat, and any ther equipment yu have brught with yu.

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23 Knwn Prblems. Extraneus light artefacts. With dim samples, in particular, rm lights and illuminatin light reflected frm the brightfield lamp husing may swamp the sample image. It is best t switch ff the rm lights, including the wall munted LED lamp. Reflectin frm the brightfield lamp can be minimized by switching in the blue/green filter, via the small lever at the rear f the lamp huse. Ask micrscpy staff if in dubt. Sftware instability. The Nikn capture sftware can be unstable, prducing lck ups and crashes. Frtunately, these are nw cmparatively rare. Shuld a lck up r crash ccur yu must perfrm a full system restart (and NOT just a sftware restart). If the prblem prevents Windws frm respnding als, hld dwn the cmputer pwer buttn fr 10 secnds t frce a system shutdwn. Wait 30 secnds, then restart the cmputer. Darren J. Paul, James Springfield, and the University f Queensland. May 2016.