"user manual"
Contents
1. Hege 86698 Endy 86007 Acquisition and analysis layout Make your own favourite layout of the right PC screen Open useful windows in view gt acquisition controls and analysis controls Save your layout in View gt Layout gt Save current layout If some of your windows are gone next time retrieve by going to View gt Layout gt Reload current layout iDu oo Oo we BB 2 r Lee Ld arent ft Grightfield Brightfeld Shared TBITC TRITC Shared Post I GORI ORG cut Save Current Layout As LI Aen Current Layo Layout Manager oe amp Q K G 4 a 5 G 5 E baal n e t a az a S raph Owa AOT 120 97 mm x 517 24 mm Scan Area 0 30 Useful floating windows Camera acquisition control ND acquisition TE2000 Pad e LUIS e XYZ navigation Hege 86698 Endy 86007 Shutter control Capture Timelapse Experiment Setup T Save to File Path C Documents and Settings MICadmini Desktopi Browse Filename G113R047 nd2 Record Data Order of Experiment E LI O Close Active Shutter when Idle Perform Time Measurement 0 ROIs Q Use Ratio Define Ratio C Switch Transmitted Lamp off when Idle 1 00 s Events Autofocus None Y Execute before Capture Execute after Capture t th2. set in the polariser A which is situated above the consensor set in the analyser B which is situated below the filter revolver on the right hand side Make sure the filters are crossed background is black image your sample D move both filters out again after imaging since each will take 50 96 of the light Hege 86698 Endy 86007 M NE Differential interference contrast In order to adjust for DIC focus and Kohler your object and lightpath to get a nice brightfield image A insert the polariser above the condensor and the analyser below the filter revolver insert the DIC prisms matching the objective s inscription in the condensor revolver and below the objective B to get the typical DIC image C Control the shadow by turning the polariser e For colour DIC D insert the plate below the polariser E Hege 86698 Endy 86007 NC Stage control Ifyou want to use the microscope without acquiring images set the stage control to MANUAL A Ifyou want to acquire images set the stage control to AUTO Ifthe stage does not react after starting the software go to Devices Manage devices B e Click on LSTEP ECO E HER STEP B and on p Connect C TG f you need to invert the XY axis on the joystick open connection parameters D and reverse X and Y E
3. User manual e MIC Molecular Imaging Center Table of content Switching ON and OFF the system The TE2000 pad Starting the software and camera selection Camera selection Available objectives Correction collar adjustment Manual multichannel acquisition Large image acquisition Timelapse acquisition Multipoint acquisition Acquisition of a 3D image Automated 5D multipoint acquisition Brightfield filters Kohler illumination Increasing contrast Adjusting phase contrast Hoffmann modulation contrast Polarization microscopy Differential interference contrast DIC Acquisition and analysis layout Stage control Shutter control Autofocus Scale bar Saving data File formats Available fluorescence filters Hege 86698 Endy 86007 Page 3 Page 4 Page 5 Page 6 Page Page 8 9 Page 10 11 Page 12 Page 13 Page 14 Page 15 Page 16 Page 17 Page 18 Page 19 Page 20 Page 21 Page 22 Page 23 Page 24 Page 25 Page 26 Page 27 Page 28 Page 29 Page 30 Do you want to take images of your sample I will analyse yes some data No just observing Pe No need to start up the pc and software Just make sure you are in manual mode on the stage control box page 24 Start up the computer Start up pc and software Log onto the server Focus your sample in the microscope Will you need the fluorescent and choose no grabber lamp Yes I have Choose DS U2
4. For the adjustment of phase contrast i bring your object in focus and ii Kohler iii Switch the condensor B to the position matching the objective s inscription In case of correct alignment C1 you will see phase contrast with homogeneous halo C2 iv Check the alignment by switching the eyepiece optics to B Bertrand lens arrow The two rings you see A1 have to overlap If not you will have strong artifacts A2 In order to adjust v move the condensor ring with an allen key D Hege 86698 Endy 86007 M i Hoffmann modulation contrast Hoffman modulation contrast creates DIC like images with less effort giving the objects a 3D appearance B In order to adjust for Hoffmann modulation contrast focus and Kohler your object and lightpath A e at the condensor switch to the filter corresponding to the objective MC1 3 e at the eyepiece switch from O to B e use either the knurled screw at the objective or at the condensor filter to make the two grey shaded objects parallel C and D Use an allen key to adjust the distance between the two grey shaded objects at the condensor filter They should overlap slightly Hege 86698 Endy 86007 MI Polarisation microscopy Polarisation microscopy is particularly useful for imaging linear structures like crystals and can also resemble dark field microscopy To adjust focus and Kohler your object and lightpath C
5. acument LM ImageProcessing M IntensityPrafile 9 Interpreted Stg IsMouseJovstickzi Stg IsNDFilterPresenti Stg IsMaosepiecePresent Stg IsPFSPresent Stg IsShutkerPresenti Stg_IsSlideLoaded Sbg_IsSlideLoaderInitialized Stg IsSlidePresentt Stg IszaomManuali Stg IszaomPresenti Stg LargeImageScan Stg LargeImageScanAreat Stg LargeImageStageScanwitkhMamer Stg_LoadslideFrom Stg OpenFirstShutter Stq OpenMicrascopeDaart M Objectives LJ Obsolete Number of Functions 2067 Reset All Interpreted Update All Interpreted Hege 86698 Endy 86007 Autofocus Especially in timelapse experiments the probe might move out of focus over time To avoid this NIS elements provides an autofocus function Click Advanced A in order to access the autofocus settings Choose either Steps in Range or Adaptive B Choose when the autofocus should be applied C e Click Define D and adjust the settings in Steps in Range you define the z stack size E in which you expect the focal region and the precision of the scanning F a eee Record Data Filename test005 nd2 Two passes Step aml 2 8004m Range s imi Crteron erortea 7 Offset Zafter aF 0 um Close Active Shutter when Idle L1 Perform Time Measurement 0 ROT j Moves 4 um down and then moves in 1 ui
6. 30 eCFP Lucifer Yellow PO PRO Alexa Fluor 430 488 BCECE Calcein CFDA Di 8 eGFP FDA FITC Fluo 4 Fluorescein FM 1 43 FM 4 64 Fura Red LysoSensor Green MitoTracker Green Rhodamine 110 Green YO PRO Alexa Fluor 488 BCECF Cy2 DIO eGFP FDA FITC Fluo 4 Fluorescein FM 1 43 MitoTracker Green Nissl Oregon Green 488 Rhodamine 110 Green YO PRO YOYO Alexa Fluor 488 BCECF BODIPY Calcein CFDA Calcium Green eGFP eYPE FIAsH FITC FM 1 43 Magnesium Green MitoTracker Green Oregon Green 488 514 Rhodamine 110 123 Green TO PRO TOTO YO PRO YOYO Alexa Fluor 555 610 BODIPY TMR X Calcium Orange d Tomato Ethidium Bromide evoglow Bs1 Bs2 Pp1 Magnesium Orange Nile Red PO PRO 3 POPO 3 Propidium Iodide Rhodamine TAMRA Tetramethylrhodamine TRITC Alexa Fluor 647 660 Atto 647 mPlum NileBlue Red Cy5 TO PRO 3 TOTO 3 Hege 86698 Endy 86007
7. Day Week Month 08 00 09 00 10 00 11 00 12 00 15 00 14 00 15 00 16 00 17 00 18 00 19 00 Reserved Simon Dankel Nikon TE 20DO 1 Check online in the booking system whether there is a user coming after you A If yes phone her him and ask whether he she needs the mercury lamp If nobody uses the mercury lamp for the following 30 minutes switch it off at the power button B 2 Close the software and shut down the PC 3 Fill out the logbook C Turn off the main switch D oO gt i gt loc m I Di EMO zu CY m LLI 1 E Hege 86698 Endy 86007 The TE2000 pad Objective switch gU Halogen light control The main switch on the power supply needs to be on A Regulate the intensity here B Halogen shutter C Shutter for the UV More objectives available dy demand 2x 40x HMC 60x light The power supply needs to be on D and lamp ignited E m TLLA n OBYE TIVE Fluorescence filter positions No Light path control Eyepiece E 6 is empty and used for imaging B W camera for fluorescence and BE Ph Pol HMC and DIC DIC F and colour camera G Hege 86698 Endy 86007 Starting the software 1 Double click the NIS AR oT button on the Desktop A AR 413 04 2 Select the camera control unit you want to use B me Nikon DS U1 For the colour camera choose Nikon DS
8. S Sh T o SATIS om 5 ER B s E E E v ti a LP el xt H GFP a m NAM ZR DABL GER MIRITC Custom j 47umjpx Sxizbit 2368 x 1895 pixels 1526 1316 MCh 343 1138 500 LUTs x mansa o9597 e Tn the Split Channel view A you o G 095 e Hold Shift key to control all channels can see during acquisition how t different channels built up You can zoom in using the middle mouse button 1 After acquisition use the LUTS to adjust the contrast for each channel individually You can also try the automatic adjustment B Hege 86698 Endy 86007 M i Large image acquisition I l Initiate stage Devices 2 saplcations Initiate stage CEU ETT A crian 2 Open the ND acquisition Ca ubican ages cit win dow A Device for Stimulation d Applications gt mmc Define Run ND ee Create HDR Image Application 3 Define the channels you want to image B 4 The image will be saved automatically Be sure to define where the image should be saved and give it a filename C 5 You need to define an editional command Hm open the UV shutter I Close active Shutter during Filter Change before the first channel Use Ratio Defne Ratio D or else your first image will be dark Find the command E Stg OpenFirstShutter ror pm Hege 86698 Endy 86007 Large image acquisition Il 5 Tick the large s
9. UI For the B W camera choose Nikon DS U2 L2 USB oe 3 Logon to the Biomiconthe Skule server to be able to Jeena 2 edodt A skule klient uib no Y save images directly onto the server Double click the biomic on m skule icon on the desktop 0 pP Enter your UiB username in i B 2 the following way username POR uibNusername and then the password D Ifyou do not have access to skule yet contact Torstein Ravnskog 7 5 86323 room 6C133cA Hege 86698 Endy 86007 Camera selection Nikon 05 2 L2 USB Nikon Ma Grabber TTET aail Le Colour BW camera camera Turn on the main switch DS Qil A colour camera B BW camera before use WS ae histological sections fluorescence acquisition BF phase contrast images DIC HMC images classical stainings Golgi timelapse imaging Nissl etc Pixels 2560 x 1920 pixels binning 2x2 4x4 Pixels 1280 x 1024 pixels binning 2x2 4x4 Cooling Peltier cooling 20 below ambient Cooling Peltier cooling 10 below ambient A D conversion 8 bit A D conversion 12 bit Exposure time 1 1000 600s Exposure time 1 1000 600 s Sensitivity Equivalent to ISO 64 Sensitivity Equivalent to ISO 800 Hege 86698 Endy 86007 Correction collar adjustment Some objectives have a correction collar A to adjust for a cover slip
10. and turn fluorescing on the fluorescent lamp samples Choose DS U1 and check the Kohler page 17 Do you need DIC or HMC If you need brightfield as well check the K hler page 17 Regular multichannel imaging Go yes to page 8 9 Timelapse Large scan Multipoint imagaing Go to page Go to page Go to page 13 12 10 11 Warm up stage Initiate stage page 24 30 min before Start the CO2 Setting up DIC page 22 mixer Setting up HMC page 20 Saving data from DS U2 This camera takes 12 bit Saving data from DS U1 images For quantification analysis keep the 12 bit Choose any format as this For localization observation save as tiff or jpeg and is a 8 bit camera change bit size to 8 Hege 86698 Endy 86007 Switch on the system Turn on the main switch A Start up the computer B Log in with your user account at the PC Ifyou do not have a user account yet ask MIC personnel to get one 4 Ifyou need the mercury lamp check online in the booking system F and in the logbook G whether it has been used in the last 30 min is it warm turn on the power switch C and press ignition D note the run time E in the logbook G 5 Start NIS AR H the NIS Elements software FRIDAY MARCH 04 2011 08 00 09 00 10 00 11 00 12 00 MIS Elements AR 4 15 04 Hege 86698 Endy 86007 Switch off the system FRIDAY MARCH 04 2011
11. can window and define the amount of images to be scanned A the first number indicating the x direction 6 Move to the centre of your object and adjust for focus and exposure time B 7 Ifyou want autofocus you need to choose define by steps C either in lambda or timelapse mode 8 Define the stepsize D and total z range E Format 12801024 no binning 3 2107s J p mm lt Range 8 Jimi creron erotea E OffsetZafterAF 0 um v Save to File Path C TEMPI Record Data Filename large scan nd2 O 60 f ABO Stitching Do Not Stitch Overlap 15 TT esting Stitching is done on PERI dr ne when the large image is acquired inside lambda loop _ Close active Shutter during Stage Movement Cams v so fens m Hege 86698 Endy 86007 Timelapse acquisition Applications 6D 1 Open the ND acquisition TB cefreunno Acqusiton AN window Applications gt M eee Device for Stimulation d Define Run ND Application 2 Define which channels you want Capture Reflection Free Image cc Capture HDR Image to image under lambda flag DOMNUM A 3 Checkthe time flag Define the interval B at which the images should be taken Define the duration of the experiment m C A 4 You can define different loops of zu timelapse experiments If y
12. e C BITPLANE SCIENTIFIC SOLUTIONS ImagePro In Photoshop you will have to adjust brightness contrast and it cannot handle two channel TIFF without problems Hege 86698 Endy 86007 Available objectives Position M Correction we Contrast mm method Plan Fluor 0 13 16 4 2 10 Plan Fluor 0 30 15 2 Phl 3 20 ELWD Plan Fluor 0 45 7 4 Phl 4 40 ELWD Plan Fluor 0 60 3 7 2 7 Ph2 5 10 HMC 0 25 6 2 MCI 6 20 HMC LWD 0 40 3 1 MC2 u f 2 Plan Apo 0 1 8 5 not recommended u r 40 HMC LWD 0 55 2 7 1 7 MC3 u f 60 Plan Apo VC 1 4 0 13 DIC N2 Abbreviations M magnification NA numerical aperture WD working distance Ph phase contrast ELWD enhanced long working distance HMC Hoffmann modulation contrast u r upon request VC violet corrected optimised for multi colour staining that includes DAPI and the visible spectrum DIC differential interference contrast All objectives work in brightfield and with polarisation microscopy All objectives except the ones dedicated for HMC can be used for fluorescence microscopy Hege 86698 Endy 86007 Available fluorescence filters DAPI CFP GFP FITC YFP TRITC Cy5 340 380 426 446 450 490 465 495 490 510 528 552 590 650 435 485 460 600 LP500 515 255 LP520 577 632 662 738 Alexa Fluor 350 BFP Coumarin DAPI Indo 1 Hoechst Marina Blue Acridine Orange Alexa Fluor 4
13. e Beginning Stg OpenFirstShutter Stg CloseFirstShutter In order to avoid bleaching the shutter has to be closed when no image is being acquired To do so automatically in multidimensional experiments untick Close Active Shutter when Idle A open Advanced gt gt B Select Execute before Capture C and Execute after Capture D Click on the arrow to the right E and select Command List F From the command list G select Advanced for fai Y B Zaa oaio saci Prise E 34202 after zach priae Load x Save x Remove w 1 time loop Run now Select Command Stg OpenFirstShutter H for Execute before Capture and Stg CloseFirstShutter for Execute after Capture F Stg_OpenFirstShutter Stg_HeatStagelsPresentt OK Cancel Help Locate Stg HeatStageSetTemperaturet Stg HeatStageSetTemperakureRater HD AdvancedAPl M Applications ne Binary a Calibration i Colocalization H Device H B Edit Sbg_HeatStageStartHeating Cooling Sbg_HeatStageStopHeatingCooling Stg_InitializeSlideLoader Stg_Is4nalyzerPresentt Stg_Is4perturePresentt Stg IsBarCodescanned Stg IsCassettePresent Stg IsCassetteScanned Stg IsCondenserPresenti Stg IsDACPresent Stg_IsFilterPresentt Stq IsLightPathPresent Stg IsLightPresenti Stg IsMicrascopePresent E d ImageD
14. l lambda large image If you need multipoint large eM EE e oe image you have to initialise the Capture HOR Image stage first Then tick all the esie modules you need A and define as described on the previous pages Do not forget to define the order of the experiment B A progress window C will inform you about the progress Use advanced D to define autofocus and usefull commands for shutter control Define Run ND Sequence Acquisition Device for Stimulation Ld HDR i FEE O Experiment overall progress v Save to File z Path ke RR Time elapsed 0 00 06 Time remaining 0 00 23 Experiment Status RA A osse Senes 303 120 350 te 20 procesan a Duration Loop v 41 KP 2hour s ES A Remaining disk space 874GB Close Active Shutter when Idle Perform Time Measuremenfl QROIs Switch Transmitted Illuminator off when Idle 1 00 s H Refocus 4 Finish X Abort Advanced Settings are selected Advanced gt gt oe e Eae Hege 86698 Endy 86007 Scale bar With NIS Elements you can add a scale bar to your images Ask MIC personnel to calibrate the objective if required is most cases it is calibrated as you find it 23 646 sec w DO Brij wane ape fy FI Orientation Type mp mmm op meme s Udemm Automatically adjust size Keep in
15. m Steps up within 8 um _ Switch Transmitted Tlluminator off when Idle oe Hege 86698 Endy 86007 Acquisition of a 3D image In order to acquire a z stack you need to define its thickness and the distance of each slide Goto Applications Define Run ND Acquisition and activate Z Series e Focus your sample and define a top A and bottom B Define the step size C The number of steps D will change accordingly It also works vice versa Furthermore you can take over the suggestion of the software for Nyquist sampling E In addition you can define the size of the image stack by moving to the centre of the stack and setting the size of the half stacks F Note that you cannot autofocus if you capture a z stack The axial shifting of the EN objective is very fast the inbuilt Fr orn w shutter is much slower so that ie ie gt there is no need for closing during axial movement G Il Save to File I Path Ci TEMP Browse Record Data Filename tE 7 close active Shutter during Z Movement Advanced gt gt Hege 86698 Endy 86007 Automated 5D multipoint acquisition Using the ND acquisition Applications gt Define Run ND Acquisition you can set up experiments that include e timelapse Applications e Multipoint XY L EM Z series Nb Define Run ND Acquisition Ctrl FAlt N e Multichanne
16. ms Pe Format i un EZ C1 ICS IDS ics Windows Bitmaps rs bmp Portable Network Graphics png OMDUS eve apr Pier Pichi it a Image Fields X Image Attributes Filename NDProgress_ConvyallariaAll nd2 Path Z Dimension 3456 x 2765 4x12bit File Size N A Memory Size 72 9 MB Filedate 29 03 2011 Image Fields Calibration um px 0 92 gt Optics Plan Fluor 10x Phi DL Type SampleID gt Author Description metadata Dimensions A 4 Camera Name DS Qi1Mc U2 12 bit Numerical Aperture 0 3 Refractive Index 1 Number of Picture Planes 4 ImageID licnc s i gt Group gt H Capturing DS Qi1Mc U2 12 bit Format 1280x1024 no binning Moise Reduction Off Offset 0 00 Contrast Linear Sampling FEE 2 Location EE gt Date 29 03 2011 14 11 03 ss Ss 2 Conclusion D Infol H Info2 E E Clear All Fill by Defaults Edit Defaults OK Cancel Hege 86698 Endy 86007 File formats Data generated on this microscope is 12 bit which is more than the Windows Photo Viewer can handle properly Apart from NIS elements use the following programmes for image visualisation and processing nigh lerreetes eJ e Tr OF Fiji Fie Edit Image Process Analyze Plugins Window Help moeone osea Oval elliptical or brush selections right click to switch FIJI Image iji MATLAB maris S
17. ou want to acquire faster imaging for an hour before doing a slower imaging then define a J c second phase D O mma 5 For long term experiments use 82 2 c autofocus under advanced E Hege 86698 Endy 86007 Multipoint acquisition Initialize the stage Be careful to not use the joystick from now on Open the ND acquisition window Applications gt proa c um 998564 7 Range 0 00 2000 00 gt mm Define Run ND Application emer um 1239 15 Range 5 00 9500 00 gt Move Use the XYZ Navigation Ee a window to move around your sample A You can go between coarse and fine B Define your multipoint positions in the XY window with a tick into an empty square C Define where the data is going to be saved D Tr TEEN If you are doing a long gs BELZ rl wird Les I poner eit timelapse use autofocus Define which channels E you want to image and the interval of your timelapse F Lr R Hege 86698 Endy 86007 Brightfield filters Neutral density Diffuser disc NCB neutral GIF green filter Use this Keep this always colour interference filter when neither in the light path balance Use Use this filter for the voltage It decreases the this filter ifthe phase contrast control of the amount of white balance microscopy This halogen lamp uneven is not filter removes nor the illumina
18. thickness between 0 and 2 mm B Me i In order to adjust Ph2 ADL 0 2 WD 3 7 2 ies e look through the eyepiece at all times turn the correction collar slightly might be somewhat stuck at the beginning refocus C turn the correction collar again slightly refocus again repeat until you get a sharp crisp image D Correction collar not adjusted Correction collar adjusted Hege 86698 Endy 86007 MI I Manual multichannel acquisition 1 Click Acquire gt Capture Multichannel Image gt Multichannel Setup 2 Click into an empty line under Optical conf A and select the channel you want to use If the channels a a eae are already there just VV tick the once you need 3 For automated saving tick Save to File B set the File Path C to an appropriate folder on the klient server and give the data to be recorded a sensible name D If your samples are bleaching a lot it is adviced to select the longest wavelength first CY5 or TRITC then green and blue last Hege 86698 Endy 86007 Manual multichannel acquisition Il ES NIS Elements AR timelapse test 02 ndz te q 4 x File Edit Acquire Calibration Image ROI Binary Measure Reference Maco View Devi indow i E E B bOO B OB B S Or Qon u Loor EY APRA SS uz c a GF EE customize v S ai WAKER EH um ae Q O As Q z n a a is TA E B ge E
19. tion satisfactory at unwanted halo exposure time caused by an low halogen effects since phase of the camera extended light voltage contrast is suffice source tungsten calculated for green wire light Hege 86698 Endy 86007 Kohler illumination Kohler illumination is imperative for all transmission microscopy techniques brightfield phase contrast HMC DIC polarisation Adjust also when changing the objective or replacing the sample 1 Close the field stop diaphragm A so that you can see the edges 2 Move the condensor up and down B until you see a sharp image of the field stop diaphragm with sharp edges 3 Centre the image of the field stop diaphragm by using the knurled condensor centring screws C 4 Open the field stop diaphragm A until the edges lie just beyond the field of view Hege 86698 Endy 86007 Increasing contrast After setting up the rA mE Kohler illumination PL Um close the aperture stop KE rr arrow The contrast depth of field will greatly increase As you close the aperture stop the resolution will decrease Diatomee in brightfield with different contrast settings A aperture open B aperture halfway closed C aperture fully closed Note that in A only a small part of the Diatomee is in focus whereas in C you see all in focus but also dirt in the image pathway Hege 86698 Endy 86007 Adjusting phase contrast
20. view Show Text Tick on the scale bar sign A on the right side of your recorded image the scale bar will appear To change the default go to scale properties B Burn scale onto image right click on scalebar Hege 86698 Endy 86007 Saving data All data should be saved on biomic and in the following two formats e nd2 is the manufacturers format that contains all the META data This is your raw data that you have to keep to be able to show what you have done e TIFF is the scientific format which can be used by almost all software but is does not contain the full META data You can adjust parts of the META data in the Image Fields dialogue The hugely popular JPEG JPG is lossy produces artefacts and is not recommended in science Use it only for simple presentation purposes Save in S biomic on skule Z gt Oo Only Selection r Selection kde 15 j TemporaryItems Selection My Recent j Trashes Documents Desktop Ne ED New folder 5 aA v Seve Color Image Fox OTHER Saye inary Image Save Annotations My Documents My Computer My Network File name NDProgress ConvallariaAllnd2 Me Save Places ND2 Image File Format nd2 JPEG2000 jp2 j2k LIM Image File Format lim NDS tid Sus Ln en L F z Save as type Compression po
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