100305 Zeiss guide
Contents
1. Laser Diode 405 Maximum Power 25 0 mw Wavelength 405 nm Status connected Lasers Laser Unit Wavelength Laser Diode 405 405 nm Argon 458 477 468 574 nm HeNel 543 nm On Hee B33 nm DH b Nimium Power 30 0 mw Wavelength 458 A77 488 514 nm B Status Ready Tube Cure EA Standby Output 2 aa a F 5 6 IN GENERAL 405 nm excites blue DAPI 488 nm excites green 543 nm excites red and 633 nm excites far red 3 Setting up configurations for sequential scanning Setting up a configuration for 3 colors blue green and red in Configuration control Always start in Channel Mode and Multi Track Configuration Control EJ Each color is set up in a separate track sequential scanning i we Channel Made Lambda Mode Online Fingerprinting Close The procedure step by step for each track color Wat Track io Switch tracks after each Line Frame Frame Fast 1 Under excitation activate the laser and choose a Mame Channels transmission Elek ch 405 2 Choose the main beam splitter HFT which will direct the laser light to your sample ES ES Sissi ie TK 3 Choose the correct beam splitters mirrors NFT for armen the emission to be directed to the chosen detector LSM 510 Came ChS 2 3 COo rm 4 Choose the correct emission filter Ee E 5 Activate the detector Ch and define the color on Rane 12. BP 420 480 l Eu iv 405nm 5 jv th2 458nm 0 1 LP 560 eis A 488 nm 0 1 Pi eh cc You only have to optimize m uL ao the intensities in the ie zn m ag e Specimen IN Mirror NNN Ready 732 x 732 1 channel 8 bit Select Display Single Track Multi Track E e d Chan xy List of Tracks Spectra Switch tracks after each Line Frame Frame Fast f gt Channels Channels Light nm 1 Laserline O Blue Ch2 405 O Green Ch3 488 Red Ch3 543 Add Track Remove Store Apply Single Track Pinhole xn p 3d Hide Beam Path and Channel Assignment Optical slice 0 8 um Pinhole 0 32 Airy Units C EM ES SR DER Detector Gain 465 4 gt E E OOOO ge Amplifier Offset 0 007 I III TI IT sacs Amplifier Gain 1 Aj m gt nove 7 excitation Line active Transmission Laser Power BP 420 480 A 405nm 01 4 yo eke sam 01 4 e Ir miner LZ LZ 48m 01 4 dle vw Ch3 Laserline on gt NFT 430 514nm 0 1 ajjpe gt e HFT 405 488 543 V4 Excitation 543 nm 20 4 Fr e 63m 01 4 u None A Specimen Ready 732 x 732 1 channel 8 bit 1 9 Finally after optimizing all colors separately I Pixel Depth Scan Direction amp Scan Average Data Depth ea 12 Bit Made Line Method Mean Scan Direction
3. e ipis 4 10 Activate all tracks under Configuration Control 1 Go to Mode and define scan average 4 is common to use 2 when doing z stacks Doing 4 will acquire the image 4 times and the result will be a mean image from those 4 In line mode each line in the image is scanned 4 times and n 27 averaged Mode Frame will Gems m average the image frame by Imm Path and Channel Assi nment F Remove Pinhole 38 4 E 1 Mad Optical slice 0 7 um Pinhole B 2l Fm U nita i Add Track 7 o fram e E aid Detector Gain E 439 4 i IIIIIIT gy Afipifie Offset 0 022 rrrrrerr c5 M AmplfieGan 1 H Averaging creates a nicer Z Benzene B Mane en gt Excitation image and reduces background ZI D c Laser ae BF 420 480 Sm noise A 8 w 45m 5 4 e p e IT 458mm D1 4 gt pre i Mirror Z Z _ 488nm 51 aj o 12 Press single and the final NFT480 r si4em 01 gt REI Val Pd Eschation 543nm 01 z gt 7 image is acquired ective Lens Image Size amp Line Step Facto Plan amp pochromat 63x 1 4 Oil DIC Light nm 5 x Y Line Step 1 Speed O OOOO i Scan Speed 8 4 gt Max Pixel Time 1 71 psec Scan Time 26 47 sec Pixel Depth Sc
4. Choose the DIC filter in the condenser in the Microscope control window For air objectives DICII for immersion objectives DICIII The DIC channel can be set up in any track in combination with any other emission Channel and laser Activate channel D ChD and inactivate the current Ch detector in the setup Press Find to find the detector gain Use FastXY to find the best focus for DIC Fine adjust the gain and offset to visually get the best DIC image Activate the other Channel in the current setup 2 2 Adjusting the Kohler illumination Essential for acquiring a nice DIC image 1 Close the felt blender until you se the polygon and focus this with the vertical adjustments screws If the polygon is not visible to begin with focus the condenser until you see it 2 Center the polygon with the centering SCrews Condenser focusing screws Polarizer D The EI 2 we Felt blender condenser in an inverted microscope Condenser centering screws 3 Open the felt blender like this and fine adjust the centering 4 Open the felt blender until you see the full view field 2 Configuration C Control BD Control Choose a DIC filter in the tab Mae Condensor for air objectives DICH l for immersion objectives DICIII mess Activate the transmission detector Y Microscope Serings X Apply Store Delete Assign Button ChD and inactivate the cur
5. Scan Control BUILD MENU Optical slice lt 0 7 pm Detector Gain dle A Scan Control Channels Channels Close OD Find o c Line Frame 077 Line Frame 77 LELY Z Stack 4l 3 EP stack Channels BE 4 a2 Nasa ipem 08332 E 4 Fast Y Single Pinhole B 4 E 1 me Pinhole 0 92 Airy Units FW 4 v us Pinhole 1 19 Airy Units Optical slice 0 8 pm Stop a 4 9 Detector Gain Amplifier Offset o1 2 Amplifier Offset 01 gt Amplifier Offset 01 gt e Cont Amplifier Gain 1 gap qp Amplifier Gain 1 u gt Amplifier Gain 1 31 gt Excitation Line active Transmission Power Line active Transmission PA Line active Transmission ae A 405nm 01 4 Yo n A PS 405mm 5 gt mr mfa 4 gt 48m 01 Ne Et v 48m 5 rjo E14 nra n1 al bl a L Optical slices thickness with the same diameter for all pinholes thickness between colors is the same 5 Optical Slice Stack Z Size Scale 2 55 um Ch3 T3 0 8 um 13 00 um Ch2 T1 0 7 um Ch3 T2 0 8 um Optimal Interval 0 37 um Optimal Pinhole Diameter M 458nm 01 4 je T 49m o1 4
6. Zeiss LSM 510 META user manual Hege Avsnes Dale Molecular Imaging Center UIB Ai Switchboard Carl Zeiss Laser Scanning Microscope LSM 510 Open the LSM software in expert mode The software opens like this PIC 510 Expert Mode OBS Before looking at the sample in the microscope press the VIS button For regular imaging you need the 4 first windows in the acquire menu marked red A Laser Control X E LSM 510 Expert Mode File Acouife Process Sb view Macro Optens Maintain Window Help oe le bu Io cp 45 4 7 488 644 nm 30 0 mw avelength 458 477 488 514 nm Status Heady n a a Li 5 bE vox 5 z Spot Eine Frame Tube Current 3 ex 2 Multi Track UPS me 4 ls i di of Tracks zj l Te Objective Lens Image Size amp Line Step Factor I ch agin aitereach Line J EANA Eras Objective Plan amp pochromat 63 1 4 Oil DIC Boe oae Dea al E Femeie tm c6 s don 2 D Elek om T Gatimal x 52 512 esee 7 MP J Apply Store Assign Butto Exe nr nr nn A Fast F l ji po Track Remove L StorezApply Single Track Speed Scan Speed j Seel Fisel Time 1 60 psec Scan Time 383 4 msec Pixel Depth Scan Direction amp S
7. amp blue pixels will not have any g 2s quantitative value m i ar 100 Save s But when adjusting the intensities you s have to consider what is important in i e 0 5 1 15 20 25 3t 35 40 your particular image If the weaker Distance un signal is what you re interested in you en might need to saturate other parts of the image This is an evaluation up to you The background should be kept around zero very few blue pixels to avoid hiding any weak signal below am 5 Paty a Ys GE s 1 US 4 4 17 Rn gt zw i gt 5 1 un oe gt 7 AT dca d 1 iiie s S v 3 Info meu c WAR A e dynamic range A OS Z5 iis n A 3 m s b E 9 Do similar adjustments Channel Mode Fe gt ome m O sn 53 08 27 AM l Bc Line Frame 277 El 7 f E LJ on the following channels ee ees xm List of Tracks en Channel Settings CO O rs Switch tracks after each tee Frame Frame Fast Ts Channels eer i Name Channels ight ten Laserline v Blue Ch2 405 Everything in the Mode Hw Os wd Add Track Remove Store Apply Single Track Config RE ER siib window is remembered by a m ee DetectorGain 23 4 1 the software zoom frame CELIIILD mem nace tr Om 4 13 rrrrrrrr amp Amplifier Gain 1 4 IT size scan speed Optical slice lt 0 7 um Pinhole 1 19 Airy Units Line active Transmission
8. Microscope Control microscope from the software Microscope Settings But these setting are most of the time more Z Anny Store Delete _ Assign Button natural to do on the microscope 2 ze m Less Except from Changing objectives Condensor Transmitted Light 4 Ww Q It s easier in the software to see TE Apetue 08 OF 00 which objectives are in the revolver and their position Prevents changing from oil to alr objectives by accident Flan Neofluar 1040 3 L pochromat 6361 2 Wi carr Plan Apaochronat 580 16 OBS Changing objectives in the revolver must be registered in the software File menu maintain objectives if not all the settings will relate to the objectives the software thinks are In 2 Switching on and off lasers switch only on the lasers you need If lasers are on when you start switch off the ones youre not using The lasers have different power ranging from 1 to 30 mW The Argon laser is the strongest laser and has Its own output 96 slider in the laser control window It is turned on in standy When status comes up as ready it can be switched on Never work in standby and always use 40 output as a starting point Turning on this laser automatically starts a fan When turning off the fan automatically stops after a while and until it stops the remote control must not be switched off All other lasers are only switched on and off and has no fan
9. 9 the channel l LP 420 f i mi Cha See details in the next pages rth Mirror NT 80 20 ZO A EE HFT Haupt Farb Tailer gt The numbers indicate which laser is vow 7 reflected towards the sample NFI Neben Farb Tailer The number indicate where the light Specimen P is split ex NFT490 all light below 490 E nm is reflected and light above 490 passes through 4 Configuration Control Channel Mode Single Track List of Tracks Ratio Multi Track Ch2 405 Store Apply Single Track Beam Path and Channel Assignment LSM 510 Camera OOO goy Prrrrrerr _ ts None Z LP 420 EE lp th LP 505 ve Al ZI 1 rm cong Mirror NT 80 20 Va None Z Specimen M hD Switch tracks after each Line Frame Frame Fast Name Channels Ligi 1 Excitation Line active Transmission 2 ia r r u r Activate laser only one for each track Hemember to turn on laser in Laser Control Typical values for 405 nm 596 488 nm 596 543 nm 80 633 nm 50 These values can be changed later during image optimization Laser Power 405nm 5 4 gt enm 01 4 gt senm a1 4 gt 514nm ET c 9 sin 01 4 m a1 4 gt 3 Close Laser 4 Configuration Control Choose transmission Channel Mode Lambd
10. 9 IEREEEEETETR stop t Remaining and total Z stack acquiring time The progression of the Z stack may be viewed in Gallery and if you see that the images contains no more information the scan can be stopped any time m 7 Saving the images 1 Press Save as on the image 2 When saving the first time you have to define the database MDB where the images should be saved A database per date you work may be a way to organize the databases Choose New MDB and define where to save it We encourage all to Save on i our external server Biomic on skule For this you need access to the server for more info contact MIC_ personnel or Torstein Ravnskog You may also Open an existing MDB oe l 3 Then enter the name of the image um Pe dum WEN AA San andre T0008 SENA ACT 123 U2 med db 100223 SRNA 1 og 4 4 The next image to be saved will automatically be wo INE saved in the previous defined database coming up dn Comms ess blue 5 All images will have the file format lsm and can only be read in LSM software or processing software that io reads Ism files like Imaris ImageJ etc A free Zeiss Ici 3 LSM browser can be download go to the MIC E Quos webpage then to Equipment and Zeiss LSM 510 LJ My Documents META At the bottom of the page you ll find the link 48 6 You can export images to all common file formats but never delete the raw data files My Network
11. Ch3 as we are doing sequential scanning of each color at the time NFT490 fits all settings because light below 490 is reflected towards Ch2 and the blue emission is filtered between 420 and 480 Light above 490 passes to Ch3 and for green emission light between 505 550 is filtered In the red setting emission light is filtered through LP560 which also works fine with NFT490 4 Configuration Control 4 z zZ Single Track Multi Track Ratio List of Tracks NC Switch tracks after each tine Frame Frame Fast T Name Channels Light nm Le E zd Green Ch3 488 v Track Ch3 543 l Store Apply Single Track Config Beam Path and Channel Assignment Add Track Remove LSM 510 Camera OCCOCOOOOO0O gy rFrrrrrrr ths nore L7 BP 420 480 EM ij Ch2 i 505 Mirror A A h3 i T NFT 490 HFT VA 405 489 543 None A Specimen A Excitation Configuration Control Channel Mode Lambda Mode Orire Fngemintina nline Fingerprinting Close Single Track Multi Track Ratio List of Tracks Beam Path and Channel Assignment LSM 510 Camera OOOO gy Frrrrrrr th None Z BP 420 480 th 505 550 Mirror A A B ae v NFT 480 f HFT 405 488 543 Z A d None Z Specimen EE 4 Configuration Control ea Channel Mode Single Track Multi Track Ratio List of Tracks Switch tracks after each Line Frame Frame Fa
12. File name Places Create type The MDB comes up with raw data information in a form The gallery will show all the images in database 100301 AIM Seles The Reuse button will help you load 777070707 all the parameters used for that Record T oft particular image into the acquiring _ LLLA Mame 11 software except the objective used Pee rem Hotes In Hl User mic Acquisition T Date Time Monday 0370172010 10 46 12 AM Scan Made Stack original data multi track 8 bit Stack Size 7324732419 71 4 um x 71 4 um 6 7 um Scaling 0 098 pm 0 098 wm 0 37 um Position x 7 4 um y 191 pm z DEO um Pixel Time 1 71 ys Objective Plan Apochromat 63x 1 4 Oil DIC Beam Splitters MBS HFT 405 488 543 eee DEST Mirror v 732 DBS MFT 490 y 735 NDD MBS None 4 49 Wavelength 405 nm 5X 488nm 30 543 nm 20 time 1 channels 3 SA Filters Ch2 BP 420 480 Ch3 BP 505 550 Ch3 LP 550 Filter Pinhole Cha 98 um Chi 98pm Ch 98 um re Delete Total 30 383 MByte Selected 30 064 MByte Current 30 054 MByte Clipboard O Bytes 26 8 Setting up for DIC transmission imaging DIC Differential Interference Imaging step by step more details on the next pages 2L o2 00 If you want to do a multicolored image with DIC do everything as described in chapter 3 and the proceed with the following steps Adjust the Kohler Illumination on the microscope
13. Offset y 13 The optimization procedure step by step see more details in the following pages 8 9 Activate only one channel at the time in Configuration Control Start with the color that frames what your interested in in the sample Go to Scan Control Mode Make a starting image with low zoom detector gain for the first image can be found with the button Find Define the zoom Use the Crop button on the side of the image If you want to be consequent and use the same zoom between images type in the zoom factor Optimize the frame size This will give the correct numbers of pixels for acquiring the image with optimal resolution dependent of chosen zoom and the objective used Adjust the scan speed by setting the pixel time to a value between 1 5 and 2 5 usec Put on the range indicator which is found under the button Palette on the side of the image oaturated pixels appear red and pixels below zero are blue Go to Channels and fine adjust the signal in the image by adjusting Detector gain and or Laser intensity then Amplifier offset while scanning continuously over the sample Never use the FastXY button always the Cont button Increasing gain and laser intensity increases the signal but first adjust the gain and when this is around 800 the laser should be increased instead Increasing amplifier offset decreases blue pixels in the image Adjust to avoid red and blue pixels in the image Do the same with the next Channels co
14. a Mode Online Fingerprinting Close Single Track Multi Track Ratio List of Tracks Switch tracks after each Line Frame Frame Fast Channels Light nm V Track Ch2 405 Store Apply Single Track Beam Path and Channel Assignment Add Track Femove Contig LSM 510 Camera OOOO gr PTTrrire E None Z LP 420 ExI lv Ch2 mal m liP505 None Wh NT 80 20 Ch3 HFT m a V4 HFT Uv 488 543 633 Excitation HFT 405 488 543 ui HFT 405 514 Specimen WA HFT 458 514 WA HFT 488 543 HFT 458 Choose the main beam splitter HFT This reflects laser light to the sample whereas the emission light will pass through on its way back The wavelength of the laser light needs to be represented in the beam splitter Ex Using 405 laser 405 needs to be present A TIP If you want to set up more than one track color pick the HFT that also reflects the lasers you plan to use in the next tracks At this point you need to know where to detect the signal This is decided based on the emission filters in front of the detector For regular imaging Ch2 and Ch3 are the most frequently used In general Ch2 can be used for blue and green Ch3 can be used for green red and far red see next page 3 Choose the beam splitters to direct the emission light to the correct emission filter and detector 14 Configuration Control MR 5 Configuration Control If al
15. an Direction amp Scan Average Data Depth Method Mean E Scan Direction gt pus Number 4 ind TT ttt 942 nove Z BP420490 EN jy th HFT 405 488 543 The range indicator can now be changed to no palette and the different colored images are created sequentially The result is 3 single images and one overlay in the same file At the bottom of the image the total and remaining acquisition time comes up 21 6 Acquisition of a Z stack Z stack step by step more details next pages 1 Always start by optimizing each color separately like shown in chapter 5 But be aware Optimize each color in the brightest optical slice focal plane This might not be the same plane for all colors For each color focus with FastXY through the stack and stop at the brightest focal plane and optimize Activate only one color when defining the stack Use the one that frames what you re interested in Activate Z stack in Scan Control and chose Mark First Last Define the borders of the Z stack Scan FastXY and focus to one end of the stack and Mark First then go back into the sample to the next border and Mark Last Go to Z Slice and click Optimal Interval to define the total number of optical slices through the stack Go to Mode and define averaging to 2 optional Start the scan The time it takes will come up under the image 22 NB You do not have to optimjze the int
16. arily use optimal frame size Perhaps you also need to increase the pixel time below the recommended 1 5 usec Its is always a good idea to minimize laser intensity on the sample and to be able to do that opening the pinhole to let more light come to the detector is an option but this will be a compromise on resolution in Z 31 When you re satisfied with the settings for acquisition open Time Series Control 5 510 Expert Mode 1 line Sirit Lamiral Define how many images the time series will consist of in Stop series which means stop series after the number of scans chosen i UO avec m D moe fe immer time between images I sen ae Ec i Grabising cycle bel S0 remaining me 000323 000247 MNNINI singled or multi colored or Unt oan sec iaee 20 sec eventually Z stacks Foam More Tagger out None d You may follow the time series coming up in lt Marker 3 the gallery You can stop the scan any time Pd In sj Asp Suse Delete 7 Start the time series Diener Tagen Tigger ene sa None Nom Sa Neve lm Follow the focus over some E LEO ell time If focus drifts stop the i E ers ames a LIE DL N scan refocus and start the Set Noe lt New ume serles over again l Wak era Lb sec Countdown until next image in the time series
17. can Average I E Data Depth 8 Bit 12 Bit Mode Line Method Mean r Scan Direction 4 imbuit Moo e Single Cond Transmitted ondensor Light nl zii em op x Q N LSM 510 Camera Iw Aperture Of 00x ifa COOCOO E 3H PIPPPPPTSES Objective t GIGS None V4 i Flan Apochromat B3x 1 4 Oi DIC LP 420 ABEL Reflected 7 FSat1h wf Light LP 505 x uz I IF j Mirror i Td MT 50 20 Z em Tube Lens A i Lens 1x None VA Specimen RE Zoom DIEITUR x 31 fi a Rotation Ea ee EEE Offset Offset 5 Offset Y Content of this user manual O O N Oo Oo gt C N Changing ODJECTIVE cece ccc cece e cece eee eee e eee eeeeeenaeeeeeeeaes p 5 Switching on and off lASEVS cece cece eee eee e eee eeeeee eens p 6 setting up configurations for sequential scanning p 7 11 Adjusting the pinhole cccc cece cece eee eee eeeeeeaeeeeeeeees p 12 Optimization and acquisition of a multicolored image p 13 21 Acquisition of a 2 SIacK ae p 22 24 SAVING the IMAGES cece ccc cece eee eee e eset ness nnne p 25 26 setting up for DIC transmission imaging p 27 30 Time series ssssssssssssssssssseeen n I Rn nnns p 31 32 1 Changing objectives Microscope Control allows you to control the TE
18. erval this will only ensure the best resolution for a potential 3D Optimize the different colors Activate the man channel color of interest Interval pm Activate Z stack H Choose Mark First Last OOO0ooco a r r Mgvefto r Focus with FastXY to one Z E border of the sample stop 1 wee Al T when the fluorescence is Am getting dim and Mark First Z Then go back through the Sis sample and decide on the other border Mark Last Total Z stack depth comes gt TR ZZ EE n Click on Z Slice and pem x _ Mark Fisttast choose Optimal Interval ee 1 n n 4 i N Mark First 920 48 um The optical slices will the penne or een overlap 50926 u d ex Last 5 Optical Slice i IT gil Stack Sid 6 67 um Scale 2 31 um e aen wo c ow B 919 40 pm gt 3 Mark First Last gt Mark First 919 40 um Keep Slice w y Kegp terval J ark Last 902 40 um 9 e e g Wd S Last INESSE Ready 512 x 512 1 channel 8 bit Ready 512 x 512 1 channel 8 bit 23 reconstruction You may define fewer slices just to get an overview in different focal planes in the sample mage 2 AIM all led Display Gallery Data Grabbing remaining time 00 03 02 of 00 04 2
19. l the light can Channel Mode Lambda Mode Online Fingerprinting Channel Mode Lambda Mode Online Fingerprinting Single Track Multi Track Ratio The NFT can also be picked to fit all planned tracks colors Close Single Track Multi Track Ratio E 1 List of Tracks E Switch tracks after each Line Frame Frame Fast Track Che 405 u 90 towards Ch2 amp 3 use a mirror 05 L In this case blue fe emission needs to be Add Track Semove Store Apply Single Track Add Track Hemove Store Apply Single Track Config direc te d towar ds Ch2 Beam Path and Channel Assignment _ Beam Path and Channel Assignment LSM 510 Camera LSM 510 Camel All NF Ts incl the mirror OOOCCOOO gem OOOO z would be ok for this PTTTTITTIRES rrrrrrrr e None Z Rane Z i LP 420 EE LP 420 EE y Che jv th2 NFT 49i uta BZ VA ae A ctr 405 498 543 Z di NFT 490 means that all But planning ahead NFT 490 is best suited more page 10 un as i Z ag ight below 490 nm is Te reflected up everything A e above 490 passes through 9 Emission filters are either Band Pass BP or Long Pass LP filters Ex BP420 480 means that the light between 420 480 passes through to the detector LP420 means that everything above 420 passes to the detector Beam Path and Channel Assignment je Beam Path and Channel Assignment gem Wh e n YO U h ave d ecid ed Descanned No
20. le E 1 01 Air Units Detector Gain 401 4 gt Amplifier Offset 0 007 4 Amplifier Gain 1 EE gt Line active Transmission 405nm 01 4 gt e C enm a1 4 je m sem 5 4 e sam a1 4 5 sim 01 4 gt eXm 01 4 Ha Laser Power 7 For fine adjusting the signal in the image use detector gain and amplifier offset while scanning continuously over the sample i It s crucial to use the Cont button not the FastXY FastXY is only used when focusing the sample Increasing the gain increases the detectors amplification of the photons from the emission the image becomes brighter Increasing the laser intensity on the sample increases the excitation creating more initial emitted photons Always exploit the detector range before increasing laser avoiding bleaching But at detector gain around 800 laser may be increased The images are generally optimal when there is little red In it In the end set the amplifier offset to a level where only a very few blue pixels in the background is visible 17 Image 2 AIM TER Select 8 The intensity adjustments done is to create an image with grey levels within the dynamic range of 256 7 8 bit image Each pixel in the image ideally should have a grey scale value of O to 256 250 i Ai z 200 CB amt Hed
21. lors point 2 5 will be remembered by the software so you only have to do adjustments in the Channel window 10 With all channels optimized activate them all in the Configuration Control 11 Go to mode and define averaging 4 is common to use in Z stacks 2 12 Press the single button and the final images are acquired 14 1 Activate only one Channel at the time an 2 CE eee m VA LE Z Configuration Conirol PI Channel Mode Lambda Mode Online Fingerprinting Single Track sa Multi Track Ratio Switch tracks after each Line Frame Frame Fast Laserline Close Spectra Name Channels Light nm O Blue Ch2 405 Green Ch3 488 O Red Ch3 543 l Store Apply Single Track Contig Beam Path and Channel Assignment Add Track Remove LSM 510 Camera OOOO gea IT 3588 873 ChS None A BP 420 480 Ch2 BP 505 550 Z Z Ch3 NFT 490 4057489 543 ZI A Erion None V4 Buen 7 0 I 2 Begin with a starting image with low zoom 0 7 will give you the largest possible image on the screen Zoom Rotation amp Offset s alt a FE Rotation e D i a Offset Sp ey wa 7 Reset Offset 0 00 um Offset r 0 00 um 3 Define the crop If you want to always keep the same zoom factor it can be typed in Zoom Rotation amp Offset Ready 512 x 512 1 channel 8 bit oom ET Rotation Ea 4 _ RM gt o Offset Re E
22. n Gescanned Camera i Descanried Non Descanmed i which emission filter to LEO gm aooi BGGa etme rrrTmrTrTrT un use and thereby the detector go back and check that the 2nd beam splitter will direct the correct part of the light Mone a LP 420 fv th Mirror NT 80 20 Va LP 560 Mirror NT 80 20 Z LP 475 LF 505 CN ESS EE a eee Ce CO eee towards the detector BP 420 400 LP 650 Mane V4 BP 470 500 SEHR e E BP 475 525 Specimen BERE BP 505 530 ien ChangePos l aan 3 Setting up for blue emission exei Use BP420 480 If LP420 is chosen you risk including emission from any autofluorescence excited with the 405 laser or any emission from cross excitation of another stain present in the sample Setting up for green emission If you plan to use a red stain in addition Use BP505 530 or BP505 550 preferably the last to include as much of the green emission as possible If only green LP505 may be used Setting up for red emission If you plan to use a far red stain in addition Use BP560 615 10 If red is the last color Use LP560 On the schemes below each color has it s own track with the corresponding settings Be aware that the path in front of the emission filters is kept the same for all settings Ch3 is chosen for green because of the emission filter BP505 550 in front not present in front of Ch2 Both green and red can go to detector
23. non t Lasti BP 425 45 2 ee Pos A mm 5 4 TA lr ma n Ar a pi Es mie dl too 7 LA Da etes a aar 01 4 7 a muera ee T re SS e e F stem Tor 4 EUN Bal em 7 Stem a2 4 He E r sie os 4 LE we ZI Specimen p UD Eabb renean tine 830 11 1 ES _ Stop A DIC image can be acquired alone and later copied into the colored images But be aware that copying will only work if the images to be copied together has exactly the same frame format 9 Time series Doing live cell imaging there are several considerations Mainly one has to keep the cells happy during the acquisition of the time series Before acquiring you need to warm up the heating chamber ideally a couple of hours before imaging to Stabilize the system Turn on the Temperature Control and the Heater When placing the cells under the microscope make sure to use the right insert If you need CO put on the COe lid on top of the stage and switch on the COe controller switch on the back of the control box This is previously set to give approx 5 96 CO at the sample Its a good idea to let the cells acclimatize for some time and to watch the potential focus drift in the beginning of a time lapse The settings has to be defined ahead of staring the time series more or less as described in chapter 3 but to keep cells happy over time you most likely need to make a compromise on image quality and thereby not necess
24. od Heset Offset AD um Offset or 3 12 um Ready 512 4 512 1 channel 8 bit 15 ee NE 4 After defining the zoom press Optimal under ae imc na i Be aware that the optimal resolution for different colors will vary So always optimize with the same color in different images Objective ee Frame Size 128 47 gu 3 512 1024 204g Optimal Dey je 72 Dir Speed Scan Speed L eee Max Pixel Time 1 71 psec en Time 2 21 sec Pixel Depth Scan Direction amp Scan Averaye Data Depth 5 Bit 12 Bit Mode Line Method Mean Scan Direction mal d Bamber E LAE L T Fast Y When combining the colors blue green and red green will again give the mean value and should be used when optimizing Single gt Shop a 5 Adjust the scan speed The pixels time should ss be somewhere between 1 5 and 2 5 usec Going Zoom Rotation amp Offset too slow will take too much time and might n3 rm 1 E bleach the sample Going too fast will not give a nice signal Offset fc Bee 6 Put on range indicator E doin found under the Palette on the side of the image Color Palette Color Palette List ange Indicator Glow Scale Rainbow Rainbow2 Scan Control Channels ae 3 4 1 Mas Optical slice lt 0 8 pm Pinho
25. rent mmm emission channel in the example Ch2 for Blue emission __ mined T an e O ia Do a Find for getting a detector An gt o TTT E gain d Ma be x Line active Transmission pst i Be ZI Fe m Sle fe Er l B Sr i IT 458mm 01 Fine adjust focus with FastX Y if pall e 2 0 ee E CES EI s B l uera Ar 514nm 01 4 p gt AH necessary bre IE RA Ein 42357 T som oT gt PEE Li A amp Z A epee T 633mm o1 REM Fine adjust detector gain and effset If necessary EIER 8 Finally activate the all the different tracks and channels colors choose averaging in Mode and do a Single Notice that the DIC image is in this example in the track for blue This means that the DIC image comes up simultaneously with the blue image and is created with the 405 nm laser The DIC image could in principle have been put in any of the tracks The DIC image comes as a separate image and in the overlay image in the Ism file wem x mw r Lin of Tracks E Soir fi boy aller nach j ume Cham Light in a E fis Ch Chi e E Green Ch3 za FA Pied Cha 543 me 9 al E 1 a p csi dice 07 m Pinhole iB 1 19 Ay Units D iee Dee Gan am 4 a LILILIII Ample Bs oir a E 4 Hr Tr De Hone Faj ei ae Line ache Tianarac
26. st Name Channels oU Blue Ch2 405 f Green Ch3 488 ME Track 4 Add Track Remove Beam Path and Channel Assignment Store Apply Single Track Config Camera OCCO gy FITITTTTRDSS Mone IN BP 420 480 NENEN r chm LP 560 ZA A ror VA ra Ch3 L NFT 490 HFT m 405 488 543 WA d None Z Specimen r thD Lambda Mode Online Fingerprinting Close 11 4 Adjusting the pinhole Go to the Scan control window in Channels Define airy unit 1 for the green channel and copy the pinhole diameter into the blue and red channel The airy unit wifNoow slightly differ between the different colors but the thickness of the optical slice will theoretically be the Same 5 Configuration Control Channel Mode Lambda Mode Single Track Multi Track List of Tracks Online Fingerprinting Ratio COO poy PTTittt Tt pe None Z BP 420 480 Ea EH BP 505 550 Mirror A A Een IA ERS Optical slices thickness with airy unit at 1 for all pinholes thickness vary between colors Stack Z Size 19 00 ym Scale 2 73 um A Ch2 T1 0 6 um Ch3 T2 0 8 um Ch3 T3 0 8 um Optimal Interval 0 32 um Optimal Pinhole Diameter Channel Settings Channels Inm iem Channels P jej B Pinhole 1 01 Airy Units Pinhole OK Optical slice lt 0 8 pm a dt M Pinhole Detector Gain R
27. ve i i i z M ssn 01 4 e B M 48m o dle Laserline The green channel is chosen as a reference because it s the channel in the middle and will represent a mean value for the pinhole diameter 5 Optimization and acquisition of a multicolored image For optimizing the image you work between Channels and Mode in the Scan Control window Optimization has to be done for each Channel color A Scan Control Channels ou Pinhole iz 4 SS a Mar a Optical slice lt 0 8 pm Pinhole B 1 01 Ain Units Detector Gain Amplifier Offset ES 0 047 ENT Amplfie Gain 1 9 4 a Excitation be m Esser d Power C dsam 01 e senm 01 4 gt 5 4 e sim 01 4 M9 sinn 01 4 gt 633nm 01 4 gt Line active Transmission X Frame Size 128 256 512 1024 2048 Method Mean Sean Direction e Number fix Scan Control Objective Lens Image Size amp Line Step Factor Objective Flan Apochromat 631 4 Oi DIC optimal x s2 v s2 Linestp 1 v Speed Scan Speed 8 4 gt Max Pixel Time 2 51 psec Scan Time 4 72 sec Pixel Depth Scan Direction amp Scan Average Data Depth 5 Bit 42 Bit Mode Line r j Zoom Fotanon amp Offset Zoom Rotation Offset Offset
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