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1. pr E ain al pe a ui e p e 4 Wi H System Preparation 1 Turn the computer ON In case of equipped concentrated power supply power on it first 2 Turn the laser ON Turning the key switch 2 1 LD559nm ON 2 2 Multi Ar 458nm 488nm 515nm 2 2 HeNe G 643nm ON 3 Turn the mercury burner ON for Fluorescence observation 4 Log on Windows ARS o Enter Password Customer name is below OLYMPUS Username Administrator Password fluoview Fv410 ASWv CS Liser D Administrator Hm Password 5 FU1O A 5 Double click this icon to log on to ASW Password OK Cancel Wait for a moment until the software is started User name Administrat lt i Password Administrator Visual Observation under the Microscope EE Observation of Fluorescence Image EE 1 Select an objective lens by using the hand switch 2 Select florescent filter cube 3 gt Click the button on the Fluoview software E lr ageAcquisitionControl Focus x2 Sn A we Depth Time Focus x4 Stop TE qv 4 Focus to the specimen a a Le Filter Mode Kalman Line Frame 2 G Analog Int lt Photon Cnt M Sequential Visual Observation under the Microscope EE Observation of Differential Interference Contrast Images Hl 1 Select the Objective Lens 2 Insert the Polarizing Plate
2. Select All ROIs Chrl 4 Cancel Selection 2 135 135 Flip Selection Delete All ROIs Paste ROI Chrl Show Profile Wert Show Profile Hori Y Show Overlay 27 27 Image Analysis Save a Z section Image as 2D file E 2D View LILY POLLEN 1 xml 28 Int 0 O Zoom Auto 119 Zoom Auto 95 Save the image in step 3 or 5 6 6 Click on the ty button 7 A2D View file name image is created Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as type xml is a file format specifically for the FV10 ASW software New Image Naming Rules Setting OIF format WMemoN File formats specifically for the FV10 ASW Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations a 28 Image Analysis Inserting the Scale Bar E 2D View LILY POLLEN xml r b 1 1 z Click on the button 2 While left clicking the image drag and drop it at a certain point 2 e ROJO 3 1 gt NIA bb bb Y E o l the ive HR 3 While clicking the right or left handle move the mouse from side to side E 2D View L
3. mu EE 3 Intensity Profile on the line is shown as intensity graph State of colocalization between each Chs is figured out apart from intensity 1 Enclose the interested region by ROI 2 Click A Histogram 3 Histogram window is shown as a graph frequency of intensity of each pixels is plotted on the region enclosed by ROI 44 45 2D Image Analysis Line Series Analysis E 2D View PTK XYZ 0if Taam rEg gt p max T Fp Backarouna i Poo Es m A INAI 1 Line on the interesting region FRHELe DIOLA 3 hahaka Line Series Analysis 2 Click 3 Intensity of Z position time on the line is shown as a graph 2D Image Analysis Co localization Ciam Cl oa 1 a a E 1 e an interesting area by E 3 2 Click MY AnnotationMode 3 Select Mest Threshold from Annotation Mode 4 According to move Thresholds of X Y axis to right and left ups and down Enclose red color X Y axis Co localization result between 2ch is changed Information of Co localization is listed under the scatter plot 45 Closing the System E OLYMPUS FLUOVIEW 1 Exit the FV10 ASW software by File Display Processing Analys 1 selecting File Exit 2
4. 1 Click Z and select 29 again then Projection image is shown on 2D View after getting XYZ image 3013 5128051251 2 y 4 3 135 INEU J 14 oom Autor 4 Yo m 2D View ivel NGadBarP6_oib Selec lt a ES fe gt b max C29 Fig aa Z e Ae AE BR Background Int o0 ma EJ 2 Click and select Ei 3 The images of Z section is shown on X axis and Y axis A RE oN According to Move to left or right side on X axis and to move to ups and down on Y axis be able to show image of Z section each position E 2D View level NGadBarPt_oib 4 The image of Z section on Y axis 5 The image of Z section on X axis 43 2D Image Analysis Intensity Profile of each Z sections 3013 S126191 2x51 2 y 4 43 13 INEU J 14 oom Autor 4 Yo Profile 2 Click md Profile and then Intensity Profile of each Z sections is shown on the X and Y axis To move to Z position be able to show Intensity Profile on each Z positions E 2D View 1evelNCadBarP6 0ib gt lt M lolx l O o E ee ce oa IN Profile H 1evelNCadBarP6 oib Z3TI37 Sze f om Auto 656 44 1 Click lt and then Automatic setting Ch Ch V Manual setting Column ich Z Int 12 Row ich T int L Int a O Multi Plane Wiew Need Projection and Merge in Tile Mode 3 Click ln to show Scale on I
5. pr E ain al A UA i ga Ad aka PONEN ERE PR ANNE lho nan Bl PREIE a ui e p e 4 Wi H System Preparation 1 Turn the computer ON In case of equipped concentrated power supply power on it first 2 Turn the laser ON Turning the key switch 2 1 LD559nm ON 2 2 Multi Ar 458nm 488nm 515nm 2 2 HeNe G 643nm ON 3 Turn the mercury burner ON for Fluorescence observation 4 Log on Windows Welcome to FV10 ASW a Enter Password Customer name is below OLYMPUS User name Administrator Password fluoview FV10 ASW ser ID Administrator Fp Password O 5 Double click this icon to log on to ASW Username Administrator Password Administrator Password OK Cancel Wait for a moment until the software is started Visual Observation under the Microscope EE Observation of Fluorescence Image EE 1 Select an objective lens by using the hand switch 2 Select florescent filter cube 3 gt Click the button on the Fluoview software E lr ageAcquisitionControl Focus x2 a ge Depth Focus x4 XYRepeat xy i 7i Stop Time y 4 Focus to the specimen I _ Sa a 3 Y S gt 5 5 Filter Mode Kalman Line Frame 2 G Analog Int Photon Cnt F Sequential C o Visual Observation under the Microscope EE Observation of Differential Inte
6. Enter StepSize Slice the recommended value can be referred to by using the Op button and check the check box 17 Image Acquisition Double Stain on XYZ Image MA 1 9 Select AutoHV and then select ScanSpeed 12 5uS Pix Slow gt gt a JE pl AutoHV P 12 FF L 7 525ms F 3 9271s 13 95 10 Select Depth 11 Press the XYZ button to acquire an image 12 Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 13 Saving the image o o Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software beAcquisitionControl gt KY EMemoi File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which he Im age cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other 4 E operations D 13 R 18 18 Image Acquisition Four Stain on XY Image EE Acquisition of 4 stain images XY fluorescence image only EE Sample Four stain of Blue fluorescence dye DAPI green fluorescence dye Alexa488 and red fluorescence dye Rhodamine far red
7. software OIF format Creates a folder that contains an image 16 Save the image as TIFF BMP JPEG bit TIFF and an accessory file which a cannot be opened separately from each other format Select Export and OIB format chose the format TIFF BMP JPEG Creates the OIF format files in a single file which is convenient for migration and other operations ay 11 11 Complement of adjusting the image E Acquisitionsetting Mode al Size Aspect Ratio 1 1 4 4 P x al bl S12byfsi2 v Rotation P E Zopm Reset butfon 7 0 13 0 6 0 488 o 515 af 543 q 633 E Microscope 60X 035 NA 1 40 p 6 Start al 1 00 ES an 50um Center Go Set A v Endal se 2 000 um StepSize um Op eto Slices 7 ll Escape X 0 00um pix 10 00um pix 2 0 00um slice 0 o 458 E P 15 0 fad lad Ld Focus Handle On TimeSscan Interval 0 sec Num 10 kake 12 1 Click Clip scan button and enclose an interesting region s image on the whole image 4 Click HA Bames 2 GOR 2 pixel setting The standard pixel is 512 x 512 3 Zoom Setting Press XY Repeat to scan and set zoom value mom ee a Lg Above image is zoomed From 1x to 2 Scan speed and pixel resolution remain even zoom value is changed asas 7 EI a Zoom scan and be able to enclose an interesting
8. 1 Click on the 2E button to O O a N select 7 z PE A 2 12 135 al O amp g TE a AZ 2 To save this image right click on the N image select Save Display and save the image with a new name WOI morer rie y pepet bihi iiini Full Screen Image e Image to Clipboard 1018 As Ares m Save Display Save As AVI Print Select All ROIs Chrl 4 Cancel Selection 2 135 135 Flip Selection Delete All ROIs Paste ROI Chrl Show Profile Wert Show Profile Hori Y Show Overlay 34 34 Image Analysis Save a Z section Image as 2D file E 2D View LILY POLLEN 1 xml 35 Int 0 O Zoom Auto 119 Zoom Auto 95 Save the image in step 3 or 5 6 6 Click on the ty button 7 A2D View file name image is created Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as type xml is a file format specifically for the FV10 ASW software New Image Naming Rules Setting OIF format WMemoN File formats specifically for the FV10 ASW Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other opera
9. jalog Int Photon Cnt ZZ 22 E ImageAcquisitionControl Filter Mode f Kalman Line M Sequential Image Acquisition Single Stain DIC on XY Image 5 ES ImageAcquisition uisitionContr Bl es 23 10 Press the XY Repeat button to start scanning Adjust the green FITC image and the differential interference contrast image Press the Stop button to stop scanning Press the XY button to acquire an image Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW HVemoN File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations Merge the images between fluorescent XY image and DIC image Edit different each files to the same file This is available for making merge image Between fluorescent image and focused DIC image eye 1 co 1 Open fluorescent image and DIC image File Device D
10. mk tiff Frames E mkbmp bmp frames Quality 1 lowest 100 highest other default 70 PNG Compression Level O none 9 highest other defau p Con 4 e me as pe an or XYZ image into a TIFF format ES 4 Right click on the Image Icon displayed on the Data Manager and select Export Set Save as type to TIFF Set Output Format to RGB Color Save the image i BMP and JPEG formats are also selectable Convert a merge image of an XY Sl 1 Right click on the Image Icon displayed on the Data Manager and select Export Set Save as type to TIFF Set Output Format to Merge Channel Save the image BMP and JPEG formats are also selectable AA AER A DASS 2D e PO a E QUE a H o naxle T Ta 8 1 F4 VA O O OU ER Full Screen Image A th Clipboard L Save Display an Save 45 AVI Select All ROIs Chril 4 Cancel Selection Flip Selection Delete All ROIs 34 135 Paste ROI Ctrl V 31 Show Profile vert Show Profile Hori Show Overlay bar inserted into a BMP format 1 Right click on the image 2 Select Save Display and save the image with a new name an AVI format 1 Right click on the image 2 Select Save as AVI and save the image with a new name 31 Image Analysis Rotating a Three dimensional animation To save a rotation file as an animated image create three 5 6 dimension
11. pA Focus x2 gt amp gt 0 gt Focus x4 xy Repeat xy izil Stop Depth Time 3 The conditions HV Offset CA and so on are reloaded F Sequential 25 Click on the Pa button to switch the display TEXT Various kinds of ROls Grid Arrow Scale bar Point Color bar 26 Overview of the 2D Operation Panel 1 Ch1 display 2 Ch2 display Display switching Frame advance Enlarge 4 4 display Adjust to the Animation window size Advance speed m 2D jew SLY SLL Ami oR MQ z D DE MAX 029 Tic TT 9 4 Oe L T 2 3D formulation Projection switching Fix the end section Fix the start section 2 21 135 Int 134 93 Zoom Auto 206 Image Analysis Opening a File 1 Double click on a file to be opened from Explorer 26 Image Analysis Acquire a Projection Images QUE a gt ml T 7 A 1 Click on the 2E button to O O a N select 7 z PE A 2 12 135 al O amp g TE a AZ 2 To save this image right click on the N image select Save Display and save the image with a new name WOI morer rie y pepet bihi iiini Full Screen Image e Image to Clipboard 1018 As Ares m Save Display Save As AVI Print
12. 1 Select the Objective Lens 2 Insert the Polarizing Plate in the Light Pass 3 Insert the DIC prism slider in the light pass E 4 gt Click the button on Fluoview software Use this knob to adjust the differential interference contrast 9 Focus to the specimen Filter Mode Kalman Line Frame x 0 aser bl Of um FA e Analog Int C Photon Cnt M Sequential N Overview of Operation Panel for Image Acquisition Scan mode Scan speed Number of pixels Zoom amp Pan Laser output adjustment Transmitted light observation visual observation Fluorescence observation visual observation DyeApply Optical path diagram TwinScanner setting Save acquisition conditions Load acquisition conditions Image display window 8 Y ImageAcquisitionControl E AcquisitionSetting Mode O amp N lt lt Fast 2 0usPixel Slow q gt Auto E P 2 0us L 105 800ms F 55 122s 46 9min Size Aspect Ratio le O AS C arbitrary x al pl 512 by gt 1 gt aj aari El _ panx A A Pany rE Ta Laser FT 458 dl Vv 488 l genie CHS CHS ail 491 pm End nm StepSizel 2 0 nm Num 51 Resolution 10 0 nm amp Microscope UAPO 40X 01 340 NA 1 35 BX A Start Go UN 0 37 um y yo _Go m lt 7 of ae M StepSize a um Op l Slices 7 Focus Handle On Escape X 0 194um pix
13. 30 l JES als alme slo al 3 Drag the mouse on the image to 2 3 observe it at a certain angle 2 21 135 Simple animation 4 Press and hold the 4 button to rotate the image around the X axis Press it again to stop rotation Press and hold the button to rotate the image around the Y axis Press it again to stop rotation Press and hold the button to rotate the image around the Z axis Press it again to stop rotation Image Control volume Single 10 100 100 32 32 Image Analysis Saving an Image oC a Aao er Convert each channel ofa an 1 XY Sie l or XYZ image into a TIFF format 1 Right click on the Image Icon displayed on the Data Manager and select Export 2 Set Save as type to TIFF Set Output Format to RGB Color 4 Save the image BMP and JPEG formats are also selectable Convert a merge image ofa an XY or XYZ image into a TIFF format Livelmage_ 3 xml data E mkmk tiff fre 5 Livelmage_ 10 bmp frames A Livelmage_ 11 tiff frames rre 1 Right click on the Image Icon E mkbmp bmp frames displayed on the Data Manager and select Export 2 Set Save as type to TIFF a 1 lowest 100 highest other default 70 3 Set Output Format to Merge Chan nel eer Level 0 none 9 highest other defau 4 n Save th e m ag e BMP
14. 4 Double click on ROI and ROI2 5 Check that the Processing Type is set to Normal and click on Execute 6 An unmixed image is obtained Indicates channel assignments of unmixed images Introductory Notes Name Color Calculate S ANA O gqi2z 1 T MS OU K K Unmixed image 28 28 Image Analysis Unmixing When each fluorescence dye point is clear sample single stain of green fluorescence dye GFP and auto fluorescence from cell ROI 2 ROI 1 Only GFP image Unmixing image between GFP and Auto fluorescence 29 1 Open the XYL image GFP auto fluorescence 2 Enclose a point dyed with GFP only and a point dyed with auto fluorescence only 3 From Processing on the menu bar select Spectral Deconvolution 4 Double click on ROI1 GFP and ROI2 Auto fluorescence 9 Check that the Processing Type is set to Normal and click on Execute 6 An unmixing image is obtained Green color is GFP Gray color is Auto fluorescence 29 Image Analysis Unmixing ll When a control sample is used From an XYL image with a single type of fluorescence dye derive the _ fluorescence spectrum of the dye and obtain an unmixed image based on the fluorescence spectrum Sample Double stain of green flu
15. 4 4 P x al bl S12byfsi2 v Rotation P E 0 488 ll p 799M Reset button 515 af 7 0 543 q 13 0 633 E 6 0 Microscope 60X 03 NA 1 40 p 6 Start al 1 00 ES 0 o 458 E P 1 el Ld an 50um Center Go Set A v Endal se 2 000 um StepSize um Op eto Slices 7 ll Escape X 0 00um pix 10 00um pix 2 0 00um slice Focus Handle On TimeSscan Interval 0 sec Num 10 kake 12 1 Click Clip scan button and enclose an interesting region s image on the whole image 4 Click 2 pixel setting The standard pixel is 512 x 512 3 Zoom Setting Press XY Repeat to scan and set zoom value aaa lt me Qmc SoS ejes zoon aa Smee gt a Above image is zoomed From 1 to 2 Scan speed and pixel resolution remain even zoom value is changed E Zoom scan and be able to enclose an interesting region s on the whole image Press XY Repeat to scan after enclose the region azeaa MERO AN SOM PA kea Scan speed and pixel resolution remain even zoom value is changed 12 Complement of adjusting the image ial 9 Pan X Y ano ie al Be able to move the field of view to set Pan X Y without stage action 6 Rotation O PanX Y and Rotation reset button Be able to rotate the whole image 7 Lata Click Auto button to acquire Optimized Conforcal aperture Conforcal a
16. amu se E A A F N i AAA j fj ii i fi i Ti 1 i il y E a y m 22 A Jf Fi j I lnieH alee sintzisls la J a i i Fi i 3 Ten e E m Ti i i i Ti l i d j i i fi Ly i i Jo ER a j e m 3 a ae a gt Se ee E a l A AAA FTO ee ee a ee ESEESE EE et se ce ee ad PEEN OE ccc if i r i i yer er as af SS A n Pm f po i FF j laz i ze i al Fl E P ie re a Y F A aT l 1 li s f F if if ii if ry A ii F F d PA dl F Pa e e re E imam nen ial Pr E O LINN Ffff fee eet ee Geet ee fees dee A al l AN i io er i A je i if ae Po y cat if PA a E 1 11 a A p F i Y I F F io F ae a a L a ae fapa ith dp uk Ee a a O a CS 24 O22 1 of ATLAS F F i Fi ee P oe A 1 li f l i i TERA T F Ti Ti j j i i l A i F F r 1 m j Lt TOA A y i j I AN a r I E I I ri d F P n y ia fi ro F Fr Ss tt JN A E py E 1 lj J E f EF ri f F F i _ FF al F i i l at UF a oo ina TPF AEF F PFa a F e FL o f a T a e aa f fy j NAAC Ti l i F a y y j Fr ipa i oF sf j Fais MVA SA e e SE HIIVA WAT ATNI NNYORAIDAA ri f a Al f a i j F Ff j k y A f pu a Se tg acer i a a a AA IN IAS j LILIA Fri F f EE rif i y Fi Af A ht ASS ha h_ 3 f J d i F Fi Z ii A L i n A i I J Tid Ls y A _ a i gt HE P E A t PA A E A
17. 35 Image Analysis Acquire a Projection Images QUE a gt ml T 7 A 1 Click on the 2E button to O O a N select 7 z PE A 2 12 135 al O amp g TE a AZ 2 To save this image right click on the N image select Save Display and save the image with a new name WOI morer rie y pepet bihi iiini Full Screen Image e Image to Clipboard 1018 As Ares m Save Display Save As AVI Print Select All ROIs Chrl 4 Cancel Selection 2 135 135 Flip Selection Delete All ROIs Paste ROI Chrl Show Profile Wert Show Profile Hori Y Show Overlay 36 36 Image Analysis Save a Z section Image as 2D file E 2D View LILY POLLEN 1 xml 37 Int 0 O Zoom Auto 119 Zoom Auto 95 Save the image in step 3 or 5 6 6 Click on the ty button 7 A2D View file name image is created Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as type xml is a file format specifically for the FV10 ASW software New Image Naming Rules Setting OIF format WMemoN File formats specifically for the FV10 ASW Creates a folder that contains an image 16 bit TIFF and an accessory file wh
18. 9 Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 10 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software EMemoi File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations 23 23 Merge the images between fluorescent XY image and DIC image Edit different each files to the same file This is available for making merge image Between fluorescent image and focused DIC image eye 1 co 1 Open fluorescent image and DIC image File Device Display Live FRET ogee A e w 2 BB 8i Fiter setting alysis Tools Windo Ml Acquisition Setting Filter E Mode Threshold O2 Image Calculation 2 S e e ct lt lt Fast 10 0us Pixel Sk Correcting Pixel Gaps y a a MES Correc ting Z Gaps Aspect Ratio 1 1 4 W Spectral Deconvolution E r Edit Experiment A a Greene Edit experiment from Processing Edit Experiment 3 Click Edit Channels and select fluo
19. Cyd This is the procedure to acquire images through Virtual Channel scan 1 Y Virtual channel Scan Select Virtual channel scan on the DyeList and then Virtual Channel Controller is automatically turned on 2 select a number of Virtual Channel from Number of phase used Setup Dyes Acridine Orange A Alexa Fluor 405 Alexa Fluor 4881473 E Alexa Fluor 488488 Fluor 546 DyeList iv Virtual Channel Scan Number of phase used Alexa Fluor 555 Selected Dyes Alexa Fluor 568 Phase1 E DAPI ea 3 Select 4dyes from DyeList 4th dye Assign Dye Manually 7 Alexa Fluor 488 473 er is registered in the Phase 2 Phase2 PM cy5 DyeList El E he Virtual Channel Scan Number of phase used 2 C3 A Selected Dyes Phasel PB pari F Alexa Fluor 488 473 RodaminRed is able to be registered on Phase2 to drag 19 19 ANA E Virtual Channel C ntroller Phase A 4 lt image Acquisition Control Focus x2 Focus x4 xy Repeat xY EST AE 9138 BP Ole Lambda Depth d o Eld 1 Mexa Fluor 4891473 HV_ Gain Offset HV Gain Offset a a Image Acquisition Four Stain on XY Image 4 Select Phase1 DAPI Alexa488 RhodaminRed are registered on ImageAquisitionControl g rt Open Save As Stop Time Slit and Filter DM are automatically set for D
20. Enclose interesting regions by ROI Line on interesting positions by ROI 2 Click El measure Zoom 100 B Region Measurement PTK XYZ oif 2 10 T 0 L 0 ROIS Image PTE XVZ of Measure All ROIs BRE 4 According to click Measure All ROIs then moen Current Measure ROlllo 5 Statistics CHS1 CHS e information ol a lt O IS calculated on Zpos 10 CenterX 150 780 Intet 121878548 000 54771708 000 i eae a CenterY 79 732 Averag 1244509 559 277 Reg ion Me a surement La 6120 813 Max 4095 000 3227 000 he eformation of FRO is Range 3 an 999 000 316 000 calcutated Orr Regit n m dDev 7175 309 W Auto enterX CenterY Area Perimeter Integration Average Max Min Range 5tdDev 3StdDew Integration Average Max Min Range StdDev um um um um CHS1 CHS1 CHS1 CH51 CHS1 CHS1 CH51 CHS CHS CHS CHS CHS CHS 57 171 49 448 3120 625 241 490 5473264 000 1107 926 4095 000 95 000 4000 000 710 261 2130 783 2952481 000 658 076 3590000 28 000 3562 000 522 518 112 522 53 402 1470 184 194 764 0620457 000 1301 724 4095 000 97 000 3998 000 883 602 2650807 1837013 000 T58 280 3468 000 23 000 3440 000 561 877 51 900 37 103 3274 688 273 2145 2573667 000 1003 410 4095 000 94 000 4001 000 700 397 2101 192 9839166 000 562504 3415 000 53000 3362 000 43 623 30 180 111 524 1732438 211 246 4386227 000 879 166 3836 00 83 000 3753 000 657 656 1972 967 7880740 000 645 072 3380 000 25 000 335
21. FV1000D Spectral Type Upright Microscope BX61 Operation Manual Contents SYSECNTINIFOGUTIOM r7r se teeter eee Aa 3 FY1000D LaserDyeList ssl 2 RAS Rei ears 4 System Preparation 7 7 7 5 Visible Observation Observation of Fluorescence image 6 Observation of Differential Interference Contrast Images T Image Acquisition Overview of Operation Panel for Image Acquisition 8 Single Stain on XY Image 9 11 Complement of adjusting the image 12 13 Double Stain on AY Image 05 222 2 ee eet ee eee 14 15 Sequential scan Line Sequential 16 Double Stain on XYZ image 17 18 FOUFStal 0n AY Image 29s2 24s se soos esas eee see Rae 19 21 single Stain DIC on XY Image 22 23 Merge the image between fluorescent XY image and DIC image 24 Spectral Image on XYZ Image 777 TT TT TT TT TT TT rnnee 25 27 UAMIXING AAA A a 28 30 Reload the image conditions 7 ne 32 Image Analysis Overview of the 2D Operation Panel Opening a file 33 Making 2D Z projection file Images 34 Saving a Z section image as 2D image 35 Inserting the Scale Bar 7777 36 Rotating a Three dimensional Image 7 37 Saving
22. Filter Mode M Kaman Line M Sequential Focus x2 lt gt L Focus x4 XY Repeat 108um Filter Mode M Kalman Line Frame 2 a Analog Int Photon Cnt M Sequential o ry 3 A 7 A AE 14 Display after DyeApply is carried out Click on the FV10 ASW software button to close the fluorescence lamp shutter Alternatively click on the PA button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click Apply button The DyeList panel can be closed by using the Close button 14 Image Acquisition Double Stain on XY Image 4 Press the XY Repeat button to start scanning 5 Adjust the green Alexa Fluor 488 image and the red Alexa Fluor 546 Analogint Photon Cut image The image adjustment is outlined below For more information refer to Appendix 1 6 Press the Stop button to stop scanning and press XY repeat to acquire the image Refer to WMemoN WMemoN Scan buttons e Continuous scan XY Repeat Q Stop scan Rough scan Line skipped 7 Saving the image Right click on the Image Icon displayed on th
23. Histogram window is shown as a graph frequency of intensity of each pixels is plotted on the area enclosed by ROI 37 38 2D Image Analysis Line Series Analysis E 2D View PTK XYZ 0if Taam rEg gt p max T Fp Backarouna i Poo Es m A INAI 1 Line on the interesting region FRHELe DIOLA 3 hahaka Line Series Analysis 2 Click 3 Intensity of Z position time on the line is shown as a graph 2D Image Analysis Co localization aaa p gt al a me al TTI TOE Bil 1 e an interesting region by i 2 Click Ez AnnotationMode 3 Select Test Threshold from Annotation Mode 4 According to move Thresholds of X Y axis to right and left ups and down Enclose red color X Y axis Co localization result between 2ch is changed Information of Co localization is listed under the scatter plot 38 Closing the System E OLYMPUS FLUOVIEW 1 Exit the FV10 ASW software by File Display Processing Analys 1 selecting File Exit 2 Open 38 Close hd Save Save As i Property 2 Exit the Windows Logout 1 Select Start Shut Down 2 On the Shut Down Window select Shut Down and click on OK 3 Turn the laser OFF Turn the key switch to the OFF position 3 1 LD559nm OFF 3 2 Mu
24. Kalman accumulation Image acquisition is repeated to the 9 specified number of times to provide Filter Mode an averaged image Consequently v Kalman i noise is averaged and roughness on the whole image is reduced Advantage The speed of each scan is fast Disadvantage Some blur occurs due to averaging of images 13 13 Image Acquisition Double Stain on XY Image WE Acquisition of a single image XY plane fluorescence image only MIN sample Double stain of green fluorescence dye Alexa 488 and red fluorescence dye Alexa 546 Simultaneous scan q Filter Mode M Kaman Line M Sequential Focus x2 lt gt L Focus x4 XY Repeat 108um Filter Mode M Kalman Line Frame 2 a Analog Int Photon Cnt M Sequential o ry 3 A 7 A AE 14 Display after DyeApply is carried out Click on the FV10 ASW software button to close the fluorescence lamp shutter Alternatively click on the PA button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click Apply button The DyeList panel can be closed by using the Close button 14 Image Acquisition Doub
25. M Kalman Line Fame 2 j 6 Analogint Photon Cnt F Sequential Display after DyeApply is carried out jalog Int Photon Cnt ZZ 22 E ImageAcquisitionControl Filter Mode Kalman Line Frame Analog Int C Photon Cnt M Sequential Image Acquisition Single Stain DIC on XY Image 5 El ImageAcquisitionControl 23 10 Press the XY Repeat button to start scanning Adjust the green FITC image and the differential interference contrast image Press the Stop button to stop scanning Press the XY button to acquire an image Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software HVemoN File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations Merge the images between fluorescent XY image and DIC image Edit different each files to the same file This is availabl
26. i AAA T oo Ti i ii F F i i FR a fr f r f i F 1 FF FR I F F j ok BD f T l AT y 2 j ri i y it F F Fa a ma ie f 1 k d y 5 AN poy i i i Pil r F a e 7 HA F T fi F F f F a E A IERNAT Lyi i Ti i F F ii ri gF oF a E m VD JP TT MA Y f Jf FN sf A a f Fy p F f F j P or EA a ET i li Tr a n EF A i l li f e TF Ti Ti 1 j i i l A i F F r 1 m j Lt TOA A y i j I AN a r I E I I ri d F P n y ia fi ro F F A Y JN A E f py E 1 lj J E f EF ri f F F r _ FF al F i i d b d i if j F i ae Fi E F i i J j A Gl 7 Y A p Pa E Po l e zya H rn HA A a A K al f TNA AC 7 E naii f iJ a oe i iF Mor i ae i i ALLA at A e 5 ia Si i F i oa H i pe a NANE QU UN o f y F E be a Vd Fa T i WF Ff a Se 1 S o i ior i ie TH f J F us HH y j Ne IAS J A rf os Fs Pd E j Fi F f Y i 7 i5 TTI a VI V S TEA a a JSSAANSAS f f F E F F Z i r 2 e Wo 3 a f j d i i F a F fy E AN pr E ain al A UA K ANNO lho nn he BERET 3 T N g M a 9 EW er H System Preparation 1 Turn the computer ON In case of equipped concentrated power supply power on it first 2 Turn the laser ON Turning the key switch 2 1 LD559nm ON 2 2 Multi Ar
27. is a file format specifically for the FV10 ASW software New Image Naming Rules Setting OIF format WMemoN File formats specifically for the FV10 ASW Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations a 30 Image Analysis Inserting the Scale Bar E 2D View LILY POLLEN xml r b 1 1 z Click on the button 2 While left clicking the image drag and drop it at a certain point 2 e ROJO 3 1 gt NIA bb bb Y E o l the ive HR 3 While clicking the right or left handle move the mouse from side to side E 2D View LILY POLLEN xml Oax Change the text size aaa a D gt pi gt max 029 Tra Fag GA 0 color style etc 4 Select Scale Bar and then right click on Scale Bar to select Format A Setting eID NI hdi Pd iiini Cut ROI Ctrl X Copy ROI Ctrl C elt Delete ROI Del Max Size Color Bar Y Format Setting ROI Manager 5 Change the setting in this window as 0 required 31 31 Image Analysis Rotating a Three dimensional Image 1 Click on the 9 button for a 2D AA A AA View file name image 2 A 3D view Is created
28. 458nm 488nm 515nm 2 2 HeNe G 643nm ON 3 Turn the mercury burner ON for Fluorescence observation 4 Log on Windows Welcome to FV10 ASW ee Enter Password Customer name is OLYMPUS below o User name Administrator FV10 ASW Password fluoview Liser D Administrator Password z z 5 Double click this icon to Password Cancel ta in Ne log on to ASW Wait for a moment until the software is started User name Administrator Password Administrator Visual Observation under the Microscope EE Observation of Fluorescence Image EE 1 Select an objective lens by using the hand switch 2 Select florescent filter cube 3 gt Click the button on the Fluoview software E lr ageAcquisitionControl Focus x2 Sn A pe Depth Time Focus x4 Stop 4 Focus to the specimen Filter Mode Kalman Line Frame 2 G Analog Int lt Photon Cnt M Sequential Visual Observation under the Microscope EE Observation of Differential Interference Contrast Images Hl 1 Select the Objective Lens 2 Insert the Polarizing Plate in the Light Pass our 3 Insert the DIC prism slider in the light pass Use this knob to adjust the differential interference contrast E 4 gt Click the button on Fluoview software 9 Focus to the specimen Filter Mode Kalman Line Frame g x Y x
29. 658 076 3590000 28 3562 000 522 518 112 522 53 402 1470 184 194 764 0620457 000 1301 724 4095 000 97 000 3998 000 883 602 2650807 1837013 000 T58 280 3468 000 23 000 3440 000 561 877 51 900 87 103 3274688 213 15 2573667 000 1003410 4095 000 94 000 4001 000 700 397 2401 192 D839166 000 569 504 3415 000 3362 000 443 623 30 180 111 524 1732 438 211 246 1386227 000 879 166 3836 00 83 000 3753 000 657 656 1972 967 7680740 000 645 072 3300 00 25000 3355 000 523 061 150 780 TAT 6120 013 313 256 1070546 000 1244509 4095 000 96 000 3999 000 125 103 2175 309 4771708 000 559 277 3227 00 H 3186 000 409 334 5 The information of all ROls Count 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Average 90 511 76 240 7145 550 246 795 6987432 600 1107 467 4043 200 93 000 3950 200 T35 404 2206 212 0656221 600 638 042 3416 000 35 000 3381 000 498 083 1494 Max 150 780 111 524 6120 813 313 258 1878548 000 1301 724 4095 000 97 000 4001 000 883 602 2650 007 4771708 000 758 280 3590 000 53 000 3562 000 561 877 1685 4 Min 51 900 49 438 1470 184 194 764 4386227 000 879 766 3836 000 83 000 3753 000 657 656 1972 967 7837013 000 559 277 3227 000 25 000 3186 000 439 334 13181 Range 98 879 62 087 4650 625 118 495 7492321 000 421 958 259 000 14 000 245 000 225 947 677 640 5934695 000 199 002 363 000 28 000 376 000 122 543 367 4 StdDev 41 309 25 569 1548 840 41 735 0699715 061 172 621 115 828 5 101 110 244 36 561 259 083 5124831 492 30 326 132 286 11 811 137 208 54 102 1
30. IX81 Spectral Type 1X81 Filter Type BX61 Spectral Type BX61 Filter Type OLYMPUS Laser Conforcal Scanning Microscope FV1000D Spectral Type invertedMicroscpel X81 Operation Manual Contents System Introqucti n eae reshoese terete eee eret ES 3 FY1000D LaserDY LISt oo ss arene ese Reise eanres 4 System Preparation 7 7 7 5 Visible Observation Observation of Fluorescence image 6 Observation of Differential Interference Contrast Images T Image Acquisition Overview of Operation Panel for Image Acquisition 8 Single Stain on XY Image 9 11 Complement of adjusting the image 12 13 DOUDIE Stall ON XY Mage a sess tess eae eee ett 14 15 Sequential scan Line Sequential 16 Double Stain On XAYZ IMAGe sssicocasopociriccc n caia tees ese se 17 18 FOUr Stain ONXA Mage 29s224sSe 46 eet Pees e 19 21 single Stain DIC on AY Image 4 lt s ses seseeeeesesceesescecs 22 23 Merge the image between fluorescent XY image and DIC image 24 Single Stain on XYZT Image ZDC XYZT image 25 26 Spectral Image on XYZ Image ccoo ont rrr rrr 21 29 Unmixing 30 33 Reload the image conditions 777777 ttt 34 Image Analysis Overview of the 2D Operation
31. Open 38 Close hd Save Save As i Property 2 Exit the Windows Logout 1 Select Start Shut Down 2 On the Shut Down Window select Shut Down and click on OK 3 Turn the laser OFF Turn the key switch to the OFF position 3 1 LD559nm OFF 3 2 Multi Ar 458 nm 488 nm 514 nm OFF 3 3 HeNe G 543 nm OFF 4 Turn the mercury burner power OFF 46 46 OLYMPUS Laser Conforcal Scanning Microscope FV1000D Filter Type Upright Microscope BX61 Operation Manual Contents System Introduction a eresnt se ters seers rire ners saree a Saree nmr 3 FVIO0CD LaSerDYCLiSt ss A Ro e 4 System Preparation 7 5 Visible Observation Observation of Fluorescence image 6 Observation of Differential Interference Contrast Images 7 Image Acquisition Overview of Operation Panel for Image Acquisition 8 Single Stain on XY Image 9 11 Complement of adjusting the image 12 13 Double Stain OM AY Mage Ys ot ee dese 14 15 Sequential scan Line Sequential 16 DOUBLE Stal ON AY ZMAGS scetcosseccesiesesGoseeheaereseee sees 17 18 Four Stalon AY Image 2 gt s224s Se5eces Sr eee as 19 21 Single Stain DIC On XY IMAJge e ssecsacesastarisasarass 22 23 Merge the image between fluorescent XY image and DIC im
32. Panel Opening a file 35 Making 2D Z projection file Images 77777 TTTTTTT CCT T TT TTTTTTTTTr 36 Saving a Z section image as 2D image 37 Inserting the Scale Bar 97777 T OTC TT TT TT rrr reer 38 Rotating a Three dimensional Image 39 Saving ah Mage ewes suscritas asas 40 Saving Rotating a Three dimensional animation file 41 2D Image Analysis Edit the image color and contrast 42 The image Of Z section RS 43 Intensity Profile of each Z sections 44 Measure 45 Line Intensity Profile on the 2D image Histogram 46 Line Series Analysis Co localization 77777777 777777777 AT TimeLapse Analysis 48 Closing ANG SyYSteM ass tonto PEO AA 48 Spectral Type Main Scanner D440nm LD473nm LD559n Ar458nm Ar488nm Ar515nn HeNe G 543nm Dye List FV1000D Lasers are available below ee A AA I PHAN aan fee ey PON a ARNS 1 f i fo if iy T a F bite i rd Ti J j E I i l F I ou E po sail e ine r ri i F f oy i i T i i ii Pi fo A Mo 7 i I d i i ri k a i T oe et E oo fi oe ee ee Ae o e E l T amu se E A A F N i AAA j f
33. Series Append Files Destination Image lonlyfluorescence oib vr Image Info Ip A 4 Click Merge CH and then Source image Image Info Sarai ageso sis the fluorescent image and the DIC A image are merged as the new file Cancel Merge CH Z se X 512 512 Axis Range Single Slice 9 Merged image between the 5 fluorescent image and the DIC image 24 Image Acquisition Single Stain on XYZT Image This is available for the Time series scan experiment a 2 Time Series Scan TimeScan Interval FreeRun Sec Num 5 Adjust the image Refer P17 18 Enter interval time to Interval Enter interval number to Num Example Acquiring time series scan images every Sminutes for 1hour is below Select Time and then click XY Tbutton to acquire Time series scan image Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 25 26 Image Acquisition Single Stain on XYZT Image El AcquisitionSetting x Mode E 2 0us Pixel Slow gt gt D AutoHV P 2 0us L 2 116ms F 1 102s S 1 1s Size Aspect Ratio 1 1 4 30 Bl 512 by aj E Tei El PanX a igs aj 458 E Pl 15 0 488 a 515 f Pl 7 0 543 a 633 ll Pl 6 0 Microscope PLAPO 60X03 NA 1 40 X et k Start 1 00 um Center Go _ Go 50um ml Iv um Op ZDC Setting ee
34. T58 280 3468 000 23 000 3440 000 561 877 51 900 87 103 3274688 213 15 2573667 000 1003410 4095 000 94 000 4001 000 700 397 2401 192 D839166 000 569 504 3415 000 3362 000 443 623 30 180 111 524 1732 438 211 246 1386227 000 879 166 3836 00 83 000 3753 000 657 656 1972 967 7680740 000 645 072 3300 00 25000 3355 000 523 061 150 780 TAT 6120 013 313 256 1070546 000 1244509 4095 000 96 000 3999 000 125 103 2175 309 4771708 000 559 277 3227 00 H 3186 000 409 334 5 The information of all ROls Count 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Average 90 511 76 240 7145 550 246 795 6987432 600 1107 467 4043 200 93 000 3950 200 T35 404 2206 212 0656221 600 638 042 3416 000 35 000 3381 000 498 083 1494 Max 150 780 111 524 6120 813 313 258 1878548 000 1301 724 4095 000 97 000 4001 000 883 602 2650 007 4771708 000 758 280 3590 000 53 000 3562 000 561 877 1685 4 Min 51 900 49 438 1470 184 194 764 4386227 000 879 766 3836 000 83 000 3753 000 657 656 1972 967 7837013 000 559 277 3227 000 25 000 3186 000 439 334 13181 Range 98 879 62 087 4650 625 118 495 7492321 000 421 958 259 000 14 000 245 000 225 947 677 640 5934695 000 199 002 363 000 28 000 376 000 122 543 367 4 StdDev 41 309 25 569 1548 840 41 735 0699715 061 172 621 115 828 5 101 110 244 36 561 259 083 5124831 492 30 326 132 286 11 811 137 208 54 102 162 3StdDew 123 928 16 107 5546 521 143 205 6099145 184 517 864 347 445 17 103 330 731 259 683 179 050 5374494 476 240 979 396 857 35
35. Tic TT 9 4 Oe L T 2 3D formulation Projection switching Fix the end section Fix the start section 2 21 135 Int 134 93 Zoom Auto 206 Image Analysis Opening a File 1 Double click on a file to be opened from Explorer 28 Image Analysis Acquire a Projection Images QUE a gt ml T 7 A 1 Click on the 2E button to O O a N select 7 z PE A 2 12 135 al O amp g TE a AZ 2 To save this image right click on the N image select Save Display and save the image with a new name WOI morer rie y pepet bihi iiini Full Screen Image e Image to Clipboard 1018 As Ares m Save Display Save As AVI Print Select All ROIs Chrl 4 Cancel Selection 2 135 135 Flip Selection Delete All ROIs Paste ROI Chrl Show Profile Wert Show Profile Hori Y Show Overlay 29 29 Image Analysis Save a Z section Image as 2D file E 2D View LILY POLLEN 1 xml 30 Int 0 O Zoom Auto 119 Zoom Auto 95 Save the image in step 3 or 5 6 6 Click on the ty button 7 A2D View file name image is created Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as type xml
36. an Image 38 Saving Rotating a Three dimensional animation file 7777777777777777 39 2D Image Analysis Edit the image color and contrast 40 The image of Z section eee SESE a RET 41 Intensity Profile of each Z sections 42 Measure te a ore ROER 43 Line Intensity Profile on the 2D image Histogram 44 Line Series Analysis Co localization 7777777777777777777777777777 45 Closing the System ee 2sosapiorit se e iio 46 Spectral Type Main Scanner Dye List FV1000D Lasers are available below HeNe G 543nm 440nm LD473nm LD559nm Ssnm Ar488nm Ar515nm i l f Ti i it i T ion T oF F j E ail a 2 j fee li F JN a oo S ew a a f 7 a A z i A A dal Pe A TT 1 r F rj rf FS E f l PFL iii i F F a j a LN rf Fi i TH 1 i i fy i n i Ti ot i A 7 e g AS py E AX a e Pa re a i j i i I i E ffs E E DE _ Tie Aa i j i i I j i j F f f r r 1 j i f F g a a P i ma E i iii JA Il f E A O ot S oe ee N N li n ee Y oe aa fay a e oD l 7 f 1 E i 1 Nx J a a E i E II I f i F ES Fg Po a 4 A rf iF F F PA i LTR S F F t y I if HA F F F E FF g i
37. and JPEG formats are also selectable x A A AA 1 Convert a an 1 image with the scale A bar inserted into a BMP format o 2 1 Right click on the image a i 2 Select Save Display and save the E Sa lt A image with a new name ai ERE a Convert an animated image into Cancel Selection s Flip Selection an AVI format Delete All ROIs a 2 34 135 Paste ROI Chrl o 1 Right click on the image L 2 Select Save as AVI and save the image with a new name 33 33 Image Analysis Rotating a Three dimensional animation To save a rotation file as an animated image create three 5 6 dimensional images according to the following procedure For example try to rotate an image by 180 degrees ice ove 4D Angle rotation stat 180 2 eng 180 lt lt lt a 5 Click on the More button EE bs E Volume Single 10 100 100 6 Click on the Angle rotation tab 7 Select the rotation axis Animation Slice move 40 Angle rotatig 8 Enter the rotation angle a Start Angle to start rotation Start 180 2 Eng 180 lt End Angle to stop rotation Frame s 5 interval 30 Frame s Rotation speed _ Interval Degrees to be rotated Sree Animation is AMI File Q at a time 9 Select AVI File and click on Create Save in Image O e Ed 1 0 O aaa tiff frames E koumura XY xml data Smkbmp bmp i a O Kidn
38. fluorescence dye Cyd This is the procedure to acquire images through Virtual Channel scan 1 Y Virtual channel Scan Select Virtual channel scan on the DyeList and then Virtual Channel Controller is automatically turned on 2 select a number of Virtual Channel from Number of phase used Setup Dyes Acridine Orange A Alexa Fluor 405 Alexa Fluor 4881473 E Alexa Fluor 488488 Fluor 546 DyeList iv Virtual Channel Scan Number of phase used Aloxa Fluor 555 Selected Dyes Alexa Fluor 568 Phaset Jj DAPI we F Assign Dye Manually F Alexa Fluor 488 473 _ Rhodamine Red X OO Select 4dyes from DyeList 4th dye is registered in the Phase 2 Phase2 J cy5 DyeList El E he Virtual Channel Scan Number of phase used 2 C3 A Selected Dyes Phasel PB pari F Alexa Fluor 488 473 RodaminRed is able to be registered on Phase2 to drag 19 19 ANA E Virtual Channel C ntroller Phase A 4 lt image Acquisition Control Focus x2 Focus x4 xy Repeat xY EST AE 9138 BP Ole Filter Mode M Kaman M Sequential HV_ Gain Offset HV Gain Offset a a Image Acquisition Four Stain on XY Image 4 Select Phase1 DAPI Alexa488 RhodaminRed are g rt Open Save As l Fa registered on ImageAquisitionControl IRE x Slit and Filter DM are automatically EE ane set fo
39. fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click on the Apply button The DyeList panel can be closed by using the Close button 4 4 Check TD1 488 Filter Mode gt M Kalman Line Fame 2 j 6 Analogint Photon Cnt F Sequential Display after DyeApply is carried out jalog Int Photon Cnt ZZ 22 Image Acquisition Single Stain DIC on XY Image E ImageAcquisitionControl Filter Mode F Kalman Line rame Analog Int C Photon Cnt M Sequential ES ImageAcquisition uisitionControl A gt lt 9 la MQ EE ll A lt lt lt HR oracle 20 ap w 1 i r 23 5 10 Press the XY Repeat button to start scanning Adjust the green FITC image and the differential interference contrast image Press the Stop button to stop scanning Press the XY button to acquire an image Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW
40. its AF ang TimeScan Interval 0 sec Num 10 M ZDC Control ZDC Shot AF Return to current pos Move to offset pos Move to AF plane Search Zone Depth 50 Offset 1 24 um Offset Search Area t Hear 400 um t Far 500 um Near Limit 1829 60 um Far Limit 10 00 um Drift value 0 00 um AF Status off Current Pos 1 Adjust the image Refer P17 18 2 Insert ZDC unit to left side 3 Check EnableZDC AF during Time Series Scan jv and click ZDC setting 4 Click Set Offset to register auto focus position Note Have to use glass bottom dish below otherwise ZDC doesn t work 5 Set Interval and Num and then click XYZT to acquire the time series image Note In case of using ZDC for Time series Scan follow below limits Interval number is more than 60 sec Rest Time is more than 30 sec otherwise ZDC doesn t work If use TimeControler Time Series Scan is able to done even interval number is within 60sec and Rest Time is within 30sec Image Acquisition Spectral Image on XYL Image EE Acquisition of a spectral image XYL EE Sample Double stain of green fluorescence dye Alexa Fluor 488 and green fluorescence dye YOYO 1 El ImageAcquisitionControl Lambda 2 9 1 Click on the FV10 ASW software button gt to close the fluorescence lamp shutter Alternatively click on the button to close the hal
41. region on the whole image Press XYRepeat to scan after enclosing the area Egon 7 Es Scan speed and pixel resolution remain even zoom value is changed 12 Complement of adjusting the image ial D Pan X Y wm m in Ej Be able to move the field of view to set Pan X Y without stage action 6 Rotation O PanX Y and Rotation reset button Be able to rotate the whole image Auto Click Auto button to acquire Optimized Conforcal aperture Conforcal aperture change conforcal aperture to larger diameter for dim fluorescence image then be able to get the more bright image But Z axis resolution gets worse 8 Laser Intensity More Laser 8 intensity is increase more bright i aS E seh More increase laser intensity is more mE pel 10 0 discoloration image is 633 ll P 50 0 9 Kalman accumulation Image acquisition is repeated to the 9 specified number of times to provide Filter Mode an averaged image Consequently Naman noise is averaged and roughness on the whole image is reduced Advantage The speed of each scan is fast Disadvantage Some blur occurs due to averaging of images 13 13 Image Acquisition Double Stain on XY Image WE Acquisition of a single image XY plane fluorescence image only MIN sample Double stain of green fluorescence dye Alexa 488 and red fluorescence dye Alexa 546 Simultaneous scan q
42. stop scanning 26 Image Acquisition Spectral Image on XYL Image a _ 10 10 Set the range of wavelength to be E TES 70 e aa acquired the slit width and the step Serta q amp gt eStart Start wavelength eEnd End wavelength eResolution Slit width eStepSize Step Select AutoHV and then select ScanSpeed As the scan speed becomes slower noise can be removed while maintaining the current brightness 1 12 Select Lambda 13 Ix Press the XYZ button to Lambda De acquire an image 14 Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 27 2l Image Analysis Unmixing When each fluorescence dye point is clear From an XYL image where fluorescence dyes with similar fluorescence spectrums are present together derive the fluorescence spectrum for each fluorescence dye and obtain an unmixed image based on the fluorescence spectrums a Sudan a o a NN ve l T ey A and green fluorescence dye YOYO 1 Mm D e Al488 yoyo 4 KUA lanos lt a a jH _ p gt max Co Fre as 8 zi 1 Open an XYL image file with both Alexa Fluor 488 and YOYO1 applied 2 Enclose a point dyed with Alexa Fluor 488 only and a point dyed with YOYO1 only 3 From Processing on the menu bar select Spectral Deconvolution
43. z aie pa E 2 E e Analog Int C Photon Cnt Sequential Overview of Operation Panel for Image Acquisition Scan mode Scan speed Number of pixels Zoom amp Pan Laser output adjustment Transmitted light observation visual observation Fluorescence observation visual observation DyeApply Optical path diagram TwinScanner setting Save acquisition conditions Load acquisition conditions Image display window 8 Y ImageAcquisitionControl E AcquisitionSetting Mode O amp N lt lt Fast 2 0usPixel Slow q gt Auto E P 2 0us L 105 800ms F 55 122s 46 9min Size Aspect Ratio le O AS C arbitrary x al pl 512 by gt 1 gt aj aari El _ panx A A Pany rE Ta Laser FT 458 dl Vv 488 l genie CHS CHS ail 491 pm End nm StepSizel 2 0 nm Num 51 Resolution 10 0 nm amp Microscope UAPO 40X 01 340 NA 1 35 BX A Start Go UN 0 37 um y yo _Go m lt 7 of ae M StepSize a um Op l Slices 7 Focus Handle On Escape X 0 194um pix Y 0 194um pix Z 0 284um slice TimeScan interval 0 sec Num 100 ial ambda Depth y en Bleach Stop Time Filter Mode Kalman fine Frame 2 e Analogint Photon Cnt M Sequential ea e O Live V iew ma ar gt Focus Objective lens Time Interval amp Time Number
44. 0 o 458 E P 15 0 fad lad Ld Focus Handle On TimeSscan Interval 0 sec Num 10 kake 12 1 Click Clip scan button and enclose an interesting region s image on the whole image Te AN LSA 4 Click HA Bames 2 GOR 2 pixel setting The standard pixel is 512 x 512 3 Zoom Setting Press XY Repeat to scan and set zoom value paul Hagens amp Ls EI gt a Above image is zoomed From 1 x to 2 Scan speed and pixel resolution remain even zoom value is changed E Zoom scan and be able to enclose an interesting region s on the whole image Press XYRepeat to scan after enclosing the region Egon 7 Es Scan speed and pixel resolution remain even zoom value is changed 12 Complement of adjusting the image ial D Pan X Y wm m in Ej Be able to move the field of view to set Pan X Y without stage action 6 Rotation O PanX Y and Rotation reset button Be able to rotate the whole image Auto Click Auto button to acquire Optimized Conforcal aperture Conforcal aperture change confocal aperture to larger diameter for dim fluorescence image then be able to get the more bright image But Z axis resolution gets worse 8 Laser Intensity More Laser 8 intensity is increase more bright id ae E see More increase laser intensity is more mE pel 10 0 discoloration image is 633 ll
45. 0 y CO I ld SEER peee Analog Int C Photon Cnt r 100 ALL SCAN COMPLETE OIF format bit TIFF and an accessory file which OIB format operations File formats specifically for the FV10 ASW Creates a folder that contains an image 16 cannot be opened separately from each other Creates the OIF format files in a single file which is convenient for migration and other lt lt cea lt lt hh _ gt vo 19 la Flopi 1 y x ipd a t me a ps Sais data P 3D KAEDE 1 PA GFP E Unmix 42 CD RW Drive E Local Disk F uto 95 WHMemoN py 11 8 10 Select AutoHV and then select ScanSpeed As the scan speed becomes slower noise can be removed while maintaining the current brightness O Press the Stop button to stop scanning gt Click on XY and 2D View Livelmage x is displayed on the window bar for the image that has been acquired Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the Image Save as Type oib or oif file format specifically for the FV10 ASW software Save the image as TIFF BMP JPEG format Select Export and chose the format TIFF BMP JPEG 11 Complement of adjusting the image E Acquisitionsetting Mode al Size Aspect Ratio 1 1
46. 0 1 633 5 0 y 5 0 E Analog Int C Photon Cnt 10 Click on XY and 2D View Livelmage x is displayed coe on the window bar for the image OS MN ee that has been acquired 1 E PA GFP nmi SAMA lt lt m jji Naf 11 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the Save as Type oib or oif file EMemoi format specifically for the FV10 ASW File formats specifically for the FV10 ASW software OIF format Creates a folder that contains an image 16 Save the Image as TIFF BMP JPEG bit TIFF and an accessory file which P cannot be opened separately from each other format Select Export and OIB format chose the format TIFF BMP JPEG Creates the OIF format files in a single file 0 which is convenient for migration and other operations ay 11 11 Complement of adjusting the image E Acquisitionsetting Mode al Size Aspect Ratio 1 1 4 4 P x al bl S12byfsi2 v Rotation P E Zopm Reset butfon 7 0 13 0 6 0 488 o 515 af 543 q 633 E Microscope 60X 035 NA 1 40 p 6 Start al 1 00 ES an 50um Center Go Set A v Endal se 2 000 um StepSize um Op eto Slices 7 ll Escape X 0 00um pix 10 00um pix 2 0 00um slice
47. 473 _ Rhodamine Red X OO Select 4dyes from DyeList 4th dye is registered in the Phase 2 Phase2 J cy5 DyeList El E he Virtual Channel Scan Number of phase used 2 C3 A Selected Dyes Phasel PB pari F Alexa Fluor 488 473 RodaminRed is able to be registered on Phase2 to drag 19 19 Image Acquisition Four Stain on XY Image ANA E Virtual Channel C ntroller Phase 1 2 4 Start Open Save As lt image Acquisition Control 0 Focus x2 Focus x4 xy Repeat xy Stop Lambda Depth Time VESES M S s 2 s Mexa Fluor 4891973 HV_ Gain Offset HV Gain Offset HV_ Gain Off FAV Gain Offset a a Laj a a ENS a Lamp ds A v v v O 665 1 1 x 4 v f Laser 4 559 7 4 0 488 v 12 0 o o lt AE 9138 BP Ole Filter Mode Kalman E5 Analogint Photon Cnt M Sequential 100 Acquistion parameters have been loaded A EM Virtual Channel C introller Phase 2 5 Start Save As Image Acquisition Control r Auto Lambda Depth Time PESE do El ro e SY EA Rane HV Gain Offset HV_ Gain Offset HV Gain Offset A Lamp al al ja PREM era o OE PP a call li el 10 1 1 339 1 38 200um 9 0V v j Xx v x Laser La Laser Laser 2 0 559 4 0 488 v 12 0 puto Filter Mode M Kalman g Analogint Photon
48. 483 411 624 162 305 456 4 il A Save Histogram Clear Close 45 45 2D Image Analysis Line Intensity Profile on the 2D image 1 Line on the 2D image by ROI _ E 2 View PTK X c f ry eco Tay foca T 2 Click 5 Intensity Profile pem badenhet badaakad Aadankad FE Intensity Profile on the line is 3 shown as intensity graph 2D Image Analysis Histogram Aa 1 Enclose interesting regions by ROI E 2D View PTK XY3 oif 2 Click E Histogram 3 Histogram window is shown as a graph frequency of intensity of each pixels is plotted on the area enclosed by ROI 46 47 2D Image Analysis Line Series Analysis E 2D View PTK XYZ 0if Taam rEg gt p max T Fp Backarouna i Poo Es m A INAI FRHELe DIOLA 3 hahaka 3 2 Click 1 Line on the 2D image me Line Series Analysis Intensity of Z position time on the line is shown as a graph 2D Image Analysis Co localization KH pri a mae j 2 3 4 Enclose an interesting regions by ROI Click E AnnotationMode Select Mest Threshold from Annotation Mode According to move Thresholds of X Y axis to right and left ups and down Encl
49. 5 AVI ane T Convert an animated image into Cancel Selection s Flip Selection a Nn AV fo FM at Delete All ROIs 2 34 135 Paste ROI Ctrl a 1 Right click on the image Show Profile Hori L 2 Select Save as AVI and save the image with a new name 40 40 ES Image Analysis Rotating a Three dimensional animation To save a rotation file as an animated image create three 5 6 dimensional images according to the following procedure For example try to rotate an image by 180 degrees ice ove 4D Angle rotation stat 180 2 eng 180 lt lt lt a 5 Click on the More button EE bs E Volume Single 10 100 100 6 Click on the Angle rotation tab 7 Select the rotation axis Animation Slice move 40 Angle rotatig 8 Enter the rotation angle a Start Angle to start rotation Start 180 2 Eng 180 lt End Angle to stop rotation Frame s 5 interval 30 Frame s Rotation speed _ Interval Degrees to be rotated Sree Animation is AMI File Q at a time 9 Select AVI File and click on Create Save in Image O e Ed 1 0 O aaa tiff frames E koumura XY xml data Smkbmp bmp i a O Kidney2 bmp frames 5 Livelmage_ 3 xml data C mkmk tiff Fre 1 0 y E n te r a fi e n a m e a n d cl l ck O n S ave E Kidney XYZ tiff frames 5 Livelmage_ 10 bmp frames al koumura pol Kidney bmp frames 5 Livelmage_ 11 ti
50. 5000 523 061 150 780 79 792 6120 813 313 256 1876548 000 1244509 4095 000 96 000 3999 000 125 103 2175 309 4771706 000 S5927 3227 000 41 000 3166 000 439 334 5 The information of all ROls Count 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Average 90 511 76 240 7145 550 246 795 6987432 600 1107 467 4043 200 93 000 3950 200 T35 404 2206 212 0656221 600 638 042 3416 000 35 000 3381 000 498 083 1494 Max 150 780 111 524 6120 813 313 258 1878548 000 1301 724 4095 000 97 000 4001 000 883 602 2650 007 4771708 000 758 280 3590 000 53 000 3562 000 561 877 1685 4 Min 51 900 49 438 1470 184 194 764 4386227 000 879 766 3836 000 83 000 3753 000 657 656 1972 967 7837013 000 559 277 3227 000 25 000 3186 000 439 334 13181 Range 98 879 62 087 4650 625 118 495 7492321 000 421 958 259 000 14 000 245 000 225 947 677 640 5934695 000 199 002 363 000 28 000 376 000 122 543 367 4 StdDev 41 309 25 569 1548 840 41 735 0699715 061 172 621 115 828 5 101 110 244 36 561 259 083 5124831 492 30 326 132 286 11 811 137 208 54 102 162 3StdDew 123 928 16 107 5546 521 143 205 6099145 184 517 864 347 445 17 103 330 731 259 683 179 050 5374494 476 240 979 396 857 35 483 411 624 162 305 456 4 il A Save Histogram Clear Close 43 43 2D Image Analysis Line Intensity Profile on the 2D image 2D View PTK X c f a RECIO E 1 Line on the 2D image by ROI _ 2 Click 5 Intensity Profile een batesbed bedanbat abad
51. 6 795 6987432 600 1107 467 4043 200 93 000 3950 200 T35 404 2206 212 0656221 600 638 042 3416 000 35 000 3381 000 498 083 1494 Max 150 780 111 524 6120 813 313 258 1878548 000 1301 724 4095 000 97 000 4001 000 883 602 2650 007 4771708 000 758 280 3590 000 53 000 3562 000 561 877 1685 4 Min 51 900 49 438 1470 184 194 764 4386227 000 879 766 3836 000 83 000 3753 000 657 656 1972 967 7837013 000 559 277 3227 000 25 000 3186 000 439 334 13181 Range 98 879 62 087 4650 625 118 495 7492321 000 421 958 259 000 14 000 245 000 225 947 677 640 5934695 000 199 002 363 000 28 000 376 000 122 543 367 4 StdDev 41 309 25 569 1548 840 41 735 0699715 061 172 621 115 828 5 101 110 244 36 561 259 083 5124831 492 30 326 132 286 11 811 137 208 54 102 162 3StdDew 123 928 16 107 5546 521 143 205 6099145 184 517 864 347 445 17 103 330 731 259 683 179 050 5374494 476 240 979 396 857 35 483 411 624 162 305 456 4 il A Save Histogram Clear Close 36 36 2D Image Analysis Line Intensity Profile on the 2D image 1 Line on the 2D image by ROI E 2D View PTK X c f ry ca Tay m T 2 Click 5 Intensity Profile een batesbed bedanbat abad mu EE Intensity Profile on the line is shown 3 as intensity graph 2D Image Analysis Histogram ag 1 Enclose an interested area by ROI E 2D View PTK XY3 oif 2 Click i Histogram 3
52. 62 3StdDew 123 928 16 107 5546 521 143 205 6099145 184 517 864 347 445 17 103 330 731 259 683 179 050 5374494 476 240 979 396 857 35 483 411 624 162 305 456 4 il A Save Histogram Clear Close 38 38 2D Image Analysis Line Intensity Profile on the 2D image 1 Line on the 2D image by ROI 2D View PTKX c f RECIO E 2 Click 5 Intensity Profile ovens badenbat bedenbed Sadaated FE 3 Intensity Profile on the line is shown as intensity graph State of colocalization between each Chs is figured out apart from intensity 1 Enclose the region by ROI 2 Click i Histogram 3 Histogram window is shown as a graph frequency of intensity of each pixels is plotted on the region enclosed by ROI 39 40 2D Image Analysis Line Series Analysis E 2D View PTK XYZ 0if Taam rEg A gt p max T Fp Backarouna i Poo Es m INAI 1 Line on the 2D image FRHELe DIOLA 3 hahaka Line Series Analysis 2 Click 3 Intensity of Z position time on the line is shown as a graph 2D Image Analysis Co localization aaa p gt al a me al TTI TOE Bil 1 e an interesting region by i 2 Click Ez AnnotationMode 3 Select Mest Threshold from
53. API Alexa488 or PhodaminRed Lamp HV_ Gain Offse e FIV_ Gain Offset Lad at Ea ee ee i ae O 665 1 1 9 0V x v f Laser r 559 y 4 0 488 12 0 haa O f5 Analogint Photon Cnt M Sequential BEM Acquistion parameters have been loaded po E Virtual Channel C introller 7J el 5 Select Phase2 Cy5 is registered on Phase 2 7 5 start Open Save As ImageAquisitionControl El image Acquisition Control x r Auto s i Slit and Filter DM are automatically ambda Depth Time set for Cy5 gos 1 1 o a SA Ea CA Lamp HV_ Gain Offset HV_ Gain Offset HV_ Gain Offset a Lagala lalea aaa Ged bed io i a call li el E fi 1 J 2 l 38 200um 9 0V Laser La Laser Laser i 2 0 559 v 4 0 488 v 120 Auto Filter Mode M Kalman Sequential Adjust the image at each phases Phase 20 Analogint Photon Cnt 100 Acquistion parameters have been loaded a Adjust the image to click gt XY Repeat at each phases Phase2 If acquire XYZ image be able to decide upper limit and bottom limit slices step size of Z axis at both phases 20 Image Acquisition Four Stain on XY Image M Virtual Channel Co u Phase fi E 7 Click 2 on Virtual Channel Controller to acquire the image Be able to start at each Phase 8 Saving t
54. Annotation Mode 4 According to move Thresholds of X Y axis to right and left ups and down Enclose red color X Y axis Co localization result between 2ch is changed Information of Co localization is listed under the scatter plot 40 2D image Analysis Series Analysis TimeLapse E 2D View FRET XYT oif DER maoan a J gt p nar 23 Fr 9 A Py olla ae Background Int po 3 l 1 Enclose interesting regions by ROI 2 Click Series Analysis 3 Series Analysis graph is shown below Y axis shows intensity X axis shows time and then be able to see time series reaction each ROIs E Series Analysis FRET XYT oif Image FRET XVT of AA Image Info Total 2 MA CESA A A eae 1N H Axis NE Time me 41 Closing the System E OLYMPUS FLUOVIEW 1 Exit the FV10 ASW software by z Display Processing Analys 1 selecting File Exit Open 7 Close hd Save Save s i Property 2 Exit the Windows Logout 1 Select Start Shut Down 2 On the Shut Down Window select Shut Down and click on OK eos 2 116ms 3 Turn the laser OFF Turn the key switch to the OFF position 3 1 LD559nm OFF 3 2 Multi Ar 458 nm 488 nm 514 nm OFF 3 3 HeNe G 543 nm OFF 4 Turn the mercury burner power OFF 42 49 OLYMPUS Laser Conforcal Scanning Microscope
55. Assign Dyes window and take step 2 again Click on the Apply button The DyeList panel can be closed by using the Close button Image Acquisition Single Stain on XY Image El ImageAcquisitionControl Focus x2 Ez A Focus x4 XY Repeat PE SEIR pelhe n e 199 Filter Mode M Kalman tp M Sequential 0 HABA tont Size 51 2x51 2 xy 498 424 Int75 Zoom 100 10 4 Press XY Repeat button click to get image e Continuous scan mode 9 Focus to the specimen 6 Adjust the green FITC image Adjust sensitivity of HV and reduce noise by offset 7 Press keyboard Ctrl H key Optimized PMT adjustment brightness intensity 2 color between white and black Maximum intensity is 4095 1 2bit if intensity is over4095 color is changed to red saturation Basically Gain value is 1 10 Image Acquisition Single Stain on XY Image E AcquisitionSetting 8 Select AutoHV and then select ScanSpeed lt lt Fast 2 0 Pixel Slow gt gt pl autom I As the scan speed becomes slower noise EE can be removed while maintaining the A E ABE current brightness e P Ec a ain se ai el ai el S am JE 9 Press the Stop button hed EN Stop l R E a Ea k E z 2 E Laser ee y A to stop scan n ng asg 5 0 1 543 10 0 1 633 5 0 y 5 0 E Analog Int C Photon
56. Cnt 10 Click on XY and 2D View Livelmage x is displayed aiaia 1 gt Z z on the window bar for the image Nh x Es E To o a ae a a thathas been acquired q nite ti y 11 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the Save as Type oib or oif file Memom format specifically for the FV10 ASW File formats specifically for the FV10 ASW software OIF format Creates a folder that contains an image 16 Save the image as TIFF BMP JPEG bit TIFF and an accessory file which a p cannot be opened separately from each other format Select Export and OIB format chose the format TIFF BMP JPEG Creates the OIF format files in a single file which is convenient for migration and other operations ay 11 11 Complement of adjusting the image E Acquisitionsetting Mode al Size Aspect Ratio 1 1 4 4 x al bl 512uyf512 v Rotation Jal 27 Zqom Reset k 7 0 13 0 6 0 488 o 515 af 543 q 633 E Microscope 60X 035 NA 1 40 p Start al 1 00 ES an 50um Center Go Set A Y Endal se 2 000 um StepSize um Op eto Slices 7 ll Focus Handle On Escape X 0 00um pix 10 00um pix 2 0 00um slice 0 o 458 E P 15 0 el lad L
57. Cnt Sequential 100 Acquistion parameters have been loaded Adjust the image at each phases Phase Phase2 20 4 Select Phase1 DAPI Alexa488 RhodaminRed are registered on ImageAquisitionControl Slit and Filter DM are automatically set for DAPI Alexa488 PhodaminRed 5 Select Phase2 Cy5 is registered on ImageAquisitionControl Slit and Filter DM are automatically set for Cy5 a Adjust the image to click gt XY Repeat at each phases If acquire XYZ image be able to decide upper limit and bottom limit slices step size of Z axis at both phases 20 Image Acquisition Four Stain on XY Image M Virtual Channel Co u Phase fi E 7 Click 2 on Virtual Channel Controller to acquire the image Be able to start at each Phase 8 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software HVemoN File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations iA 21 21 Image Acquisition Sin
58. F A fF A i A S 5 E F EF 3 T N g M a 9 EW er H System Preparation 1 Turn the computer ON In case of equipped concentrated power supply power on it first 2 Turn the laser ON Turning the key switch 2 1 LD559nm ON 2 2 Multi Ar 458nm 488nm 515nm 2 2 HeNe G 643nm ON 3 Turn the mercury burner ON for Fluorescence observation 4 Logon Windows Malcom AA Enter Password Customer name is OLYMPUS below User name Administrator FV10 ASW Password fluoview User ID Administrator Fy Password DEA 5 Double click this icon to oe cence D Password ok Cancel log on to ASW Username Administrator Password Administrator Wait for a moment until the software is started Visual Observation under the Microscope EE Observation of Fluorescence Image EE 1 Select an objective lens by using the hand switch 2 Select florescent filter cube 3 gt Click the button on the Fluoview software E lr ageAcquisitionControl Focus x2 a ge Depth Focus x4 XYRepeat xy i 7i Stop Time q 4 Focus to the specimen I _ Sa a 3 Y S gt 5 5 Filter Mode Kalman Line Frame 2 G Analog Int Photon Cnt F Sequential C o Visual Observation under the Microscope EE Observation of Differential Interference Contrast Images MM
59. FV1000MPE FV1000 User s Manual Laser Safety Guide FV1000MPE User s Manual Safety Manual or Safety Guide FV1000 User s Manual Safety Guide FV1000MPE User s Manual Operation Manual or Operation FV1000 User s Manual Hardware Manual FV1000 FV10 ASW User s Manual Quick Start User s manual constitution of FV1000 e FV1000MPE FV1000 User s Manual Laser Safety Guide e FV1000 User s Manual Safety Guide e FV1000 User s Manual Hardware Manual e FV1000 FV10 ASW User s Manual Quick Start Also we have prepared one service manual for this system as below Technical personnels who perform the service require to take the service training e FV1000MPE FV1000 Service Manual IN In case of purchasing the laser simultaneously we have prepared the following manual for the laser o MaiTai Series User s Manual Quick Start In addition we have prepared one service manual for the laser as below Technical personnels who perform the laser service require to take the service training eo MailTai Series Service Manual Part or whole of this software as well as manual shall not be used or duplicated without consent Registred trademark Microsoft Microsoft Windows are registered trademarks of Microsoft Corporation Other brand names and product names are trademarks or registered trademarks of their respective owners CONTENTS pe This Quick Start has divided into the volume by the following system configurations
60. G seee KEEN 2 Contrast 0 00 Yelow O Hlo H Save As Load Reset Close 2 Edit contrast to drag to left or right side and another way to edit contrast is entering value on of Image Js edited Max and Min Max4095 Min0 According to gein value up be able to reduce noise of the image AAA ur S a Ti ELA 3 Min and Max value are changed and contrast Edit each Ch 0 Bo N 4 Orange The i J contrast fan Re Jue CT Gamma 1 00 A Fall i RA Spec B rare FITC Spec3 Intensity 0 00 cray Contrast 0 00 Yelow Hito CO I or Save As Load Reset Close 4 To click another color be able to Edit a color Above example Change Green to Red to click Red 33 33 TILE Ch Size 640x640 xy 575 362 Int 125 zoom Auto 46 The i 2D Image Analysis the image of Z section m 2D View level NGadBarPb oib 1 Click Z and select 29 again then Projection image is shown on 2D View after getting XYZ image 3013 5128051251 2 y 4 3 135 INEU J 14 oom Autor 4 Yo m 2D View ivel NGadBarP6_oib Selec lt a ES fe gt b max C29 Fig aa Z e Ae AE BR Background Int o0 ma EJ 2 Click and select Ei 3 The images of Z section is shown on X axis and Y axis A RE oN According to Move to left or right side on X axis
61. ILY POLLEN xml Oax Change the text size aaa a D gt pi gt max 029 Tra Fag GA 0 color style etc 4 Select Scale Bar and then right click on Scale Bar to select Format A Setting eID NI hdi Pd iiini Cut ROI Ctrl X Copy ROI Ctrl C elt Delete ROI Del Max Size Color Bar Y Format Setting ROI Manager 5 Change the setting in this window as 0 required 29 29 Image Analysis Rotating a Three dimensional Image 1 Click on the 9 button for a 2D AA A AA View file name image 2 A 3D view Is created 30 l JES als alme slo al 3 Drag the mouse on the image to 2 3 observe it at a certain angle 2 21 135 Simple animation 4 Press and hold the 4 button to rotate the image around the X axis Press it again to stop rotation Press and hold the button to rotate the image around the Y axis Press it again to stop rotation Press and hold the button to rotate the image around the Z axis Press it again to stop rotation Image Control volume Single 10 100 100 30 30 Image Analysis Saving an Image Bana lt Al lt Livelmage_ 3 xml data E mkmk tiff fre 5 Livelmage_ 10 bmp frames A Livelmage_ 11 tiff frames A Livelmage_ 11 xml data
62. OLYMPUS Users Manual Quick Start FLUOVIEW FV 1000 LASER SCANNING BIOLOGICAL MICROSCOPE FV10 ASW Ver2 0 Thank you for your purchase of Olympus microscope at this time Hold this manual by your side when using this microscope all the time and keep it with care after reading AXT197 Caution pd Caution FV1000MPE is a CLASS 4 laser product FV1000 is a CLASS 3B laser product The procedures for using this system are classified as follows e Service Service means any adjustment or repair performed by highly trained and skilled technical personnels who are provided the service training following to the service manual for this system The performance has influence on the feature of this system and there is a risk which unintended CLASS 3B or CLASS 4 laser light is emitted eo Maintenance Maintenance means adjustment or other procedures performed by customers to maintain that this system functions properly o Operation Operation means all performance described in the user s manuals in this system CLASS 3B or CLASS 4 laser light is only emitted from the objective lens during the actual execution The User s Manuals of this system consist of the following In order to maintain the full performance of this system and ensure your safety be sure to read these user s manuals and the operating instructions for the laser unit and light source unit before use User s manual constitution of FV1000MPE
63. P 50 9 Kalman accumulation Image acquisition is repeated to the 9 specified number of times to provide Filter Mode an averaged image Consequently Naman noise is averaged and roughness on the whole image is reduced Advantage The speed of each scan is fast Disadvantage Some blur occurs due to averaging of images 13 13 Image Acquisition Double Stain on XY Image WE Acquisition of a single image XY plane fluorescence image only MIN sample Double stain of green fluorescence dye Alexa 488 and red fluorescence dye Alexa 546 Simultaneous scan q Filter Mode M Kaman Line M Sequential Focus x2 lt gt L Focus x4 XY Repeat 108um Filter Mode M Kalman Line Frame 2 a Analog Int Photon Cnt M Sequential o ry 3 A 7 A AE 14 Display after DyeApply is carried out Click on the FV10 ASW software button to close the fluorescence lamp shutter Alternatively click on the PA button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click Apply button The DyeList panel can be closed by using the Close button 14 Image Acqu
64. Press the XY button to acquire an image 5 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software The image is acquired EMemol File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations 16 py 16 Image Acquisition Double Stain on XYZ Image WE Acquisition of 3D images XYZ fluorescence image only EE sample Double stain of green fluorescence dye FITC and red fluorescence dye Rhodamine This is the procedure to acquire images through Line Sequential scanning 1 Click on the 4 Take steps 1 to 7 described on pages 13 and 14 Press the XY Repeat button to start scanning and A buttons to shift the focal point Refer to WMemoN When the sample upper limit is displayed on the image accept it using the Set button Click on the and Mbuttons to shift the focal point Refer to WMemoN When the sample lower limit is displayed on the image accept it using the Set button Press the Stop button to stop scanning
65. Specimen ye a Select Mirror XA N BF 492 _ 537 565 665 nm i nm nm BA650IF T DM405 488 543 None HS1 FHS2 150 1408 30 um Dye iv Dye CA x Y atic Static2 Select BS20 80 or Select CHS1 only DM405 488 25 25 Image Acquisition Spectral Image on XYL Image El ImageAcquisitionControl 0 gt Focus x2 Focus x4 XY Repeat xy PEs PE HE 3p E Ya HV J4 E VA e vaj v v v v v v v v v v v w rey 680 1 0 588 1 0 613 1 51 142 1 0 150um v xX v X v x v x Laser Laser Laser Laser 488 v 5 0 543 v 26 0 r 633 v 5 0 488 v 5 0 Auto 07 LJ Fitter Mode M Kalman ce Gain Offset HV_ Gain Offset HV_ Gain Offset HV Gain Offset c ja ad EE ES 2 EE E E 24 224 ed MN a Analog Int Photon Cnt M Sequential 0 E Spectral Setting 400rnm 500mm G00ntm TOO S00nm 26 5 4 Click on the button and the Spectral Setting window appears Set the slit width for CHS1 to 20 nm for example 6 Press the XY Repeat button to start scanning While observing the image Click the left side of slit and drag to the point which the highest brightness is achieved Note Move the slit position only while keeping the slit width at 20 nm Adjust the image on the highest brightness Press the Stop button to
66. Y 0 194um pix Z 0 284um slice TimeScan interval 0 sec Num 100 ial ambda Depth y en Bleach Stop Time Filter Mode Kalman fine Frame 2 e Analogint Photon Cnt M Sequential ea e O Live V iew ma ar gt Focus Objective lens Time Interval amp Time Number for acquisition of XYT or XT image Scan buttons Select XYZ XYT or XYL Adjustment of each channel Confocal aperture Light intensity adjustment for halogen bulb Kalman Image file thumbnail Display of files in the memory Image Acquisition Single Stain on XY Image EE Acquisition of a single image XY plane fluorescence image only MIN Sample Single stain of green fluorescence dye FITC Fitter Mode M Kalman Line Frame 2 ES Analog int Photon Cnt Sequential E ImageAcquisitionControl Focus x2 a Focusx4 xy Repeat xy i 71 v Laser 11633 5 0 Filter Mode Kalman Line Frame 2 4 ie Analogint Photon Cnt Sequential 1 Click on the FV10 ASW software button gt to close the fluorescence lamp shutter Alternatively click on the gt button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different r
67. able for the Time series scan experiment a 2 Time Series Scan TimeScan Interval FreeRun Sec Num 5 Adjust the image Refer P17 18 Enter interval time to Interval Enter interval number to Num Example Acquiring time series scan images every Sminutes for 1hour is below Select Time and then click XY Tbutton to acquire Time series scan image Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 25 26 Image Acquisition Single Stain on XYZT Image El AcquisitionSetting x Mode E 2 0us Pixel Slow gt gt D AutoHV P 2 0us L 2 116ms F 1 102s S 1 1s Size Aspect Ratio 1 1 4 3 y Bl 512 by Eo sl ae des El PanX a igs aj 458 E Pl 15 0 488 a 515 f Pl 7 0 543 a 633 ll Pl 6 0 Microscope PLAPO 60X03 NA 1 40 X et k Start 1 00 um Center Go _ Go 50um ml iv um Op ZDC Setting ee its AF ang TimeScan Interval 0 sec Num 10 M ZDC Control ZDC Shot AF Return to current pos Move to offset pos Move to AF plane Search Zone Depth 50 Offset 1 24 um Offset Search Area t Hear 400 um t Far 500 um Near Limit 1829 60 um Far Limit 10 00 um Drift value 0 00 um AF Status off Current Pos 1 Adjust the image Refer P17 18 2 Insert ZDC unit to left side 3 Check Enab
68. age 24 Reload the image conditions gt 77700 ttre 25 Image Analysis Overview of the 2D Operation Panel Opening a file 26 Making 2D Z projection file Images 7777 TTTTTTT Tren 21 Saving a Z section image as 2D image 28 Inserting the Scale Bar 777 TT TT TT Tere 29 Rotating a Three dimensional Image 30 Saving ah Mage tesiussica liado das 31 Saving Rotating a Three dimensional animation file 32 2D Image Analysis Edit the image color and contrast 33 The image of Z section tooo ccoo rrr rrr rrr rrr rrr 34 Intensity Profile of each Z sections 35 Measure retainer Sense AA re SOS 36 Line Intensity Profile on the 2D image Histogram 37 Line Series Analysis Co localization 777777777777777 7777777777777 38 Closing the SYSICM sitecoluslrasccedeba session leete 39 Filter Type Main Scanner Filter type scanner D440nm LD473nm LD559n Ar458nm Ar488nm Ar515nn HeNe G 543nm Dye List FV1000D Lasers are available below ee A A AA I PHAN aan fee ey PON a ARNS 1 f i fo if iy T a F bite i rd Ti J j E I i l F I ou E po sail e ine r ri i F f oy i i T i i ii Pi fo A Mo 7 i I d i i ri k a i T oe et E oo fi oe ee ee Ae o e E l T
69. al images according to the following procedure For example try to rotate an image by 180 degrees ice ove 4D Angle rotation stat 180 2 eng 180 lt lt lt a 5 Click on the More button EE bs E Volume Single 10 100 100 6 Click on the Angle rotation tab 7 Select the rotation axis Animation Slice move 40 Angle rotatig 8 Enter the rotation angle a Start Angle to start rotation Start 180 2 Eng 180 lt End Angle to stop rotation Frame s 5 interval 30 Frame s Rotation speed _ Interval Degrees to be rotated Sree Animation is AMI File Q at a time 9 Select AVI File and click on Create Save in Image O e Ed 1 0 O aaa tiff frames E koumura XY xml data Smkbmp bmp i a O Kidney2 bmp frames 5 Livelmage_ 3 xml data C mkmk tiff Fre 1 0 y E n te r a fi e n a m e a n d cl l ck O n S ave E Kidney XYZ tiff frames 5 Livelmage_ 10 bmp frames al koumura pol Kidney bmp frames 5 Livelmage_ 11 tiff frames Kidney tiff Frames A Livelmage_ 11 xml data O koumura xY tiff Frames 5 mk tiff Frames File name Save as type AVI Files avi l Cancel 32 2D Image Analysis Edit the image color and contrast 1 Click LUT and then LUT table appears below TILE Ch Size 640x640 x y 629 4 Into Zoom Auto 46 7 or f e 1 00 E cans 000 M
70. and to move to ups and down on Y axis be able to show image of Z section each position E 2D View level NGadBarPt_oib 4 The image of Z section on Y axis 5 The image of Z section on X axis 34 2D Image Analysis Intensity Profile of each Z sections 1 Click and then 3013 S126191 24512 x y 4 3 13 INEU J 14 oom Autor 4 Yo Automatic setting Ch Ch V Manual setting Column ich Z Int 12 Row ich T int L Int a O Multi Plane Wiew Profile 2 Click wi Profile and then Intensity Profile of each Z sections is shown on the X and Y axis Need Projection and Merge in Tile Mode To move 1O Z Pn be able to show EE mG zana lt max C2 E EPS 3 Click ln to show Scale on Intensity Profile Profile H 1eyel NCadBarP6_oib b zaar due 4 According to click be able to show as equal scale of Profile window as 2Dimage 35 35 2D Image Analysis Measure DFA px AE i JUE A i 1 Enclose interesting regions by ROI Line on interesting positions by ROI 2 Click El measure Z101 Size 800x600 xy 621 12 Int 108 89 Zoom 100 B Region Measurement PTK XYZ oif 2 10 T 0 L 0 ROIS Image PTE YYZ oif Measure All ROIs vieasure All ROIS J e n Image Info 4 According to
71. ck boxes Click on three boxes when three types of fluorescence dyes are used Check that Processing Type is set to Blind and click on Execute An unmixed image is obtained 31 32 Reload the image conditions 1 Open the file and click i 2 21 135 Int 134 93 Zoom Auto 206 7D Control Panel ERE Al E 2 _ a AS 2 clio EP E ImageAcquisitionControl pA Focus x2 gt amp gt 0 gt Focus x4 xy Repeat xy izil Stop Depth Time 3 The conditions HV Offset CA and so on are reloaded F Sequential 32 Click on the Pa button to switch the display TEXT Various kinds of ROls Grid Arrow Scale bar Point Color bar 33 Overview of the 2D Operation Panel 1 Ch1 display 2 Ch2 display Display switching Frame advance Enlarge 4 4 display Adjust to the Animation window size Advance speed m 2D jew SLY SLL Ami oR MQ z D DE MAX 029 Tic TT 9 4 Oe L T 2 3D formulation Projection switching Fix the end section Fix the start section 2 21 135 Int 134 93 Zoom Auto 206 Image Analysis Opening a File 1 Double click on a file to be opened from Explorer 33 Image Analysis Acquire a Projection Images QUE a gt ml T 7 A
72. clic AA ala L EES DN A Leal a Current Measure ROIMo 5 Statistics CHS1 CHS F CALIU JI O v ATS d U Zpos 10 i Tpos 0 CenterX F 121878548 000 54771708 000 CenterY 19 732 Averag 1244 509 559 277 Reg ION Measure nent maar Area 6120 813 Max 4095 000 3227 000 hetrformation of 1154 IS Range 3999 000 7186 000 calculated orr Re n I5tdDev 2175 309 W Dicnlay Table 7pos 10 Tpos 0 Lpos 0 enterX CenterY Area Perimeter Integration Average Max Min Range 5tdDev 3StdDew Integration Average Max i Range StdDev um um um um CHS1 CHS1 CHS1 CH51 CHS1 CHS1 CH51 CHS CHS CHS CHS CHS 57 171 49 448 3120 625 241 490 5473264 000 1107 926 4095 000 95 000 4000 000 710 261 2130 783 2952481 000 658 076 3590000 28 3562 000 522 518 112 522 53 402 1470 184 194 764 0620457 000 1301 724 4095 000 97 000 3998 000 883 602 2650807 1837013 000 T58 280 3468 000 23 000 3440 000 561 877 51 900 87 103 3274688 213 15 2573667 000 1003410 4095 000 94 000 4001 000 700 397 2401 192 D839166 000 569 504 3415 000 3362 000 443 623 30 180 111 524 1732 438 211 246 1386227 000 879 166 3836 00 83 000 3753 000 657 656 1972 967 7680740 000 645 072 3300 00 25000 3355 000 523 061 150 780 TAT 6120 013 313 256 1070546 000 1244509 4095 000 96 000 3999 000 125 103 2175 309 4771708 000 559 277 3227 00 H 3186 000 409 334 5 The information of all ROls Count 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Average 90 511 76 240 7145 550 24
73. d TimeSscan Interval 0 sec Num 10 kake 12 1 Click Clip scan button and enclose a interesting region s image on the whole image 2 pixel setting The standard pixel is 512 x 512 3 Zoom Setting tton Fress XY Repeat to scan and set zoom value paul Hagens amp PI Ls mN E Above image is zoomed From 1x to 2 Scan speed and pixel resolution remain even zoom value is changed 4 Click a Zoom scan and be able to enclose an interesting regions on the whole image Press XYRepeat to scan after enclosing the area Egon 7 Es Scan speed and pixel resolution remain even zoom value is changed 12 Complement of adjusting the image ial 3 PanX Y y i E ln Ej Be able to move the field of view to set Pan X Y without stage action 6 Rotation O PanX Y and Rotation reset button Be able to rotate the whole image Auto Click Auto button to acquire Optimized Conforcal aperture Conforcal aperture change conforcal aperture to larger diameter for dim fluorescence image then be able to get the more bright image But Z axis resolution gets worse g 8 Laser Intensity More Laser _ intensity is increase more bright T 458 ll 5 0 Image IS i 488 ll 5 0 k TARIS 515 al 5 0 More increase laser intensity is more 543 il 10 0 discoloration image is m 633 alll 5 0 viv iY Y le 9
74. displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software beAcquisitionControl gt KY EMemoi File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which 0 cannot be opened separately from each other he Image j A OIB format Creates the OIF format files in a single file which is convenient for migration and other 4 E operations D 18 18 Image Acquisition Four Stain on XY Image EE Acquisition of 4 stain images XY fluorescence image only EE Sample Four stain of Blue fluorescence dye DAPI green fluorescence dye Alexa488 and red fluorescence dye Rhodamine far red fluorescence dye Cyd This is the procedure to acquire images through Virtual Channel scan 1 Y Virtual channel Scan Select Virtual channel scan on the DyeList and then Virtual Channel Controller is automatically turned on 2 select a number of Virtual Channel from Number of phase used Setup Dyes Acridine Orange A Alexa Fluor 405 Alexa Fluor 4881473 E Alexa Fluor 488488 Fluor 546 DyeList iv Virtual Channel Scan Number of phase used Aloxa Fluor 555 Selected Dyes Alexa Fluor 568 Phaset Jj DAPI we F Assign Dye Manually F Alexa Fluor 488
75. e Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software 15 15 Image Acquisition Double Stain on XY Image EE Acquisition of a single image XY plane fluorescence image only Em sample Double stain of green fluorescence dye Alexa 488 and red fluorescence dye Alexa 546 Sequential scan Line Sequential is introduced here seen 1 Click on the DyeList button On the Depth Time Bleach start stop by Key DyeList panel double click on a I fluorescence reagent to be used for A observation a aw 2 Check Sequential and select Line Filter Mode Kalman ol E Analog Int C Photon Cnt 3 Adjust the green Alexa Fluor 488 image and the red Alexa Fluor 546 image Group 1 Group 2 Group 3 Group 4 4 Press the XY button to acquire an image 5 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software The image is acquired EMemol File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operatio
76. e dye derive the _ fluorescence spectrum of the dye and obtain an unmixed image based on the fluorescence spectrum Sample Double stain of green fluorescence dye Alexa Fluor 488 and green fluorescence dye YOYO 1 T EJ a j oo pe man C3 TT Ta 0 El Am De al Pro Deco 0 0 ROI ListZ 1 T 1 Image Al488 yoyo1 2 xml pereme is Spectral Profile 32 Close 8 Open an XYL image file with both Alexa Fluor 488 and YOYO1 applied 9 From Processing on the menu bar select Spectral Deconvolution 10 Double click on Alexa Fluor 488 and YOYO1 which have been registered in the database of fluorescence spectrums 11 Check that the Processing Type is set to Normal and click on Execute 12 An unmixed image is obtained 32 Image Analysis Unmixing lil When only the number of types of fluorescence dyes is known Blind Unmixing From an XYL image where fluorescence dyes with similar fluorescence spectrums are present together obtain an unmixed image based on only the number of types of fluorescence dyes 2D View Al488 yoyo a ES f DAE Lama z sa a H m gt max C2 Wiz Ta 29 E 1 4 E e Al4B8 yoyo1 2 xml Bpectral Profile Unmixed image 33 Open an XYL image file
77. e for making merge image Between fluorescent image and focused DIC image eye 1 co 1 Open fluorescent image and DIC image File Device Display Live FRET ogee A e w 2 BB 8i Fiter setting alysis Tools Windo Ml Acquisition Setting Filter E Mode Threshold O2 Image Calculation 2 S e e ct lt lt Fast 10 0us Pixel Sk Correcting Pixel Gaps y a a MES Correc ting Z Gaps Aspect Ratio 1 1 4 W Spectral Deconvolution xs Se es IE Edit Experiment oo a Edit experiment from Processing Edit Experiment 3 Click Edit Channels and select fluorescent image file at select DIC oo rs image at Source Image Image Info X 512 Y 512 Axis Range Single Slice v4 Image Info Check Image Info M 1 2 to make the merge file If all channels are checked all El Edit Experiment E e Edit Type Channels are reflected in the new merged file Edit Channels Append Series Extract Series Append Files Destination Image lonlyfluorescence oib vr Image Info Ip A 4 Click Merge CH and then Source image Image Info Sarai ageso sis the fluorescent image and the DIC A image are merged as the new file Cancel Merge CH Z se X 512 512 Axis Range Single Slice 9 Merged image between the 5 fluorescent image and the DIC image 24 Image Acquisition Single Stain on XYZT Image This is avail
78. e with both Alexa Fluor 488 and YOYO1 applied 2 Enclose a point dyed with Alexa Fluor 488 only and a point dyed with YOYO1 only 3 From Processing on the menu bar select Spectral Deconvolution 4 Double click on ROI and ROI2 5 Check that the Processing Type is set to Normal and click on Execute 6 An unmixed image is obtained Indicates channel assignments of unmixed images Introductory Notes Name Color Calculate S WARS B gqi2z 1 T MS U K K Unmixed image 30 30 Image Analysis Unmixing When each fluorescence dye point is clear sample single stain of green fluorescence dye GFP and auto fluorescence from cell 1 Open the XYL image GFP auto fluorescence hia 2 Enclose a point dyed with GFP only 2 and a point dyed with ROI 1 auto fluorescence only 3 From Processing on the menu bar select Spectral Deconvolution 4 Double click on ROI1 GFP and ROI2 Auto fluorescence 9 Check that the Processing Type is set to Normal and click on Execute 6 An unmixing image is obtained Green color is GFP Gray color is Auto fluorescence 31 zi Image Analysis Unmixing ll When a control sample is used From an XYL image with a single type of fluorescenc
79. eagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click on the Apply button The DyeList panel can be closed by using the Close button Image Acquisition Single Stain on XY Image El ImageAcquisitionControl Focus x2 Ez Focus x4 XY Repeat PE 4 Press XY Repeat button click to get image SEIR pelhe e Continuous scan mode 9 Focus to the specimen CB 199 Filter Mode M Kalman tp 6 Adjust the green FITC image M Sequential 0 Adjust sensitivity of HV and reduce noise by offset 7 Press keyboard Ctrl H key Optimized PMT adjustment brightness intensity 2 color between white and black Maximum intensity is 4095 12bit if intensity is over4095 color is changed to red saturation HABA tont Basically Gain value is 1 Size 51 2x51 2 xy 498 424 Int75 Zoom 100 10 10 Image Acquisition Single Stain on XY Image E AcquisitionSetting 8 Select AutoHV and then select ScanSpeed lt lt Fast 2 0 Pixel Slow gt gt pl autom I As the scan speed becomes slower noise EE can be removed while maintaining the A E ABE current brightness e P Efc a ain se ai el ai el S am JE 9 Press the Stop button hed EN Stop l R E a Ea k E z 2 E Laser ee y A to stop scan n ng asg 5 0 1 543 10
80. erted into a BMP format 1 Right click on the image 2 Select Save Display and save the image with a new name an AVI format 1 Right click on the image 2 Select Save as AVI and save the image with a new name 38 Image Analysis Rotating a Three dimensional animation To save a rotation file as an animated image create three 5 6 dimensional images according to the following procedure For example try to rotate an image by 180 degrees ice ove 4D Angle rotation stat 180 2 eng 180 lt lt lt a 5 Click on the More button EE bs E Volume Single 10 100 100 6 Click on the Angle rotation tab 7 Select the rotation axis Animation Slice move 40 Angle rotatig 8 Enter the rotation angle a Start Angle to start rotation Start 180 2 Eng 180 lt End Angle to stop rotation Frame s 5 interval 30 Frame s Rotation speed _ Interval Degrees to be rotated Sree Animation is AMI File Q at a time 9 Select AVI File and click on Create Save in Image O e Ed 1 0 O aaa tiff frames E koumura XY xml data Smkbmp bmp i a O Kidney2 bmp frames 5 Livelmage_ 3 xml data C mkmk tiff Fre 1 0 y E n te r a fi e n a m e a n d cl l ck O n S ave E Kidney XYZ tiff frames 5 Livelmage_ 10 bmp frames al koumura pol Kidney bmp frames 5 Livelmage_ 11 tiff frames Kidney tiff Frames A Livelmage_ 11 x
81. ey2 bmp frames 5 Livelmage_ 3 xml data C mkmk tiff Fre 1 0 y E n te r a fi e n a m e a n d cl l ck O n S ave E Kidney XYZ tiff frames 5 Livelmage_ 10 bmp frames al koumura pol Kidney bmp frames 5 Livelmage_ 11 tiff frames Kidney tiff Frames A Livelmage_ 11 xml data O koumura xY tiff Frames 5 mk tiff Frames File name Save as type AVI Files avi l Cancel 34 2D Image Analysis Edit the image color and contrast 1 Click LUT and then LUT table appears below TILE Ch Size 640x640 x y 629 4 Into Zoom Auto 46 7 or f e 1 00 E cans 000 MG sees KEEN 2 Contrast 0 00 Yelow O Hlo H Save As Load Reset Close 2 Edit contrast to drag to left or right side and another way to edit contrast is entering value on of Image Is edited Max and Min Max4095 MinQ According to gX Min value up be able to reduce noise of the image E 2D View Image0006 NM Bec im E oo PJ e 4 m5 SORSe S 3 Min and Max value are changed and contrast Edit each Ch 0 SS DU Orange Min 76 Max 3733 Auto a Te Gamma 1 00 A Fall gt ARA Spec B CG FITC Spec3 Intensity 0 00 cray Contrast 0 00 Yelow Hito CO I or Save As Load Reset Close 4 To click another color be able to Edit a color Above example Change Green to Red
82. ff frames Kidney tiff Frames A Livelmage_ 11 xml data O koumura xY tiff Frames 5 mk tiff Frames File name Save as type AVI Files avi l Cancel 41 2D Image Analysis Edit the image color and contrast 1 Click LUT and then LUT table appears below TILE Ch Size 640x640 x y 629 4 Into Zoom Auto 46 26 ET S rea 1 00 Cyan Fall E oo Specs A E o 2 Contrast 0 00 Yellow Hi Lo A Intensity Save As Load Reset Close 2 Edit contrast to drag to left or right side and another way to 3 Min and Max value are changed and contrast edit contrast is entering value on of image_is edited a GRA According t yet Min value up be able to reduce noise of the imaylax and Min Max4095 Min0 2D View Image0006 NM Bec PT A as PY 4 me Da REA YZ Edit each Ch 0 SS DU Orange Min 76 Max 3733 Auto a Te Gamma 1 00 A Fall gt ARA Spec B CG FITC Spec3 Intensity 0 00 cray Contrast 0 00 Yelow Hito CO l or Save As Load Reset Close 4 4 To click another color be able to Edit a color Above example Change Green to Red to click Red 42 42 The im ontrast TILE Ch Size 640x640 xy 575 362 Int 125 zoom Auto 46 The i 2D Image Analysis the image of Z section m 2D View level NGadBarPb oib
83. for a sample that has two unknown types of fluorescence dyes From Processing on the menu bar select Spectral Deconvolution Click on two Calculate check boxes Click on three boxes when three types of fluorescence dyes are used Check that Processing Type is set to Blind and click on Execute An unmixed image is obtained 33 34 Reload the image conditions 1 Open the file and click 14 2 21 135 Int 134 93 Zoom Auto 206 E 2D Control Panel ERE Al E 2 _ a AS 2 clio EP E ImageAcquisitionControl pA Focus x2 gt amp gt 0 gt Focus x4 xy Repeat xy izil Stop Depth Time 3 The conditions HV Offset CA and so on are reloaded F Sequential 34 Click on the Pa button to switch the display TEXT Various kinds of ROls Grid Arrow Scale bar Point Color bar 35 Overview of the 2D Operation Panel 1 Ch1 display 2 Ch2 display Display switching Frame advance Enlarge 4 4 display Adjust to the Animation window size Advance speed m 2D jew SLY SLL Ami oR MQ z D DE MAX 029 Tic TT 9 4 Oe L T 2 3D formulation Projection switching Fix the end section Fix the start section 2 21 135 Int 134 93 Zoom Auto 206 Image Analysis Opening a File 1 Double click on a file to be opened from Explorer
84. for acquisition of XYT or XT image Scan buttons Select XYZ XYT or XYL Adjustment of each channel Confocal aperture Light intensity adjustment for halogen bulb Kalman Image file thumbnail Display of files in the memory Image Acquisition Single Stain on XY Image EE Acquisition of a single image XY plane fluorescence image only MIN Sample Single stain of green fluorescence dye FITC Fitter Mode M Kalman Line Frame 2 ES Analog int Photon Cnt Sequential E ImageAcquisitionControl Focus x2 a Focusx4 xy Repeat xy i 71 v Laser 11633 5 0 Filter Mode Kalman Line Frame 2 4 ie Analogint Photon Cnt Sequential 1 Click on the FV10 ASW software button gt to close the fluorescence lamp shutter Alternatively click on the gt button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click on the Apply button The DyeList panel can be closed by using the Close button Image Acquisition Single Stain on XY Image El ImageAcquisitionControl Focus x2 Ez A Focus x4 XY Repeat PE SEIR pelhe n e 199 F
85. gle Stain DIC on XY Image WE Acquisition of a single image XY plane fluorescence image and differential interference contrast image MMM Sample Green fluorescence dye FITC and differential interference contrast image ty Filter Mode Sequential 1 Click on the FV10 ASW software button z to close the fluorescence lamp shutter Alternatively click on the l button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click on the Apply button The DyeList panel can be closed by using the Close button 4 4 Check TD1 488 Filter Mode gt M Kalman Line Fame 2 j 6 Analogint Photon Cnt F Sequential Display after DyeApply is carried out jalog Int Photon Cnt ZZ 22 Image Acquisition Single Stain DIC on XY Image 5 Press the XY Repeat button to start eee scanning 6 Adjust the green FITC image and the differential interference contrast image F kaiman Line Frame fp ie Analogint Photon Cnt 7 Press the Stop button to stop scanning 8 Press the XY button to acquire an A a a image
86. he image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software HVemoN File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations iA 21 21 Image Acquisition Single Stain DIC on XY Image WE Acquisition of a single image XY plane fluorescence image and differential interference contrast image MMM sample Green fluorescence dye FITC and differential interference contrast image ty Filter Mode Sequential 1 Click on the FV10 ASW software button z to close the fluorescence lamp shutter Alternatively click on the l button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click on the Apply button The DyeList panel can be closed by using the Close button 4 4 Check TD1 488 Filter Mode gt
87. hold the button to rotate the image around the Y axis Press it again to stop rotation Press and hold the button to rotate the image around the Z axis Press it again to stop rotation Image Control volume Single 10 100 100 39 39 Image Analysis Saving an Image Pil ie nla Bahl 4 Right click on the Image Icon displayed on the Data Manager and select Export 2 Set Save as type to TIFF 3 Set Output Format to RGB Color 4 Save the image BMP and JPEG formats are also selectable Convert a merge image of an XY ers or XYZ image into a TIFF format 5 Livelmage_ 10 bmp frames A Livelmage_ 11 tiff frames 1 Right click on the Image Icon displayed on the Data Manager and select Export 2 Set Save as type to TIFF Set Output Format to Merge Channel a aaa sO none 9 highest other defau 4 Save the image BMP and JPEG formats are also selectable Quality 1 lowest 100 highest other default 70 Kkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkk ETA 1 f Convert an image with the scale _ _ bar inserted into a BMP format _ 2 1 Right click on the image 2 Select Save Display and save the E Full Screen Image i d 5 a image with a new name th Clipboard 2 POO NAE Y Save Display Save 4
88. ich cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations a 37 Image Analysis Inserting the Scale Bar E 2D View LILY POLLEN xml r b 1 1 z Click on the button 2 While left clicking the image drag and drop it at a certain point 2 e ROJO 3 1 gt NIA bb bb Y E o l the ive HR 3 While clicking the right or left handle move the mouse from side to side E 2D View LILY POLLEN xml Oax Change the text size aaa a D gt pi gt max 029 Tra Fag GA 0 color style etc 4 Select Scale Bar and then right click on Scale Bar to select Format A Setting eID NI hdi Pd iiini Cut ROI Ctrl X Copy ROI Ctrl C elt Delete ROI Del Max Size Color Bar Y Format Setting ROI Manager 5 Change the setting in this window as 0 required 38 38 Image Analysis Rotating a Three dimensional Image 1 Click on the 9 button for a 2D AA A AA View file name image 2 A 3D view Is created 30 l JES als alme slo al 3 Drag the mouse on the image to 2 3 observe it at a certain angle 2 21 135 Simple animation 4 Press and hold the 4 button to rotate the image around the X axis Press it again to stop rotation Press and
89. ick on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click Apply button The DyeList panel can be closed by using the Close button 14 Image Acquisition Double Stain on XY Image 4 Press the XY Repeat button to start scanning 5 Adjust the green Alexa Fluor 488 image and the red Alexa Fluor 546 Analogint Photon Cut image The image adjustment is outlined below For more information refer to Appendix 1 6 Press the Stop button to stop scanning and press XY repeat to acquire the image Refer to WMemoN WMemoN Scan buttons e Continuous scan XY Repeat Q Stop scan Rough scan Line skipped 7 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software 15 15 Image Acquisition Double Stain on XY Image EE Acquisition of a single image XY plane fluorescence image only Em sample Double stain of green fluorescence dye Alexa 488 and red fluorescence dye Alexa 546 Sequential scan Line Sequential is introduced here seen 1 Click on
90. ilter Mode M Kalman tp M Sequential 0 HABA tont Size 51 2x51 2 xy 498 424 Int75 Zoom 100 10 4 Press XY Repeat button click to get image e Continuous scan mode 9 Focus to the specimen 6 Adjust the green FITC image Adjust sensitivity of HV and reduce noise by offset 7 Press keyboard Ctrl H key Optimized PMT adjustment brightness intensity 2 color between white and black Maximum intensity is 4095 12bit if intensity is over4095 color is changed to red saturation Basically Gain value is 1 10 Image Acquisition Single Stain on XY Image E AcquisitionSetting 8 Select AutoHV and then select _ ScanSpeed a AO pato BE As the scan speed becomes slower noise apr oh P J can be removed while maintaining the sa Uc E current brightness 9 KJ Press the Stop button to stop scanning POE SEE peee Analog Int C Photon Cnt 10 gt Click on XY and 2D View Livelmage x is displayed an Tee ei on the window bar for the image al Soe thathas been acquired Image 11 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the Save as Type oib or oif file Memom format specifically for the FV10 ASW File formats specifically for the FV10 ASW
91. in the Light Pass our 3 Insert the DIC prism slider in the light pass Use this knob to adjust the differential interference contrast E 4 gt Click the button on Fluoview software 9 Focus to the specimen Filter Mode Kalman Line Frame g x Y x z aie pa E 2 E e Analog Int C Photon Cnt Sequential Overview of Operation Panel for Image Acquisition Scan mode Scan speed Number of pixels Zoom amp Pan Laser output adjustment Transmitted light observation visual observation Fluorescence observation visual observation DyeApply Optical path diagram TwinScanner setting Save acquisition conditions Load acquisition conditions Image display window 8 Y ImageAcquisitionControl E AcquisitionSetting Mode O amp N lt lt Fast 2 0usPixel Slow q gt Auto E P 2 0us L 105 800ms F 55 122s 46 9min Size Aspect Ratio le O AS C arbitrary x al pl 512 by gt 1 gt aj aari El _ panx A A Pany rE Ta Laser FT 458 dl Vv 488 l genie CHS CHS ail 491 pm End nm StepSizel 2 0 nm Num 51 Resolution 10 0 nm amp Microscope UAPO 40X 01 340 NA 1 35 BX A Start Go UN 0 37 um y yo _Go m lt 7 of ae M StepSize a um Op l Slices 7 Focus Handle On Escape X 0 194um pix Y 0 194um pix Z 0 284um slice TimeScan interval 0
92. isition Double Stain on XY Image 4 Press the XY Repeat button to start scanning 5 Adjust the green Alexa Fluor 488 image and the red Alexa Fluor 546 Analogint Photon Cut image The image adjustment is outlined below For more information refer to Appendix 1 6 Press the Stop button to stop scanning and press XY repeat to acquire the image Refer to WMemoN WMemoN Scan buttons e Continuous scan XY Repeat Q Stop scan Rough scan Line skipped 7 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software 15 15 Image Acquisition Double Stain on XY Image EE Acquisition of a single image XY plane fluorescence image only Em sample Double stain of green fluorescence dye Alexa 488 and red fluorescence dye Alexa 546 Sequential scan Line Sequential is introduced here seen 1 Click on the DyeList button On the Depth Time Bleach start stop by Key DyeList panel double click on a I fluorescence reagent to be used for A observation a aw 2 Check Sequential and select Line Filter Mode Kalman ol E Analog Int C Photon Cnt 3 Adjust the green Alexa Fluor 488 image and the red Alexa Fluor 546 image Group 1 Group 2 Group 3 Gro
93. isplay Live FRET ogee A e w 2 BB 8i Fiter setting alysis Tools Windo Ml Acquisition Setting Filter E Mode Threshold O2 Image Calculation 2 S e e ct lt lt Fast 10 0us Pixel Sk Correcting Pixel Gaps y a a MES Correc ting Z Gaps Aspect Ratio 1 1 4 W Spectral Deconvolution xs Se es IE Edit Experiment oo a Edit experiment from Processing Edit Experiment 3 Click Edit Channels and select fluorescent image file at select DIC oo rs image at Source Image Image Info X 512 Y 512 Axis Range Single Slice v4 Image Info Check Image Info 1 2 to make the merge file If all channels are checked all El Edit Experiment E e Edit Type Channels are reflected in the new merged file Edit Channels Append Series Extract Series Append Files Destination Image lonlyfluorescence oib vr Image Info Ip A 4 Click Merge CH and then Source image y 4 Image Info ra ageso sis the fluorescent image and the DIC A image are merged as the new file Cancel Merge CH Z se X 512 512 Axis Range Single Slice 9 Merged image between the 5 fluorescent image and the DIC image 24 25 Reload the image conditions 1 Open the file and click 14 2 21 135 Int 134 93 Zoom Auto 206 E 2D Control Panel ERE Al E 2 _ a AS 2 clio EP E ImageAcquisitionControl
94. ity Profile on each Z positions m 2D View level NCadBarP6 oib 3 Click lao to show Scale on Intensity Profile Profile H 1eyel NCadBarP6_oib b zaar due 4 According to click be able to show as equal scale of Profile window as 2Dimage 37 37 2D Image Analysis Measure DFA px AE i JUE A i 1 Enclose interesting regions by ROI Line on interesting positions by ROI 2 Click El measure Z101 Size 800x600 xy 621 12 Int 108 89 Zoom 100 B Region Measurement PTK XYZ oif 2 10 T 0 L 0 ROIS Image PTE YYZ oif Measure All ROIs vieasure All ROIS Image Info 4 According to clic y At ss AZ SN TY ey I Current Measure ROIHo 5 Statistics CHS CHS v U AUU JI O v O J Zpos 10 i Tpos 0 CenterX i 421878548 000 54771708 000 CenterY 19 732 Averag 1244 509 559 277 Reg ION Measure nent maar Area 6120 813 Max 4095 000 3227 000 hetrformation of 1154 IS Range 3999 000 7186 000 calculated orr Re n I5tdDev 2175 309 W Dicnlay Table 7pos 10 Tpos 0 Lpos 0 enterX CenterY Area Perimeter Integration Average Max Min Range 5tdDev 3StdDew Integration Average Max i Range StdDev um um um um CHS1 CHS1 CHS1 CH51 CHS1 CHS1 CH51 CHS CHS CHS CHS CHS 57 171 49 448 3120 625 241 490 5473264 000 1107 926 4095 000 95 000 4000 000 710 261 2130 783 2952481 000
95. j ii i fi i Ti 1 i il y E a y m 22 A Jf Fi j I lnieH alee sintzisls a 1 E _ _ A 4 llas ti gt a T Fi i 3 Ten e E m Ti i i i Ti l i d j i i fi Ly i i Jo ER a j e a ae a ee ee a ee a te a A a EE an Y Vagus ical MN ccc if i r i i yer er as af SS A n Pm f po i FF j laz i ze i al Fl E P ie re a Y F A aT 1 li s f F if if ii if ry A ii F F d PA dl F Pa e e rs on lal Pr E O i i oy fF fF fF fF Ff Y A A Sf A Of SF l AN i ao io i A je i if ae Po y cat if y IE NAT oy i ra i ra F E di Ti a A L E fee ees ee fe ee ee ee E ee ee es Gr dee O ee er dee ee i of FF ee 1 of ATAR fof F i y j ee P oe A i l li f e TF Ti Ti 1 j i i l A i F F r 1 m j Lt TOA A y i j I AN a r I E I I ri d F P n y ia fi ro F F A Y JN A E f py E 1 lj J E f EF ri f F F r _ FF al F i i E i j j j if E an i F i F r j i i k r A f F E ff a i f j i T rs on a A Ti f F me Le E pem a les ae Veni NNE F 1 I i j ER l F ioe j lf ff SE a e Ny a 5 7 yA EN j y y f ff y i a AID EF j a sali Y O E BR FI F A F j A F Ni AY LL ALAA SS f d i F j F F Z j r di Wo F d f j F id d on Fi A FIN F a NE de
96. le Stain on XY Image 4 Press the XY Repeat button to start scanning 5 Adjust the green Alexa Fluor 488 image and the red Alexa Fluor 546 Analogint Photon Cut image The image adjustment is outlined below For more information refer to Appendix 1 6 Press the Stop button to stop scanning and press XY repeat to acquire the image Refer to WMemoN WMemoN Scan buttons e Continuous scan XY Repeat Q Stop scan Rough scan Line skipped 7 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software 15 15 Image Acquisition Double Stain on XY Image EE Acquisition of a single image XY plane fluorescence image only Em sample Double stain of green fluorescence dye Alexa 488 and red fluorescence dye Alexa 546 Sequential scan Line Sequential is introduced here seen 1 Click on the DyeList button On the Depth Time Bleach start stop by Key DyeList panel double click on a I fluorescence reagent to be used for A observation a aw 2 Check Sequential and select Line Filter Mode Kalman ol E Analog Int C Photon Cnt 3 Adjust the green Alexa Fluor 488 image and the red Alexa Fluor 546 image Group 1 Group 2 Group 3 Group 4 4
97. leZDC AF during Time Series Scan m nd click ZDC setting 4 Click Set Offset to register auto focus position Note Have to use glass bottom dish below otherwise ZDC doesn t work 5 Set Interval and Num and then click XYZT to acquire the time series image Note In case of using ZDC for Time series Scan follow below limits Interval number is more than 60 sec Rest Time is more than 30 sec otherwise ZDC doesn t work If use TimeControler Time Series Scan is able to done even interval number is within 60sec and Rest Time is within 30sec 2 Reload the image conditions 1 Open the file and click i 2 21 135 Int 134 93 Zoom Auto 206 7D Control Panel ERE Al E 2 _ a AS 2 clio EP E ImageAcquisitionControl pA Focus x2 gt amp gt 0 gt Focus x4 xy Repeat xy izil Stop Depth Time 3 The conditions HV Offset CA and so on are reloaded F Sequential 2l Click on the Pa button to switch the display TEXT Various kinds of ROls Grid Arrow Scale bar Point Color bar 28 Overview of the 2D Operation Panel 1 Ch1 display 2 Ch2 display Display switching Frame advance Enlarge 4 4 display Adjust to the Animation window size Advance speed m 2D jew SLY SLL Ami oR MQ z D DE MAX 029
98. left side of slit Q and drag to the point which the highest brightness is achieved Note Move the slit position only while keeping the slit width at 20 nm Adjust the image on the highest brightness Press the Stop button to stop scanning 28 Image Acquisition Spectral Image on XYL Image a _ 10 10 Set the range of wavelength to be E TES 70 e aa acquired the slit width and the step Serta q amp gt eStart Start wavelength eEnd End wavelength eResolution Slit width eStepSize Step Select AutoHV and then select ScanSpeed As the scan speed becomes slower noise can be removed while maintaining the current brightness 1 12 Select Lambda 13 EC Press the XYZ button to Lambda De acquire an image 14 Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 29 29 Image Analysis Unmixing When each fluorescence dye point is clear From an XYL image where fluorescence dyes with similar fluorescence spectrums are present together derive the fluorescence spectrum for each fluorescence dye and obtain an unmixed image based on the fluorescence spectrums a Sudan a o a NN ve l T ey A and green fluorescence dye YOYO 1 Mm D e Al488 yoyo 4 KUA lanos lt a a jH _ p gt max Co Fre as 8 zi 1 Open an XYL image fil
99. lti Ar 458 nm 488 nm 514 nm OFF 3 3 HeNe G 543 nm OFF 4 Turn the mercury burner power OFF 39 39 OLYMPUS OLYMPUS CORPORATION Shinjuku Monolith 3 1 Nishi Shinjuku 2 chome Shinjuku ku Tokyo Japan OLYMPUS LIFE SCIENCE EUROPA GMBH Postfach 10 49 08 20034 Hamburg Germany OLYMPUS AMERICA INC 3500 Corporate Parkway P O Box 610 Center Valley PA 18034 0610 U S A OLYMPUS SINGAPORE PTE LTD 491B River Valley Road 12 01 04 Valley Point Office Tower Singapore 248373 OLYMPUS AUSTRALIA PTY LTD 31 Gilby Road Mount Waverley Victoria 3149 Australia OLYMPUS LATIN AMERICA INC 5301 Blue Lagoon Drive Suite 290 Miami FL 33126 U S A Printed in Japan 200810 1 1
100. mation file 34 2D Image Analysis Edit the image color and contrast 35 The image of Z secon 997889884 ns 36 Intensity Profile of each Z sections 3 Measure 38 Line Intensity Profile on the 2D image Histogram 39 Line Series Analysis Co localization 7777777777777777777777777777 40 TimeLapse Analysis 41 Closing the System 41 Filter Type Main Scanner Filter type scanner 440nm LD473nm LD559 nn Sgnm Ar488nm Ar515nm Dye List FV1000D Lasers are available below HeNe G 543nm f fi F j j ji j T i F i Y l i i ATREA ii int ane i F F G Pp a mo i F F ri t al E J r pul A F F Ti l j i f f f i i A i a F i j I l Ti F Pi a a r i 1 i 7 lz F L ee E ain ig i d j Ti FE f l Ti F F F d F d Ta k i g i j J F F a Ti ri ii ar d r o Ma gt un hl al E li i i oe F A I F F 1 l enn aaa dl Ma ad 000 li f f r _ y d i To m i i it i l Foa j i fr ss AY al I de a j fi f F pi F Fi Ey e LAs j i l i E i Fi a a F i j F j F i T i Ma i a Fl A L i Fj F j Y j A La S tt ame a Ae f
101. ml data O koumura xY tiff Frames 5 mk tiff Frames File name Save as type AVI Files avi l Cancel 39 2D Image Analysis Edit the image color and contrast 1 Click LUT and then LUT table appears below TILE Ch Size 640x640 x y 629 4 Into Zoom Auto 46 26 ae IC 1 00 E cans 000 MG sees KEEN gt 2 Contrast 0 00 Yelow Hilo H Save As Load Reset Close 2 Edit contrast to drag to left or right side and another way to dit contrast is entering value on 3 M d M lue are changed and contrast ON mel eg 9 Max and Min Max4095 MinQ According aget Min value up be able to reduce noise of the image SATA OIX IN mm ur wit ne el T a EME 3 n Edit each Ch The im ontrast Gamma r Fall TILE Ch Size 640x640 xy 575 362 Int 125 Zoom Auto 46 B Spec Pean E 0 00 Yellow HiLo The i color Save As Load Reset Close 4 To click another color be able to Edit a color Above example Change Green to Red to click Red 40 40 2D Image Analysis the image of Z section m 2D View level NGadBarPb oib 1 Click Z and select 29 again then Projection image is shown on 2D View after getting XYZ image 3013 5128051251 2 y 4 3 135 INEU J 14 oom Aut
102. n Escape X 0 194um pix Y 0 194um pix Z 0 284um slice TimeScan interval 0 sec Num 100 ial ambda Depth y en Bleach Stop Time Filter Mode Kalman fine Frame 2 e Analogint Photon Cnt M Sequential ea e O Live V iew ma ar gt Focus Objective lens Time Interval amp Time Number for acquisition of XYT or XT image Scan buttons Select XYZ XYT or XYL Adjustment of each channel Confocal aperture Light intensity adjustment for halogen bulb Kalman Image file thumbnail Display of files in the memory Image Acquisition Single Stain on XY Image EE Acquisition of a single image XY plane fluorescence image only MIN Sample Single stain of green fluorescence dye FITC Fitter Mode M Kalman Line Frame 2 ES Analog int Photon Cnt Sequential E ImageAcquisitionControl Focus x2 a Focusx4 xy Repeat xy i 71 v Laser 11633 5 0 Filter Mode Kalman Line Frame 2 4 ie Analogint Photon Cnt Sequential 1 Click on the FV10 ASW software button gt to close the fluorescence lamp shutter Alternatively click on the gt button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selecti
103. nning Enter StepSize Slice the recommended value can be referred to by using the Op button and check the check box 17 Image Acquisition Double Stain on XYZ Image QQ 9 Select AutoHV and then select ScanSpeed 12 5us5 Pix Slow gt gt a JE pl AutoHV P 12 FF L 7 525ms F 3 9271s 13 95 10 Select Depth 11 Press the XYZ button to acquire an image 12 Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 13 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software beAcquisitionControl KY EMemoi File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separatel from each other he Image re OIB format Creates the OIF format files in a single file 13 R which is convenient for migration and other 4 E operations D 18 18 Image Acquisition Four Stain on XY Image EE Acquisition of 4 stain images XY fluorescence image only EE Sample Four stain of Blue fluorescence dye DAPI green fluorescence dye Alexa488 and red fluorescence dye Rhodamine far red fluorescence dye
104. ns 16 py 16 Image Acquisition Double Stain on XYZ Image WE Acquisition of 3D images XYZ fluorescence image only EE sample Double stain of green fluorescence dye FITC and red fluorescence dye Rhodamine This is the procedure to acquire images through Line Sequential scanning 1 Click on the 4 Take steps 1 to 7 described on pages 13 and 14 Press the XY Repeat button to start scanning and A buttons to shift the focal point Refer to WMemoN When the sample upper limit is displayed on the image accept it using the Set button Click on the and buttons to shift the focal point Refer to WMemoN When the sample lower limit is displayed on the image accept it using the Set button Press the Stop button to stop scanning Enter StepSize Slice the recommended value can be referred to by using the Op button and check the check box 17 Image Acquisition Double Stain on XYZ Image MA 1 9 Select AutoHV and then select ScanSpeed 12 5uS Pix Slow gt gt a JE pl AutoHV P 12 FF L 7 525ms F 3 9271s 13 95 10 Select Depth 11 Press the XYZ button to acquire an image 12 Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 13 Saving the image o o Right click on the Image Icon
105. ntensity Profile 4 According to click a be able to show as equal scale of Profile window as 2Dimage 44 2D Image Analysis Measure DFA px AE i JUE A i 1 Enclose interesting regions by ROI Line on interesting positions by ROI 2 Click El measure Z101 Size 800x600 xy 621 12 Int 108 89 Zoom 100 B Region Measurement PTK XYZ oif 2 10 T 0 L 0 ROIS Image PTE YYZ oif Measure All ROIs vieasure All ROIS J e n Image Info 4 According to clic AA ala L EES DN A Leal a Current Measure ROIMo 5 Statistics CHS1 CHS F CALIU JI O v ATS d U Zpos 10 i Tpos 0 CenterX F 121878548 000 54771708 000 CenterY 19 732 Averag 1244 509 559 277 Reg ION Measure nent maar Area 6120 813 Max 4095 000 3227 000 hetrformation of 1154 IS Range 3999 000 7186 000 calculated orr Re n I5tdDev 2175 309 W Dicnlay Table 7pos 10 Tpos 0 Lpos 0 enterX CenterY Area Perimeter Integration Average Max Min Range 5tdDev 3StdDew Integration Average Max i Range StdDev um um um um CHS1 CHS1 CHS1 CH51 CHS1 CHS1 CH51 CHS CHS CHS CHS CHS 57 171 49 448 3120 625 241 490 5473264 000 1107 926 4095 000 95 000 4000 000 710 261 2130 783 2952481 000 658 076 3590000 28 3562 000 522 518 112 522 53 402 1470 184 194 764 0620457 000 1301 724 4095 000 97 000 3998 000 883 602 2650807 1837013 000
106. ocus x2 Focus x4 xy Repeat xY Stop Lambda Depth Time VESES M S s 2 s Mexa Fluor 4891973 HV_ Gain Offset HV Gain Offset HV_ Gain Off FAV Gain Offset a a Laj a a ENS a Lamp d IE AE 9138 BP Ole O 665 1 1 9 0 V xX v f Laser 559 y 4 0 488 v 12 0 Filter Mode Kalman E5 Analogint Photon Cnt M Sequential BEM Acquistion parameters have been loaded A E Virtual Channel C introller Phase 2 5 Start Open Save As lt El image Acquisition Control X r i i Lambda Depth Time PES gt dos alla o Tas EA EA HV Gain Offset HV_ Gain Offset HV Gain Offset A Lamp Ca a a Zanj es 24 2 L2 Lol v v v v v bi ae 10 1 1 339 1 38 200um 9 0V y i x v x Laser La Laser Laser 2 0 559 4 0 488 v 12 0 puto Filter Mode M Kalman g Analogint Photon Cnt Sequential 100 Acquistion parameters have been loaded Adjust the image at each phases Phase Phase2 20 4 Select Phase1 DAPI Alexa488 RhodaminRed are registered on ImageAquisitionControl Slit and Filter DM are automatically set for DAPI Alexa488 or PhodaminRed 5 Select Phase2 Cy5 is registered on ImageAquisitionControl Slit and Filter DM are automatically set for Cy5 a Adjust the image to click gt XY Repeat a
107. ogen bulb shutter Filter Mode Kalman E Analog Int C Photon Cnt M Sequential 2 Click on the gt button to view the optical path diagram 3 3 Make settings as shown below LightPathtDyes Trans Lamp T ASU i TD M 25 Specimen Ye a Select Mirror XA N BF 492 _ 537 565 665 nm i nm nm BA650IF T DM405 488 543 None HS1 FHS2 150 1408 30 um Dye iv Dye CA x Y atic Static2 Select BS20 80 or Select CHS1 only DM405 488 27 Za Image Acquisition Spectral Image on XYL Image El ImageAcquisitionControl Pa Focus x2 Focus x4 XY Repeat xy dos pella e ela e o pS E SS ee J Nur JE m ee ea S gt gt v gt v v v v gt v k rey 680 1 0 588 1 0 613 1 5 142 1 0 150um v 5 v x v x v X Laser Laser Laser Laser 488 v 5 0 543 v 26 0 r 633 v 5 0 488 v 5 0 Auto g Filter Mode M Kalman co f Analog Int Photon Cnt Gain Offset HV_ Gain Offset HV_ Gain Offset HV Gain Offset ja ad Led 4 2 ed ed E 24 224 ed E M Sequential 0 e A 400rnm 500mm G00ntm TOO S00nm 28 Click on th PF button and the Spectral Setting window appears Set the slit width for CHS1 to 20 nm for example Press the XY Repeat button to start scanning While observing the image Click the
108. on and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click on the Apply button The DyeList panel can be closed by using the Close button Image Acquisition Single Stain on XY Image n 4 Press XY Repeat button click to El ImageAcquisitionControl pA Focus x2 Ez lt gt Focus x4 xY Repeat Stop L g et m ag e Ea i A E irs n oa gt Continuous scan mode Te 9 Focus to the specimen auto E Holman co Es pp Analog Int Photon Cnt M Sequential 6 Adjust the green FITC image 0 reduce noise by offset _ 7 Press keyboard Ctrl H key Optimized PMT adjustment brightness intensity 2 color between white and black Maximum intensity is 4095 1 2bit if intensity is over 4095 color is changed to red saturation HABA tont Basically Gain value is 1 Size 51 2x51 2 xy 498 424 Int75 Zoom 100 10 10 Image Acquisition Single Stain on XY Image E AcquisitionSetting Slow gt gt amonv J 2 005 Pixel lt lt Fast Size Aspect Ratio x al f 1 1 f 4 3 G b 512 by ESS do BE o o ME HV_ Gain Offset Lagala v v v z v v v v af v 746 1 0 650 1 0 650 1 v x x v x Laser Laser Laser Laser 5 0 543 10 0 533 5
109. or 4 Yo m 2D View ivel NGadBarP6_oib Selec lt a ES fe gt b max C29 Fig aa Z e Ae AE BR Background Int o0 ma EJ 2 Click and select Ei 3 The images of Z section is shown on X axis and Y axis A RE oN According to Move to left or right side on X axis and to move to ups and down on Y axis be able to show image of Z section each position E 2D View level NGadBarPt_oib 4 The image of Z section on Y axis 5 The image of Z section on X axis 41 2D Image Analysis Intensity Profile of each Z sections 1 Click lt and then 3013 S126191 2X51 2 y 4 43 13 INEU J 14 oom Autor 4 Yo Automatic setting Ch Ch V Manual setting Column ich Z Int 12 Row ich T int L Int a O Multi Plane Wiew Profile 2 Click md Profile and then Intensity Profile of each Z sections is shown on the X and Y axis Need Projection and Merge in Tile Mode To move to Z position be able to show Intensity Profile on each Z positions E 2D View level NCadBar P6 oib 3 Click lao to show Scale on Intensity Profile Profile H 1eyel NCadBarP6_oib b zaar due 4 According to click be able to show as equal scale of Profile window as 2Dimage 42 42 2D Image Analysis Measure ox Fo o ua Ca A Background Int 0 O 3U 1
110. orescence dye Alexa Fluor 488 and green fluorescence dye YOYO 1 T EJ a j oo pe man C3 TT Ta 0 El Am De al Pro Deco 0 0 ROI ListZ 1 T 1 Image Al488 yoyo1 2 xml pereme is Spectral Profile 30 Close 8 Open an XYL image file with both Alexa Fluor 488 and YOYO1 applied 9 From Processing on the menu bar select Spectral Deconvolution 10 Double click on Alexa Fluor 488 and YOYO1 which have been registered in the database of fluorescence spectrums 11 Check that the Processing Type is set to Normal and click on Execute 12 An unmixed image is obtained 30 Image Analysis Unmixing lil When only the number of types of fluorescence dyes is known Blind Unmixing From an XYL image where fluorescence dyes with similar fluorescence spectrums are present together obtain an unmixed image based on only the number of types of fluorescence dyes 2D View Al488 yoyo a ES f DAE Lama z sa a H m gt max C2 Wiz Ta 29 E 1 4 E e Al4B8 yoyo1 2 xml Bpectral Profile Unmixed image 31 Open an XYL image file for a sample that has two unknown types of fluorescence dyes From Processing on the menu bar select Spectral Deconvolution Click on two Calculate che
111. ose red color X Y axis Co localization result between 2ch is changed Information of Co localization is listed under the scatter plot 47 2D image Analysis Series Analysis TimeLapse E 2D View FRET XYT oif DER maoan a J gt p nar 23 Fr 9 A Py olla ae Background Int po 3 l 1 Enclose interesting regions by ROI 2 Click Series Analysis 3 Series Analysis graph is shown below Y axis shows intensity X axis shows time and then be able to see time series reaction each ROIs E Series Analysis FRET XYT oif Image FRET XVT of AA image info Total 2 i eee eee ate Ch H Axis ra 0000 15000 20 000 Time me 48 Closing the System E OLYMPUS FLUOVIEW 1 Exit the FV10 ASW software by z Display Processing Analys 1 selecting File Exit Open 7 Close hd Save Save s i Property 2 Exit the Windows Logout 1 Select Start Shut Down 2 On the Shut Down Window select Shut Down and click on OK eos 2 116ms 3 Turn the laser OFF Turn the key switch to the OFF position 3 1 LD559nm OFF 3 2 Multi Ar 458 nm 488 nm 514 nm OFF 3 3 HeNe G 543 nm OFF 4 Turn the mercury burner power OFF 49 49 OLYMPUS Laser Conforcal Scanning Microscope FV1000D Filter Type invertedMicroscpel X81 Operation Manual 2 Content
112. perture change conforcal aperture to larger diameter for dim fluorescence image then be able to get the more bright image But Z axis resolution gets worse 8 Laser Intensity More Laser ER 8 intensity is increase more bright 458 ll P 5 0 image IS e Dia E pe More increase laser intensity is more 543 fl pel 10 0 discoloration image is 633 ll b 5 0 9 Kalman accumulation Image acquisition is repeated to the 9 specified number of times to provide Filter Mode an averaged image Consequently Y Kalman noise is averaged and roughness on the whole image is reduced Advantage The speed of each scan is fast Disadvantage Some blur occurs due to averaging of images 13 13 Image Acquisition Double Stain on XY Image WE Acquisition of a single image XY plane fluorescence image only MIN sample Double stain of green fluorescence dye Alexa 488 and red fluorescence dye Alexa 546 Simultaneous scan q Filter Mode M Kaman Line M Sequential Focus x2 lt gt L Focus x4 XY Repeat 108um Filter Mode M Kalman Line Frame 2 a Analog Int Photon Cnt M Sequential o ry 3 A 7 A AE 14 Display after DyeApply is carried out Click on the FV10 ASW software button to close the fluorescence lamp shutter Alternatively click on the PA button to close the halogen bulb shutter Cl
113. r DAPI Alexa488 PhodaminRed exa Fluor 4981473 2 HV_ Gain Offse e FIV_ Gain Offset Lad at Ea ee ee i ae 100 Acquistion parameters have been loaded A EM Virtual Channel C introller Phase 2 5 Start Image Acquisition Control PESE HV_ Gain Offset Laj_ 10 y e Laser La 2 0 Filter Mode M Kaman M Sequential Adjust the image at each phases Phase1 20 Lambda Depth x ni 1 1 9 0 V 4 0 lla88 v 12 0 j2 4 Analog Int C Photon Cnt 141 7J be 5 Select Phase2 Cy5 is registered on Open Save As ImageAquisitionControl r Auto n n a Slit and Filter DM are automatically set for Cy5 Pe ee SY pr le TT ll 1 J sig l i 200 um 9 0 V Analogint Photon Cnt 100 Acquistion parameters have been loaded a Adjust the image to click gt XY Repeat at each phases Phase2 If acquire XYZ image be able to decide upper limit and bottom limit slices step size of Z axis at both phases 20 Image Acquisition Four Stain on XY Image M Virtual Channel Co u Phase fi E 7 Click 2 on Virtual Channel Controller to acquire the image Be able to start at each Phase 8 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib o
114. r oif file format specifically for the FV10 ASW software HVemoN File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations iA 21 21 Image Acquisition Single Stain DIC on XY Image WE Acquisition of a single image XY plane fluorescence image and differential interference contrast image MMM Sample Green fluorescence dye FITC and differential interference contrast image ty Filter Mode Sequential 1 Click on the FV10 ASW software button z to close the fluorescence lamp shutter Alternatively click on the l button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the Assign Dyes window and take step 2 again Click on the Apply button The DyeList panel can be closed by using the Close button 4 4 Check TD1 488 Filter Mode gt M Kalman Line Fame 2 j 6 Analogint Photon Cnt F Sequential Display after DyeApply is carried out
115. rescent image file at Destination Image select DIC Source Image only dic oib i m ag e at S O u rce m a g e Image Info X 512 Y 512 Axis Range Single Slice v4 Image Info Check Image Info M 1 2 to make the merge file If all channels are checked all El Edit Experiment E e Edit Type Channels are reflected in the new merged file Edit Channels Append Series Extract Series Append Files Destination Image lonlyfluorescence oib vr Image Info Ip A 4 Click Merge CH and then Source image Image Info Sarai ageso sis the fluorescent image and the DIC A image are merged as the new file Cancel Merge CH Z se X 512 512 Axis Range Single Slice 9 Merged image between the 5 fluorescent image and the DIC image 24 Image Acquisition Spectral Image on XYL Image EE Acquisition of a spectral image XYL EE Sample Double stain of green fluorescence dye Alexa Fluor 488 and green fluorescence dye YOYO 1 El ImageAcquisitionControl Lambda 2 9 1 Click on the FV10 ASW software button gt to close the fluorescence lamp shutter Alternatively click on the button to close the halogen bulb shutter Filter Mode Kalman E Analog Int C Photon Cnt M Sequential 2 Click on the gt button to view the optical path diagram 3 3 Make settings as shown below LightPathtDyes Trans Lamp T ASU i TD M 25
116. rference Contrast Images MM 1 Select the Objective Lens 2 Insert the Polarizing Plate in the Light Pass 3 Insert the DIC prism slider in the light pass E 4 gt Click the button on Fluoview software Use this knob to adjust the differential interference contrast 9 Focus to the specimen Filter Mode Kalman Line Frame x 0 aser bl Of um FA e Analog Int C Photon Cnt M Sequential N Overview of Operation Panel for Image Acquisition Scan mode Scan speed Number of pixels Zoom amp Pan Laser output adjustment Transmitted light observation visual observation Fluorescence observation visual observation DyeApply Optical path diagram TwinScanner setting Save acquisition conditions Load acquisition conditions Image display window 8 Y ImageAcquisitionControl E AcquisitionSetting Mode O amp N lt lt Fast 2 0usPixel Slow q gt Auto E P 2 0us L 105 800ms F 55 122s 46 9min Size Aspect Ratio le O AS C arbitrary x al pl 512 by gt 1 gt aj aari El _ panx A A Pany rE Ta Laser FT 458 dl Vv 488 l genie CHS CHS ail 491 pm End nm StepSizel 2 0 nm Num 51 Resolution 10 0 nm amp Microscope UAPO 40X 01 340 NA 1 35 BX A Start Go UN 0 37 um y yo _Go m lt 7 of ae M StepSize a um Op l Slices 7 Focus Handle O
117. s System introduction a 3 FVY 10000 Laser DyeList Rss sass R eases 4 System Preparation 9 ar sse Sars seme reese ete oe SaaS ae SSeS ees 5 Visible Observation Observation of Fluorescence image 6 Observation of Differential Interference Contrast Images T Image Acquisition Overview of Operation Panel for Image Acquisition 8 Single Stain on XY Image 9 11 Complement of adjusting the image 12 13 DOUDIE Stall ON XY Mage a sess tess eae eee ett 14 15 Sequential scan Line Sequential 16 Double Stain On XAYZ IMAGe sssicocasopociriccc n caia tees ese se 17 18 FOUr Stain ONXA Mage 29s224sSe 46 eet Pees e 19 21 single Stain DIC on AY Image 4 lt s ses seseeeeesesceesescecs 22 23 Merge the image between fluorescent XY image and DIC image 24 Single Stain on XYZT Image ZDC XYZT image 25 26 Reload the image conditions 77777 TT TT ttre 21 Image Analysis Overview of the 2D Operation Panel Opening a file 28 Making 2D Z projection file Images 7777777777777777777777777777777 29 Saving a Z section image as 2D image 30 Inserting the Scale Bar 97777 T OTC TT TT TT rrr reer 31 Rotating a Three dimensional Image 39 Saving aM Mage esietenssiuealasssia std de 33 Saving Rotating a Three dimensional ani
118. s Press it again to stop rotation Image Control volume Single 10 100 100 37 37 Image Analysis Saving an Image HCE a Livelmage_ 3 xml data E mkmk tiff fre 5 Livelmage_ 10 bmp frames A Livelmage_ 11 tiff frames A Livelmage_ 11 xml data mk tiff Frames E mkbmp bmp frames Quality 1 lowest 100 highest other default 70 r PNG Compression Level O none 9 highest other defau Convert each channel ofa an XY l or XYZ image into a TIFF format 14 Right click on the Image Icon displayed on the Data Manager and select Export 2 Set Save as type to TIFF 3 Set Output Format to RGB color 4 Save the image BMP and JPEG formats are also selectable cea ge age Taa DE XYZ Image into a TIFF format 1 Right click on the Image Icon displayed on the Data Manager and select Export 2 Set Save as type to TIFF Set Output Format to Merge Channel 4 Save the image BMP and JPEG formats are also selectable mari A a V4 A VA O O 134 135 38 A jH gt p nax R Ta TA Full Screen Image th Clipboard Save Display Save Os AVI Select All ROIs Ctrl 4 Cancel Selection Flip Selection Delete All ROIs Paste ROI Ctrl y Show Profile vert Show Profile Hori Show Overlay 1 2 bar ins
119. s in a single file which is convenient for migration and other 4 E operations D 18 18 Image Acquisition Four Stain on XY Image EE Acquisition of 4 stain images XY fluorescence image only EE Sample Four stain of Blue fluorescence dye DAPI green fluorescence dye Alexa488 and red fluorescence dye Rhodamine far red fluorescence dye Cyd This is the procedure to acquire images through Virtual Channel scan 1 Y Virtual channel Scan Select Virtual channel scan on the DyeList and then Virtual Channel Controller is automatically turned on 2 select a number of Virtual Channel from Number of phase used Setup Dyes Acridine Orange A Alexa Fluor 405 Alexa Fluor 4881473 E Alexa Fluor 488488 Fluor 546 DyeList iv Virtual Channel Scan Number of phase used Alexa Fluor 555 Selected Dyes Alexa Fluor 568 Phase1 E DAPI ea 3 Select 4dyes from DyeList 4th dye Assign Dye Manually 7 Alexa Fluor 488 473 er is registered in the Phase 2 Phase2 PM cy5 DyeList El E he Virtual Channel Scan Number of phase used 2 C3 A Selected Dyes Phasel PB pari F Alexa Fluor 488 473 RodaminRed is able to be registered on Phase2 to drag 19 19 Image Acquisition Four Stain on XY Image ANA E Virtual Channel C ntroller Open Save As Phase A 4 Start lt image Acquisition Control x F
120. sec Num 100 ial ambda Depth y en Bleach Stop Time Filter Mode Kalman fine Frame 2 e Analogint Photon Cnt M Sequential ea e O Live V iew ma ar gt Focus Objective lens Time Interval amp Time Number for acquisition of XYT or XT image Scan buttons Select XYZ XYT or XYL Adjustment of each channel Confocal aperture Light intensity adjustment for halogen bulb Kalman Image file thumbnail Display of files in the memory Image Acquisition Single Stain on XY Image EE Acquisition of a single image XY plane fluorescence image only MIN Sample Single stain of green fluorescence dye FITC Fitter Mode M Kalman Line Frame 2 ES Analog int Photon Cnt Sequential E ImageAcquisitionControl Focus x2 a Focusx4 xy Repeat xy i 71 v Laser 11633 5 0 Filter Mode Kalman Line Frame 2 4 ie Analogint Photon Cnt Sequential 1 Click on the FV10 ASW software button gt to close the fluorescence lamp shutter Alternatively click on the gt button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a fluorescence reagent to be used for observation To cancel the selection and select a different reagent double click on the fluorescence dye listed on the
121. software EMemoi File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations Merge the images between fluorescent XY image and DIC image Edit different each files to the same file This is available for making merge image Between fluorescent image and focused DIC image eye 1 co 1 Open fluorescent image and DIC image File Device Display Live FRET ogee A e w 2 BB 8i Fiter setting alysis Tools Windo Ml Acquisition Setting Filter E Mode Threshold O2 Image Calculation 2 S e e ct lt lt Fast 10 0us Pixel Sk Correcting Pixel Gaps y a a MES Correc ting Z Gaps Aspect Ratio 1 1 4 W Spectral Deconvolution xs Se es IE Edit Experiment oo a Edit experiment from Processing Edit Experiment 3 Click Edit Channels and select fluorescent image file at select DIC oo rs image at Source Image Image Info X 512 Y 512 Axis Range Single Slice v4 Image Info Check Image Info M 1 2 to make the merge file If all channels are checked all El Edit Experiment E e Edit Type Channels are reflected in the new merged file Edit Channels Append Series Extract
122. t scanning and A buttons to shift the focal point Refer to WMemoN When the sample upper limit is displayed on the image accept it using the Set button Click on the and buttons to shift the focal point Refer to WMemoN When the sample lower limit is displayed on the image accept it using the Set button Press the Stop button to stop scanning Enter StepSize Slice the recommended value can be referred to by using the Op button and check the check box 17 Image Acquisition Double Stain on XYZ Image MA 1 9 Select AutoHV and then select ScanSpeed 12 5uS Pix Slow gt gt a JE pl AutoHV P 12 FF L 7 525ms F 3 9271s 13 95 10 Select Depth 11 Press the XYZ button to acquire an image 12 Click on SeriesDone and 2D View Livelmage x is displayed on the window bar for the image that has been acquired 13 Saving the image o o Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software beAcquisitionControl gt KY EMemoi File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format file
123. t each phases If acquire XYZ image be able to decide upper limit and bottom limit slices step size of Z axis at both phases 20 Image Acquisition Four Stain on XY Image M Virtual Channel Co u Phase fi E 7 Click 2 on Virtual Channel Controller to acquire the image Be able to start at each Phase 8 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software HVemoN File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations iA 21 21 Image Acquisition Single Stain DIC on XY Image WE Acquisition of a single image XY plane fluorescence image and differential interference contrast image MMM Sample Green fluorescence dye FITC and differential interference contrast image ty Filter Mode Sequential 1 Click on the FV10 ASW software button z to close the fluorescence lamp shutter Alternatively click on the l button to close the halogen bulb shutter Click on the DyeList button On the DyeList panel double click on a
124. the DyeList button On the Depth Time Bleach start stop by Key DyeList panel double click on a I fluorescence reagent to be used for A observation a aw 2 Check Sequential and select Line Filter Mode Kalman ol E Analog Int C Photon Cnt 3 Adjust the green Alexa Fluor 488 image and the red Alexa Fluor 546 image Group 1 Group 2 Group 3 Group 4 4 Press the XY button to acquire an image 5 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software The image is acquired EMemol File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations 16 py 16 Image Acquisition Double Stain on XYZ Image WE Acquisition of 3D images XYZ fluorescence image only EE sample Double stain of green fluorescence dye FITC and red fluorescence dye Rhodamine This is the procedure to acquire images through Line Sequential scanning 1 Click on the 4 Take steps 1 to 7 described on pages 13 and 14 Press the XY Repeat button to star
125. tions a 35 Image Analysis Inserting the Scale Bar E 2D View LILY POLLEN xml r b 1 1 z Click on the button 2 While left clicking the image drag and drop it at a certain point 2 e ROJO 3 1 gt NIA bb bb Y E o l the ive HR 3 While clicking the right or left handle move the mouse from side to side E 2D View LILY POLLEN xml Oax Change the text size aaa a D gt pi gt max 029 Tra Fag GA 0 color style etc 4 Select Scale Bar and then right click on Scale Bar to select Format A Setting eID NI hdi Pd iiini Cut ROI Ctrl X Copy ROI Ctrl C elt Delete ROI Del Max Size Color Bar Y Format Setting ROI Manager 5 Change the setting in this window as 0 required 36 36 Image Analysis Rotating a Three dimensional Image 1 Click on the 9 button for a 2D AA A AA View file name image 2 A 3D view Is created 30 l JES als alme slo al 3 Drag the mouse on the image to 2 3 observe it at a certain angle 2 21 135 Simple animation 4 Press and hold the 4 button to rotate the image around the X axis Press it again to stop rotation Press and hold the button to rotate the image around the Y axis Press it again to stop rotation Press and hold the button to rotate the image around the Z axi
126. to click Red 35 35 The ima ontrast TILE Ch Size 640x640 x y 575 362 Int 125 Zoom Auto 46 The i 2D Image Analysis the image of Z section m 2D View level NGadBarPb oib 1 Click Z and select 29 again then Projection image is shown on 2D View after getting XYZ image 3013 5128051251 2 y 4 3 135 INEU J 14 oom Autor 4 Yo m 2D View ivel NGadBarP6_oib Selec lt a ES fe gt b max C29 Fig aa Z e Ae AE BR Background Int o0 ma EJ 2 Click and select Ei 3 The images of Z section is shown on X axis and Y axis A RE oN According to Move to left or right side on X axis and to move to ups and down on Y axis be able to show image of Z section each position E 2D View level NGadBarPt_oib 4 The image of Z section on Y axis 5 The image of Z section on X axis 36 2D Image Analysis Intensity Profile of each Z sections 1 Click and then 3013 S126191 2x51 2 y 4 43 13 INEU J 14 oom Autor 4 Yo Automatic setting Ch Ch V Manual setting Column ich Z Int 12 Row ich T int L Int a O Multi Plane Wiew Profile 2 Click md Profile and then Intensity Profile of each Z sections is shown on the X and Y axis Need Projection and Merge in Tile Mode To move to Z position be able to show Intens
127. up 4 4 Press the XY button to acquire an image 5 Saving the image Right click on the Image Icon displayed on the Data Manager and select Save As to save the image Save as Type oib or oif file format specifically for the FV10 ASW software The image is acquired WWVemoN File formats specifically for the FV10 ASW OIF format Creates a folder that contains an image 16 bit TIFF and an accessory file which cannot be opened separately from each other OIB format Creates the OIF format files in a single file which is convenient for migration and other operations 16 py 16 Image Acquisition Double Stain on XYZ Image WE Acquisition of 3D images XYZ fluorescence image only EE sample Double stain of green fluorescence dye FITC and red fluorescence dye Rhodamine This is the procedure to acquire images through Line Sequential scanning 1 Click on the 4 Take steps 1 to 7 described on pages 13 and 14 Press the XY Repeat button to start scanning and A buttons to shift the focal point Refer to WMemoN When the sample upper limit is displayed on the image accept it using the Set button Click on the and buttons to shift the focal point Refer to WMemoN When the sample lower limit is displayed on the image accept it using the Set button Press the Stop button to stop sca

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