CKX-CCSW Confluency checker INSTRUCTIONS. Software for research/education This software is designed to use in research and or education fields.

Transcription

1 INSTRUCTIONS Confluency checker NOTE: This instruction mnul is for the Olympus confluency checker. To ensure the sfety, obtin optimum performnce nd to fmilirize yourself fully with the use of this softwre, we recommend tht you study this mnul thoroughly before using the softwre, nd lwys keep this mnul t hnd when using the softwre. Keep this instruction mnul in sfe plce. Softwre for reserch/eduction This softwre is designed to use in reserch nd or eduction fields. Opticl Microscope Accessory

2 Contents Introduction... Hndling Precutions... Cutions in use... System comptibility... PC nd softwre... 3 System digrm... 4 Instlltion Instlltion Activtion Uninstlltion Bsic opertions Initil Settings... 9 Strting the softwre...9 Exiting the softwre Initil settings Mesuring while cquiring the imge with DP/DP7...3 Mesuring the cell count in the culture vessel while cquiring the imge with DP/DP Mesuring the confluency in the culture vessel while cquiring the imge with DP/DP Mesuring from the cquired imge... Mesuring the cell count from the cquired imge... Mesuring the confluency from the cquired imge Mesuring while cquiring the imge with cmers other thn DP/DP Mesuring the cell count in the culture vessel while cquiring the imge with cmers other thn DP/DP7...8 Mesuring the confluency in the culture vessel while cquiring the imge with cmers other thn DP/DP Mesuring the cell count nd the confluency t the sme time Re-using the mesurement settings Cmer connection (when using DP/DP7)...37 ii

3 5 Screen flow [Min] screen Displying the submenu Displying the [Setting] screen Vrious settings [Min] screen...40 Setting the source Submenu... 4 Setting the re of culture vessel...4 Setting the objective mgnifiction Setting the dpter mgnifiction Setting the size of imge sensor Setting the number of imges [Setting] screen...43 Setting the file formt used for sving results...44 Setting the count color Setting the confluency color Displying the focus indictor Setting the working folder Setting the file nme Setting the lnguge Setting the DP imge formt Setting the clibrtion of DP/DP Displying About [Property] screen Sving the settings Loding the settings...5 iii

4 7 Effective mesurement of cell count/confluency Zoom re Using the cell scle br effectively...53 Setting the cell scle br Specifictions Troubleshooting...56 iv

5 Introduction The confluency checker is product to mesure the cell count nd the confluency from the imge where the culture cells re cquired with the Olympus microscope digitl cmer DP/DP7 or other cmers. Contents in this instruction mnul This instruction mnul covers only the contents concerning the confluency checker. For procedures to use the microscope digitl cmer DP/DP7 or other cmers, refer to the instruction mnul of the respective cmer. Trdemrk Microsoft, Excel, Internet Explorer nd Windows re registered trdemrks or trdemrks of U.S. Microsoft Corportion in the United Sttes nd other countries. Intel nd Intel Core re trdemrks of Intel Corportion in the United Sttes nd/or other countries. Compny nmes nd/or product nmes described in this mnul re trdemrks or registered trdemrks of the respective compnies. Softwre license greement If you use this product, you shll be deemed to hve consented to the terms nd conditions in OLYMPUS END- USER LICENSE AGREEMENT. "OLYMPUS END-USER LICENSE AGREEMENT" is included in the product. Plese red it crefully before using the product.

6 Hndling Precutions Cutions in use Imge dt. The recorded imge dt my be lost (destroyed) in the following cses. Olympus is not lible for the loss (destruction) of the recorded dt. When the user or third prty crried out repir to the product. When the system is terminted or the power cord is unplugged while PC is recording or deleting (initilizing). When the cble is disconnected while cquiring still imges or movies. When the dt hve been kept sved in the recording medi such s USB memory beyond its dt storge life (pprox. from one to severl yers). When the product is dmged.. If following phenomen occur even though the cmer is working properly, imges cnnot be restored nd the dmges re not compensted. The imge is bnorml. File properties such s file nmes, file dte, etc. re bnorml. The imge ws lost. }} In generl, s there is dt storge lifetime in the recording medi, such s USB memory, HDD, CD-R, DVD-R, etc., the sved files my be lost fter severl yers. }} The imge dt my be lost (corrupted) unexpectedly, so mke frequent bckups of the dt you cquired. OLYMPUS shll hve no libility for ny dmge (including compenstion for the corrupted imge dt) from the use or incpble use of this product. }} Mke sure tht there is no problem in cquiring the specimen imges before cquisition. Even though the problem my occur by the cquired imges, Olympus is not lible for these problems. Connection to network If you use this product connected to the network, the product my not work properly due to following problems.. Infection to the computer virus through network. Mlfunction cused by pplying OS service pck, security ptch, or updting third-prty softwre including runtime librries 3. The cquired imges re missed or the cquisition process is delyed or stopped due to the overloded system which is cused by running the ntivirus softwre, etc. System comptibility Restrictions in use } } Plese lso confirm the restrictions described in the instruction mnuls of the digitl cmer DP/DP7 or other cmers you will use.

7 PC nd softwre Recommended configurtions Common in Desktop PC / Lptop PC / Tblet PC No. Item Operting Environments CPU Intel Atom, Intel Core.i3, Intel Core.i5, Intel Core.i7, Intel Xeon, (CPU with the processing cpbility of Intel Core i3 or more is recommended.) RAM 4GB 3 HDD GB or more hrd disk spce for instlltion 4 Disply Desktop PC / Lptop PC: Resolution 80 x 04 (t lest 04 x 768), grphic crd vilble for 3 bit color. Tblet PC: 0. inch 90 x 00 (t lest 8.3 inch) 5 Communiction USB3.0 port (when using DP/DP7) port 6 OS Microsoft Windows 8. Professionl (3bit/64bit) Microsoft Windows 8 Professionl (3bit/64bit) Microsoft Windows 7 Ultimte/Professionl (3bit/64bit) SP 7 Lnguge English, Jpnese, Simplified Chinese Even though the bove mentioned recommended operting environments re stisfied, it does not necessrily gurntee the functions of combintion use with ll PCs commercilly vilble. Refer to Olympus website to check the PCs whose performnces in combintion use with this system were confirmed. PC. The PC dt my be lost (corrupted) unexpectedly, so mke frequent bckups of the dt you cquired. Olympus is not lible for ny dmge due to the use or disbility in use of this product, including compenstion for lost dt.. When the free spce in the hrd disk drive (HDD) is reduced, the dt processing speed my be extremely slow or errors my occur frequently. To prevent this, delete unnecessry dt files frequently. 3

8 System digrm The system configurtion for using this product is shown below. DP-CU/DP7-CU This product Cmer hed Interfce cble ** PC * (Desktop PC / Lptop PC / Tblet PC) (Downlod from Olympus web site.) Cmer DP-CU * DP7-CU * (Hereinfter referred to s DP/DP7) Cmer dpter U-TV0.5XC U-TVX U-CMAD Microscope CKX53 system, etc. }} Consult Olympus for the comptible microscope nd cmer dpter. * The cmer hed my not work properly in combintion with some PCs. Refer to Olympus website to check the PCs whose performnces in combintion use with this system were confirmed. ** Do not use the interfce cble for other use. 4

9 Instlltion Before instlling softwre. Close ll running pplictions before instlling softwre.. Softwre cnnot be instlled unless the user ccount logging on the Windows is registered s Administrtor. If it is registered s User, chnge it to Administrtor. (For the chnge of user ccount, refer to the Help of Windows.) - Instlltion The procedures to instll this product is described below. 3 Open (decompress) the file downloded from the Olympus web site on the desktop screen of PC where this product will be instlled. Click "Setup.exe". Select the lnguge to use. Select the lnguge from followings. English Chinese (simplified chrcters) Jpnese The procedures described below re for the cse when [Jpnese] is selected. 4 Click [Next]. 5 Select [Yes, I ccept ll terms of the License Agreement.] nd click [Next]. 5

10 6 Input [User nme] nd [Compny]. These fields my hve lredy been filled. 7 Select the folder to instll the softwre nd click [Next]. The following folder is specified s the initil setting. 3-bit version C:\Progrm Files\Olympus\Micro\ 64-bit version C:\Progrm Files (x86)\olympus\micro\ 8 Click [Instll]. The instlltion strts. During the instlltion process, if "Would you like to instll this device softwre?] regrding the instlltion of the USB driver of DP/DP7 ppers, click [Instll]. 6

11 9 Click [Finish]. This is the end of the instlltion. 7

12 - Activtion If pssing 30 dys of more fter the dte of instlltion, you must ctivte this product using the seril code. Without ctivtion, you will not be ble to use this softwre. The procedures to ctivte this product is described below. 3 Click on the desktop. This product strts nd the screen to input the license key ppers. Input the seril number in the [Seril No.] field, nd input the license key in the [License Key] field b. Click [Register] c. Clicking [Finish] will close this screen without ctivtion. b c TIP Within 30 dys fter the dte of instlltion, the bove screen ppers when exiting the softwre. -3 Uninstlltion The procedures to uninstll this product is described below. TIP If "Setup.exe" is deleted during instlltion, use Windows OS functions to uninstll the product. 3 Click "setup.exe". Click [Next]. Select [Delete] nd click [Next]. The uninstlltion strts. 4 Click [Finish]. 8

13 3 Bsic opertions This section describes the bsic operting procedures of this product. After initil settings re completed, you cn mesure the cell count nd the confluency ccording to the procedures described below. Mesure the cell count/confluency in the culture vessel while cquiring the imge using the cmer (DP/DP7, etc.). Mesure the cell count/confluency in the culture vessel from the cquired imges. 3- Initil Settings Strting the softwre Strt this product ccording to the procedures described below. Windows 7 Click on the desktop screen. Or click in the order of [Strt] button - [All progrms] - [Olympus] - []. If the error or the wrning screen ppers fter the softwre strts, click [OK]. Windows 8/8. Disply the desktop screen nd click. Or on the strt screen, click [.exe] in [All pplictions]. If the error or the wrning screen ppers fter the softwre strts, click [OK]. 9

14 Entire view When you strt this product, the following screen ppers first. g h b c e f d Item Description Control re Are to perform min opertions of this product You cn select from three types of screens: [Min] / [Setting] / [Property] by the tb in the upper prt of the re. b Zoom re Are to zoom in/out the imge in the preview re c Preview re Are to disply the imge to be mesured d Thumbnil re * Are to disply the imge in the specified folder by thumbnil nd to select the imge to be mesured e Focus indictor * Displys the contrst level of the live imge s focusing guide when cquiring with DP/DP7. f Cell scle br Scle to check whether the cell size is mesurble or not g Button to minimize Minimizes the screen. h Button to close Closes * These fields my not be displyed depending on the cmer to be used nd softwre settings. Exiting the softwre Exit this product ccording to the procedures described below. Click Click [OK]. in the title br. 0 If the softwre hs not been ctivted, the ctivtion screen ppers.

15 3 Initil settings This section describes the initil setting procedures of this product. This opertion is necessry when you strt this product for the first time. After the second time, the product strts in the sttus reflecting the lst settings when exiting the softwre. When you strt this product, the messge "Unble to disply due to no recorded picture. Set the folder contining the cquired imge." my pper, but it is not mlfunction. (Mesuring while cquiring the imge with the cmer) Confirm tht powers of PC nd the cmer re turned OFF nd connect PC to the cmer. For the connection procedures when using DP/DP7, see pge 37. When using other cmers, refer to the instruction mnul of the respective cmer. b c (Mesuring while cquiring the imge with the cmer) Confirm tht the light pth selector of the microscope is set to the cmer side. For procedures to operte the light pth selector of the microscope, refer to the instruction mnul of the microscope in use. Strt this product ccording to the procedures described on pge 9. The [Min] screen ppers in the control re. Click [Setting]. The [Setting] screen ppers. Click [Generl] b. Click c. The screen to select the folder ppers. Select the folder to use s working folder nd click [Select Folder]. TIP The selected folder is used for sving the imge cquired with this product or for selecting the imge to be used for mesurement. Imges in the folder re displyed in the thumbnil re of this product. (These imges re not displyed when mesuring while cquiring the imge with DP/DP7.)

16 7 Click [Min] d. The [Min] screen ppers. 8 In [Source], click e nd select the source. Select the source from followings. d f g e [DP]: Appers when DP/DP7 is connected. (Keep this sttus when mesuring while cquiring the imge with DP/ DP7.) [Folder]: Appers when DP/DP7 is not connected. (Keep this sttus when mesuring while cquiring the imge with other cmers.) [Select Imge]: Select this field when mesuring from the cquired imge. h i 9 0 If the submenu is not opened, click [Configurtion] f to open the submenu. Click g in [Adpter Mgnifiction] to disply the items, nd select the cmer dpter connected to the microscope. Items to be displyed re described below. No. Mgnifiction X0.5 (Initil setting) X.0 Click h in [Size of Imge Sensor] to disply the items, nd select the size of the imge sensor of the cmer connected to the microscope. If DP/DP7 is connected, settings re not necessry. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the i field. No. Sensor type [mm] (Horizontl) [mm] (Verticl) DP DP7 3 4/ Custom Direct input Direct input This is the end of initil settings.

17 3- Mesuring while cquiring the imge with DP/DP7 You cn mesure the cell count nd the confluency in the culture vessel while cquiring the imge with DP/DP7. Mesuring the cell count in the culture vessel while cquiring the imge with DP/DP7 Strt this product ccording to the procedures described on pge 9. The [Min] screen ppers in the control re. Click in [Source] nd select [DP]. 3 Plce the culture vessel of the specimen to be mesured on the stge of the microscope. 4 While viewing the live imge of the cmer displyed in the preview re, rotte the focusing knob of the microscope to bring the cell in the culture vessel into focus. At this time, the blue br of the focus indictor must be 80% or more of the entire length of the focus indictor. (If the blue br is not 80% or more of the entire length, refer to the instruction mnul of the microscope nd set ech units of the microscope, djust the brightness, clen lenses, etc. properly.) Focus indictor 5 While viewing the live imge of the cmer displyed in the preview re, rotte the nosepiece of the microscope nd select the objective with the mgnifiction where the cell size mtches with the size displyed in the cell scle br. * If the cell is defocused due to chnging the objective, perform 4 gin to bring the cell into focus. Cell scle br For procedures to confirm the cell size while looking t the cell scle br, see pge 53. * Set the size of one cell to the size between "MIN" nd "MAX" from the left end of the cell scle br. 3

18 b c d 6 7 If the submenu is not opened, click [Configurtion] b to open the submenu. Click c of [Are of Culture Vessel] to disply the items, nd select the re of the culture vessel to be used. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the d field. No. Type of culture vessel [mm ] (Are) 35mm Dish 96 60mm Dish, mm Dish 7, mm Dish 7,67 5 T-5 Flsk (Initil setting),500 6 T-75 Flsk 7,500 f g e 7 T-60 Flsk 6, well well well well well 3 3 Custom Direct input 8 Click e in [Objective Mgnifiction] to disply the items, nd select the mgnifiction of the objective. Items to be displyed re described below. No. Mgnifiction X X4 (Initil setting) 3 X0 9 0 Click the [Count] check box f to tick it. Click [Clibrtion] g. The [Cell Count Setting] screen ppers. 4

19 While viewing the cell imge displyed in the preview re, djust the threshold on the [Cell Count Setting] screen so tht the point (shown in red s defult) is displyed one by one in the cell. There re two wys to djust the threshold: one is to click h or drg i to djust the slider nd the other is to input the numericl vlue into j directly. When the threshold is determined, click [OK] k. The [Cell Count Setting] screen is closed. h j h i k On the [Min] screen, set the number of imges to be mesured while cquiring. In [Number of Imges], input the numericl vlue into the l field directly or click [+] or [-] in the m field. TIP The more the number of imges is incresed, the higher the mesurement ccurcy becomes, but it tkes longer time. It is recommended to strt from pprox. 5 imges s guide nd set the number of imges ccording to the mesurement conditions. 3 Click [Strt] n. The [Mesurement Result View] screen ppers. l m n 5

20 p 4 5 Bring the cell into focus gin ccording to the procedure in 4. Click [Snp] o on the [Mesurement Result View] screen. The imge is cquired nd the mesurement results re displyed in p. o 6 7 Move the culture vessel with your hnd to chnge the cquisition position, nd repet 4 nd 5. Ech time you cquire the imge, the vlue displyed in p chnges. Single: Displys the cell count of the imge cquired lst. Averge: Displys the verge of the cell count of imges cquired so fr. Estimtion: Displys the estimtion of the cell count of the entire culture vessel clculted from the verge. When the number of imge cquisitions set in [Number of Imges] is finished, the [FINISH] button ppers. The vlue in p when the [FINISH] button is pressed is the finl result. Click [FINISH]. The mesurement results re sved nd the [Mesurement Result View] screen is closed. This is the end of the mesurement of cell count. TIP To select the procedures to sve mesurement results (CSV file or overly imge), see pge 44. 6

21 Mesuring the confluency in the culture vessel while cquiring the imge with DP/DP7 Strt this product ccording to the procedures described on pge 9. The [Min] screen ppers in the control re. Click in [Source] nd select [DP]. 3 Plce the culture vessel of the specimen to be mesured on the stge of the microscope. 4 While viewing the live imge of the cmer displyed in the preview re, rotte the focusing knob of the microscope to bring the cell in the culture vessel into focus. At this time, the blue br of the focus indictor must be 80% or more of the entire length of the focus indictor. (If the blue br is not 80% or more of the entire length, refer to the instruction mnul of the microscope nd set ech units of the microscope, djust the brightness, clen lenses, etc. properly.) Focus indictor 5 While viewing the live imge of the cmer displyed in the preview re, rotte the nosepiece of the microscope nd select the objective with the mgnifiction where the cell size mtches with the size displyed in the cell scle br. * If the cell is defocused due to chnging the objective, perform 4 gin to bring the cell into focus. Cell scle br For procedures to confirm the cell size while looking t the cell scle br, see pge 53. * Set the size of one cell to the size between "MIN" nd "MAX" from the left end of the cell scle br. 7

22 b c d 6 7 If the submenu is not opened, click [Configurtion] b to open the submenu. Click c of [Are of Culture Vessel] to disply the items, nd select the re of the culture vessel to be used. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the d field. No. Type of culture vessel [mm ] (Are) 35mm Dish 96 60mm Dish, mm Dish 7, mm Dish 7,67 5 T-5 Flsk (Initil setting),500 6 T-75 Flsk 7,500 f g e 7 T-60 Flsk 6, well well well well well 3 3 Custom Direct input 8 Click e in [Objective Mgnifiction] to disply the items, nd select the mgnifiction of the objective. Items to be displyed re described below. No. Mgnifiction X X4 (Initil setting) 3 X0 9 0 Click the [Confluency] check box f to tick it. Click [Clibrtion] g. The [Step ] screen is displyed. 8

23 h Click [OK] h on the [Step ] screen nd drg the cell prt in the imge displyed on the preview re. The drgged line (shown in blue s defult) ppers nd the [Step ] screen is displyed. i Click [OK] i on the [Step ] screen nd drg the non-cell prt (bckground prt) in the imge displyed on the preview re. The drgged line (shown in green s defult) ppers nd the [Step 3] screen is displyed. Here, the imge displyed in the preview re is divided in different colors: cell prt (shown in blue s defult) nd bckground prt (shown in green s defult). Exmple of good color clssifiction result Exmple of bd color clssifiction result j k 3 If the color clssifiction result in is not good, drg the dditionl cell prts or the bckground prts. l To dd the cell prt, click [Add Cell Are] j on the [Step 3] screen nd perform the sme opertion s. To dd the non-cell prt (bckground prt), click [Add Bckground Are] k on the [Step 3] screen nd perform the sme opertion s. 4 When the color clssifiction of the cell prt nd the bckground prt is determined, click [OK] l on the [Step 3) screen. The [Step 3] screen is closed. 9

24 5 On the [Min] screen, set the number of imges to be mesured while cquiring. In [Number of Imges], input the numericl vlue into the m field directly or click [+] or [-] in the n field. TIP The more the number of imges is incresed, the higher the mesurement ccurcy becomes, but it tkes longer time. It is recommended to strt from pprox. 5 imges s guide nd set the number of imges ccording to the mesurement conditions. 6 Click [Strt] o. The [Mesurement Result View] screen ppers. m n o q 7 8 Bring the cell into focus gin ccording to the procedure described in 4. Click [Snp] p on the [Mesurement Result View] screen. The imge is cquired nd the mesurement result is displyed in q. p 9 0 Move the culture vessel with your hnd to chnge the cquisition position, nd repet 7 nd 8. Ech time you cquire the imge, the vlue displyed in q chnges. Single: Displys the confluency of the imge cquired lst. Averge: Displys the verge of the confluency of imges cquired so fr. Estimtion: Displys the estimtion of the confluency of the entire culture vessel clculted from the verge. When the number of imge cquisitions set in [Number of Imges] is finished, the [FINISH] button ppers. The vlue in q when the [FINISH] button is pressed is the finl result. Click [FINISH]. The mesurement results re sved nd the [Mesurement Result View] screen is closed. This is the end of the mesurement of confluency. TIP To select the procedure to sve mesurement results (CSV file or overly imge), see pge 44. 0

25 3-3 Mesuring from the cquired imge You cn mesure the cell count nd the confluency from the cquired imge. Mesuring the cell count from the cquired imge b c 3 Strt this product ccording to the procedures described on pge 9. The [Min] screen ppers in the control re. Click [Setting]. The [Setting] screen ppers. Click [Generl] b. 4 Click c. The screen to select the folder ppers. 5 Select the folder where the imge to be used for mesurement is sved nd click [Select Folder]. d 6 7 Click [Min] d. The [Min] screen ppers. Click e in [Source] nd select [Select Imge]. e 8 Select the imge to be used for mesurement from imges displyed in the thumbnil re. The imge is displyed in the preview re. (If you select the imge where the cell is not focused, too bright imge or too drk imge, the correct mesurement results my not be obtined.) 9 Look t the imge displyed in the preview re nd check tht the cell size mtches with the size displyed in the cell scle br. * For procedures to check the cell size while looking t the cell scle br, see pge 53. Cell scle br * Set the size of one cell to the size between "MIN" nd "MAX" from the left end of the cell scle br. TIP If the cell size is not mtched with the size displyed in the cell scle br, the correct mesurement results my not be obtined. In this cse, use the commercilly-vilble imge editing softwre to chnge the imge size, etc. nd perform mesurement.

26 f g h 0 If the submenu is not opened, click [Configurtion] f to open the submenu. Click g of [Are of Culture Vessel] to disply the items, nd select the re of culture vessel used for cquiring the imge. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the h field. No. Type of culture vessel [mm ] (Are) 35mm Dish 96 60mm Dish, mm Dish 7, mm Dish 7,67 5 T-5 Flsk (Initil setting),500 j k i 6 T-75 Flsk 7,500 7 T-60 Flsk 6, well well well well well 3 3 Custom Direct input TIP If these settings re not pproprite, the correct mesurement results my not be obtined. Click i in [Objective Mgnifiction] to disply the items, nd select the mgnifiction of the objective used for cquiring the imge. Items to be displyed re described below. No. Mgnifiction X X4 (Initil setting) 3 X0 TIP If these settings re not pproprite, the correct mesurement results my not be obtined. 3 4 Click the [Count] check box j to tick it. Click [Clibrtion] k. The [Cell Count Setting] screen ppers.

27 5 While viewing the cell imge displyed in the preview re, djust the threshold on the [Cell Count Setting] screen so tht the point (shown in red s defult) is displyed one by one in the cell. There re two wys to djust the threshold: one is to click l or drg m to djust the slider nd the other is to input the numericl vlue into n directly. When the threshold is determined, click [OK] o. The [Cell Count Setting] screen is closed. l n l m o 6 When mesuring multiple imges, select ll imges to be used for mesurement from imges displyed in the thumbnil re. Clicking the imge in the thumbnil re will chnge between the selected sttus (frmed in blue) nd the unselected sttus (not frmed in blue). TIP For the imge to be used for mesurement, be sure to select the imge cquired using the sme specimen nd the sme mgnifiction. q p 7 8 Click [Strt] p. The [Mesurement Result View] screen ppers nd ll imges selected re mesured. When the [FINISH] button ppers, the mesurement process is finished nd the results re displyed in q. Single: Displys the cell count of the imge mesured lst. Averge: Displys the verge of the cell count of ll imges mesured. Estimtion: Displys the estimtion of the cell count of the entire culture vessel clculted from the verge. Click [FINISH]. The mesurement results re sved nd the [Mesurement Result View] screen is closed. This is the end of the mesurement of cell count. TIP To select the procedure to sve mesurement results (CSV file or overly imge), see pge 44. 3

28 Mesuring the confluency from the cquired imge b c 3 Strt this product ccording to the procedures described on pge 9. The [Min] screen ppers in the control re. Click [Setting]. The [Setting] screen ppers. Click [Generl] b. 4 Click c. The screen to select the folder ppers. 5 Select the folder where the imge to be used for mesurement is sved nd click [Select Folder]. d 6 7 Click [Min] d. The [Min] screen ppers. Click e in [Source] nd select [Select Imge]. e 8 Select the imge to be used for mesurement from imges displyed in the thumbnil re. The imge is displyed in the preview re. (If you select the imge where the cell is not focused, too bright imge or too drk imge, the correct mesurement results my not be obtined.) 9 Look t the imge displyed in the preview re nd check tht the cell size mtches with the size displyed in the cell scle br. * For procedures to check the cell size while looking t the cell scle br, see pge Cell scle br * Set the size of one cell to the size between "MIN" nd "MAX" from the left end of the cell scle br. TIP If the cell size is not mtched with the size displyed in the cell scle br, the correct mesurement results my not be obtined. In this cse, use the commercilly-vilble imge editing softwre to chnge the imge size, etc. nd perform mesurement.

29 f g h 0 If the submenu is not opened, click [Configurtion] f to open the submenu. Click g of [Are of Culture Vessel] to disply the items, nd select the re of culture vessel used for cquiring the imge. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the h field. No. Type of culture vessel [mm ] (Are) 35mm Dish 96 60mm Dish, mm Dish 7, mm Dish 7,67 5 T-5 Flsk (Initil setting),500 j k i 6 T-75 Flsk 7,500 7 T-60 Flsk 6, well well well well well 3 3 Custom Direct input TIP If these settings re not pproprite, the correct mesurement results my not be obtined. Click i in [Objective Mgnifiction] to disply the items, nd select the mgnifiction of the objective used for cquiring the imge. Items to be displyed re described below. No. Mgnifiction X X4 (Initil setting) 3 X0 TIP If these settings re not pproprite, the correct mesurement results my not be obtined. 3 4 Click the [Confluency] check box j to tick it. Click [Clibrtion] k. The [Step ] screen is displyed. 5

30 l 5 Click [OK] l on the [Step ] screen nd drg the cell prt in the imge displyed on the preview re. The drgged line (shown in blue s defult) ppers nd the [Step ] screen is displyed. m 6 Click [OK] m on the [Step ] screen nd drg the non-cell prt (bckground prt) in the imge displyed on the preview re. The drgged line (shown in green s defult) ppers nd the [Step 3] screen is displyed. Here, the imge displyed in the preview re is divided in different colors: cell prt (shown in blue s defult) nd bckground prt (shown in green s defult). Exmple of good color clssifiction result Exmple of bd color clssifiction result n o 7 If the color clssifiction result in 6 is not good, drg the dditionl cell prts or the bckground prts. p To dd the cell prt, click [Add Cell Are] n on the [Step 3] screen nd perform the sme opertion s 5. To dd the non-cell prt (bckground prt), click [Add Bckground Are] o on the [Step 3] screen nd perform the sme opertion s When the color clssifiction of the cell prt nd the bckground prt is determined, click [OK] p on the [Step 3) screen. The [Step 3] screen is closed.

31 9 When mesuring multiple imges, select ll imges to be used for mesurement from imges displyed in the thumbnil re. Clicking the imge in the thumbnil re will chnge between the selected sttus (frmed in blue) nd the unselected sttus (not frmed in blue). TIP For the imge to be used for mesurement, be sure to select the imge cquired using the sme specimen nd the sme mgnifiction. r q 0 Click [Strt] q. The [Mesurement Result View] screen ppers nd ll imges selected re mesured. When the [FINISH] button ppers, the mesurement process is finished nd the results re displyed in r. Single: Displys the confluency of the imge mesured lst. Averge: Displys the verge of the confluency of ll imges mesured. Estimtion: Displys the estimtion of the confluency of the entire culture vessel clculted from the verge. Click [FINISH]. The mesurement results re sved nd the [Mesurement Result View] screen is closed. This is the end of the mesurement of confluency. TIP To select the procedure to sve mesurement results (CSV file or overly imge), see pge 44. 7

32 3-4 Mesuring while cquiring the imge with cmers other thn DP/DP7 You cn mesure the cell count nd the confluency in the culture vessel while cquiring the imge with cmers other thn DP/DP7. Mesuring the cell count in the culture vessel while cquiring the imge with cmers other thn DP/DP7 Strt this product ccording to the procedures described on pge 9. The [Min] screen ppers in the control re. Click in [Source] nd select [Folder]. 3 Plce the culture vessel of the specimen to be mesured on the stge of the microscope. 4 While looking into the finder of the cmer in use, rotte the focusing knob of the microscope to bring the cell in the culture vessel into focus. 5 Acquire one imge of the specimen ccording to the operting procedures of the cmer in use. 6 Select the imge cquired in 5 from imges displyed in the thumbnil re. The imge is displyed in the preview re. 7 Look t the imge displyed in the preview re nd check tht the cell size mtches with the size displyed in the cell scle br. * For procedures to confirm the cell size while looking t the cell scle br, see pge 53. If the cell size is not mtched with the size displyed in the cell scle br, chnge the objective nd perform from 4 to 7 gin. Cell scle br 8 * Set the size of one cell to the size between "MIN" nd "MAX" from the left end of the cell scle br.

33 b c d 8 9 If the submenu is not opened, click [Configurtion] b to open the submenu. Click c of [Are of Culture Vessel] to disply the items, nd select the re of the culture vessel to be used. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the d field. No. Type of culture vessel [mm ] (Are) 35mm Dish 96 60mm Dish, mm Dish 7, mm Dish 7,67 5 T-5 Flsk (Initil setting),500 f g e 6 T-75 Flsk 7,500 7 T-60 Flsk 6, well well well well well 3 3 Custom Direct input 0 Click e in [Objective Mgnifiction] to disply the items, nd select the mgnifiction of the objective. Items to be displyed re described below. No. Mgnifiction X X4 (Initil setting) 3 X0 Click the [Count] check box f to tick it. Click [Clibrtion] g. The [Cell Count Setting] screen ppers. 9

34 3 While viewing the cell imge displyed in the preview re, djust the threshold on the [Cell Count Setting] screen so tht the point (shown in red s defult) is displyed one by one in the cell. There re two wys to djust the threshold: one is to click h or drg i to djust the slider nd the other is to input the numericl vlue into j directly. When the threshold is determined, click [OK] k. The [Cell Count Setting] screen is closed. h j h i k 4 On the [Min] screen, set the number of imges to be mesured while cquiring. In [Number of Imges], input the numericl vlue into the l field directly or click [+] or [-] in the m field. TIP The more the number of imges is incresed, the higher the mesurement ccurcy becomes, but it tkes longer time. It is recommended to strt from pprox. 5 imges s guide nd set the number of imges ccording to the mesurement conditions. l m n 5 Click [Strt] n. o The [Mesurement Result View] screen ppers nd mesurement results of the imge currently displyed re displyed in o. 30

35 o 6 7 Move the culture vessel with your hnd to chnge the cquisition position nd bring the cell into focus ccording to the procedure in 4. Then, cquire the imge of the specimen ccording to the operting procedures of the cmer in use. Repet this opertion. Do not chnge the file formt (TIFF or JPEG) of the imge to be cquired during this opertion. Otherwise, the mlfunction of the softwre my be cused. Ech time you cquire the imge, the vlue displyed in o chnges. Single: Displys the cell count of the imge cquired lst. Averge: Displys the verge of the cell count of imges cquired so fr. Estimtion: Displys the estimtion of the cell count of the entire culture vessel clculted from the verge. When the number of imge cquisitions set in [Number of Imges] is finished, the [FINISH] button ppers. The vlue in o when the [FINISH] button is pressed is the finl result. Click [FINISH]. The mesurement results re sved nd the [Mesurement Result View] screen is closed. This is the end of the mesurement of cell count. TIP To select the procedure to sve mesurement results (CSV file or overly imge), see pge 44. 3

36 Mesuring the confluency in the culture vessel while cquiring the imge with cmers other thn DP/DP7 Strt this product ccording to the procedures described on pge 9. The [Min] screen ppers in the control re. Click in [Source] nd select [Folder]. 3 Plce the culture vessel of the specimen to be mesured on the stge of the microscope. 4 While looking into the finder of the cmer in use, rotte the focusing knob of the microscope to bring the cell in the culture vessel into focus. 5 Acquire one imge of the specimen ccording to the operting procedures of the cmer in use. 6 Select the imge cquired in 5 from imges displyed in the thumbnil re. The imge is displyed in the preview re. 7 Look t the imge displyed in the preview re nd check tht the cell size mtches with the size displyed in the cell scle br. * For procedures to confirm the cell size while looking t the cell scle br, see pge 53. If the cell size is not mtched with the size displyed in the cell scle br, chnge the objective nd perform from 4 to 7 gin. Cell scle br * Set the size of one cell to the size between "MIN" nd "MAX" from the left end of the cell scle br. 3

37 b c d 8 9 If the submenu is not opened, click [Configurtion] b to open the submenu. Click c of [Are of Culture Vessel] to disply the items, nd select the re of the culture vessel to be used. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the d field. No. Type of culture vessel [mm ] (Are) 35mm Dish 96 60mm Dish, mm Dish 7, mm Dish 7,67 5 T-5 Flsk (Initil setting),500 f g e 6 T-75 Flsk 7,500 7 T-60 Flsk 6, well well well well well 3 3 Custom Direct input 0 Click e in [Objective Mgnifiction] to disply the items, nd select the mgnifiction of the objective. Items to be displyed re described below. No. Mgnifiction X X4 (Initil setting) 3 X0 Click the [Confluency] check box f to tick it. Click [Clibrtion] g. The [Step ] screen is displyed. 33

38 h 3 Click [OK] h on the [Step ] screen nd drg the cell prt in the imge displyed on the preview re. The drgged line (shown in blue s defult) ppers nd the [Step ] screen is displyed. i 4 Click [OK] i on the [Step ] screen nd drg the non-cell prt (bckground prt) in the imge displyed on the preview re. The drgged line (shown in green s defult) ppers nd the [Step 3] screen is displyed. Here, the imge displyed in the preview re is divided in different colors: cell prt (shown in blue s defult) nd bckground prt (shown in green s defult). Exmple of good color clssifiction result Exmple of bd color clssifiction result j k 5 If the color clssifiction result in 4 is not good, drg the dditionl cell prts or the bckground prts. l To dd the cell prt, click [Add Cell Are] j on the [Step 3] screen nd perform the sme opertion s 3. To dd the non-cell prt (bckground prt), click [Add Bckground Are] k on the [Step 3] screen nd perform the sme opertion s When the color clssifiction of the cell prt nd the bckground prt is determined, click [OK] l on the [Step 3) screen. The [Step 3] screen is closed.

39 7 On the [Min] screen, set the number of imges to be mesured while cquiring. In [Number of Imges], input the numericl vlue into the m field directly or click [+] or [-] in the n field. TIP The more the number of imges is incresed, the higher the mesurement ccurcy becomes, but it tkes longer time. It is recommended to strt from pprox. 5 imges s guide nd set the number of imges ccording to the mesurement conditions. m n o 8 Click [Strt] o. The [Mesurement Result View] screen ppers nd mesurement results of the imge currently displyed re displyed in p. p 9 Move the culture vessel with your hnd to chnge the cquisition position nd bring the cell into focus ccording to the procedure in 4. Then, cquire the imge of the specimen ccording to the operting procedures of the cmer in use. Repet this opertion. Do not chnge the file formt (TIFF or JPEG) of the imge to be cquired during this opertion. Otherwise, the mlfunction of the softwre my be cused. Ech time you cquire the imge, the vlue displyed in p chnges. Single: Displys the confluency of the imge cquired lst. Averge: Displys the verge of the confluency of imges cquired so fr. Estimtion: Displys the estimtion of the confluency of the entire culture vessel clculted from the verge. When the number of imge cquisitions set in [Number of Imges] is finished, the [FINISH] button ppers. The vlue in p when the [FINISH] button is pressed is the finl result. 0 Click [FINISH]. The mesurement results re sved nd the [Mesurement Result View] screen is closed. This is the end of the mesurement of confluency. TIP To select the procedure to sve mesurement results (CSV file or overly imge), see pge

40 3-5 Mesuring the cell count nd the confluency t the sme time You cn mesure the cell count nd the confluency t the sme time by ticking both [Count] nd [Confluency] on the [Min] screen in the control re. Refer to the respective operting procedures for detils. 3-6 Re-using the mesurement settings When you mesure other culture cells under the sme conditions, you don't need to set [Are of Culture Vessel] or [Objective Mgnifiction] specified during mesurement nd to clibrte [Count] or [Confluency] gin. Sving the mesurement settings nd the clibrtion results llows you to lod them when mesuring under the sme conditions. For detils, see pge 5. 36

41 4 Cmer connection (when using DP/DP7) b Be sure to turn OFF the power of PC before connecting interfce cbles. Alwys use the interfce cble (USB 3.0 cble) provided by Olympus. Using commercilly vilble USB 3.0 cbles or hubs does not gurntee the opertion of the equipment. Keep the interfce cbles well wy from the equipment generting het, such s the lmp housing of microscope. The interfce cble is vulnerble to bend or twist. Be creful not to pply excess force. Connect the interfce cble in the correct orienttion pying ttention to the shpe of the connector. d ss TIP For detils of cmer connection, refer to the instruction mnul provided with DP/DP7. Connect the connector of the interfce cble to the connector b of the cmer hed. c Connect the connector c of the other end of the interfce cble to the USB3.0 connector d of PC. }} USB3.0 connector: Depending on PC, the terminl re is blue or "SS" is printed. }} If the interfce cble is connected to the USB.0 connector, the cmer hed does not work.. 37

42 5 Screen flow 5- [Min] screen The flow from the [Min] screen of this product is shown below. [Min] screen [Cell Count Setting] screen Zoom re [Property] screen [Mesurement Result View] screen Re-clibrtion 38 [Setting] screen, [Generl] tb [Setting] screen, [Confluency Checker] tb

43 5- Displying the submenu This section describes the procedure to disply the submenu of the [Min] screen. Disply the [Min] screen. Click [Configurtion]. The submenu is displyed. Clicking gin closes the submenu. 5-3 Displying the [Setting] screen b This section describes the procedure to disply the [Setting] screen. Click [Setting]. TIP To disply the [Property] screen, click [Property] b. 39

44 6 Vrious settings 6- [Min] screen This section describes the settings vilble on the [Min] screen. You cn disply the [Min] screen by clicking the [Min] tb in the control re. Minimize Sve Exp. time Source Sve destintion Select imge source Configurtion Are of Culture Vessel Objective Mgnifiction Adpter Mgnifiction Size of Imge Sensor Clibrtion Clibrtion Strt Count Confluency Number of Imges Setting the source This section describes the procedures to select the folder contining the imge to be used when mesuring by using the cquired imges. Disply the [Min] screen. Click nd set the source. Select the source from followings. [DP]: Appers when DP/DP7 is connected. (Keep this sttus when mesuring while cquiring the imge with DP/ DP7.) [Folder]: Appers when DP/DP7 is not connected. (Keep this sttus when mesuring while cquiring the imge with other cmers.) [Select Imge]: Select this field when mesuring from the cquired imge. 40

45 6- Submenu Setting the re of culture vessel b This section describes the procedures to set the re of culture vessel of the cell. Disply the [Min] screen. Click, nd select from the items displyed. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the b field. No. Type of culture vessel [mm ] (Are) 35mm Dish 96 60mm Dish, mm Dish 7, mm Dish 7,67 5 T-5 Flsk (Initil setting),500 6 T-75 Flsk 7,500 7 T-60 Flsk 6, well well well well well 3 3 Custom Direct input Setting the objective mgnifiction This section describes the procedures to set the objective mgnifiction. Disply the [Min] screen. Click, nd select from the items displyed. Items to be displyed re described below. No. Mgnifiction X X4 (Initil setting) 3 X0 4

46 3 Setting the dpter mgnifiction This section describes the procedures to set the dpter mgnifiction. Disply the [Min] screen. Click, nd select from the items displyed. Items to be displyed re described below. No. Mgnifiction X0.5 (Initil setting) X.0 4 Setting the size of imge sensor b This section describes the procedures to set the size of imge sensor. Disply the [Min] screen. Click, nd select from the items displyed. Items to be displyed re described below. If [Custom] is selected, input the numericl vlue in the b field. No. Sensor type [mm] (Verticl) [mm] (Horizontl) DP * DP7 * 3 4/3 * Custom Direct input Direct input * : If DP is connected, DP is displyed s n initil setting. * : If DP7 is connected, DP7 is displyed s n initil setting. * 3: If DP/DP7 is not connected, 4/3 is displyed s n initil setting. 4

47 5 Setting the number of imges This section describes the procedures to set the number of imges to be mesured. Disply the [Min] screen. In [Number of Imges], input the numericl vlue into the field directly or click b to disply the vlue in the left field, nd set it. You cn set the numericl vlue in the re from to 50. b 6-3 [Setting] screen This section describes the settings vilble on the [Setting] screen. The [Setting] screen cn be displyed by clicking the [Setting] tb in the control re. [Confluency Checker] screen Click [Confluency Checker] b to disply the [Confluency Checker] screen. b Sve Result Count Color Confluency Color Cell Scle Br Focus Indictor 43

48 [Generl] screen Click [Generl] c to disply the [Generl] screen. b Working Folder File Nme Lnguge DP Imge Formt Clibrtion About Setting the file formt used for sving results b This section describes the procedures to set the file formt to be used for sving the results. 3 Disply the [Setting] screen. Click the [Confluency Checker] tb. Click the [CSV] or [Overly Imge] check box in b to tick it. If the [CSV] is ticked, the cell count is sved in CSG formt. If the [Overly Imge] is ticked, the cell count is sved in TIFF formt. 44

49 Setting the count color b This section describes the procedures to set the color used for mesuring the cell count. Disply the [Setting] screen. Click the [Confluency Checker] tb. 3 Click b, nd select from the list displyed. You cn select from following colors. Red (R:55, G:0, B:0) Green (R:0, G:55, B:0) Blck (R:0, G:0, B:0) Blue (R:0, G:0, B:55) White (R:55, G:55, B:55) Mgent (R:55, G:0, B:55) Yellow (R:55, G:55, B:0) Cyn (R:0, G:55, B:55) 45

50 3 Setting the confluency color b c This section describes the procedures to set the cell color nd the bckground color used when mesuring the confluency. Disply the [Setting] screen. Click the [Confluency Checker] tb. 3 Click b in [Cell] nd select from the list displyed. You cn select from following colors. Red (R:55, G:0, B:0) Green (R:0, G:55, B:0) Blck (R:0, G:0, B:0) Blue (R:0, G:0, B:55) White (R:55, G:55, B:55) Mgent (R:55, G:0, B:55) Yellow (R:55, G:55, B:0) Cyn (R:0, G:55, B:55) 4 Click c in [Bckground], nd select from the list displyed. You cn select the sme colors s [Cell]. 46

51 4 Displying the focus indictor The focus indictor is displyed when DP/DP7 is connected. It shows the contrst level of the live imge s focusing guide. This section describes the procedure to disply the focus indictor. 3 Disply the [Setting] screen. Click the [Confluency Checker] tb. Click the [Disply] check box in b to tick it. Focus indictor b As focusing guide, the focused live imge cn be shown on the disply. The focus indictor is displyed s following br in the lower re of the window. The meter s mximum level is displyed utomticlly ccording to the contrst of the specimen. Norml view The current level is displyed in blue br. The mximum level is displyed in red. Current level Mximum level When the imge is focused, the blue br moves closer to the mximum level. When the imge is not focused, the blue br will be short. As focusing guide, djust the microscope so tht the level is closer to the mximum level. Level 0, unevluble view Mximum evlution level view }} The current level is updted every pprox. 0. sec. }} The mximum level is updted when the higher mximum level is identified. 47

52 5 Setting the working folder b This section describes the procedures to set the working folder. Disply the [Setting] screen. 3 Click [Generl] tb. Click b. The screen to select the folder ppers. 4 Select the working folder nd click [Select Folder]. 6 Setting the file nme b This section describes the procedures to set the file nmes of the cquired imges or mesurement results. 3 Disply the [Setting] screen. Click [Generl] tb. Input the file nme in the b field. The file nme differs depending on the file formt s shown below. CSV file XXX_ddmmyyyy_hhmmss.csv When [DP] is selected in [Source] XXX_nn_ddmmyyyy_hhmmss.xxx In cse of Overly Imge XXX_nn_ddmmyyyy_hhmmss_TAG.tif "XXX": Texts inputted to b "dd": Dte, "mm": Month, "yyyy": Yer "hh": Hour, "mm": Minute, "ss": Second, "nn": Seril Number 48

53 7 Setting the lnguge b This section describes the procedures to chnge the lnguge of displys such s menus to different lnguge. Disply the [Setting] screen. Click [Generl] tb. 3 Click b, nd select from the list displyed. The selected lnguge is displyed in the field on the right. You cn select from following lnguges. No. Lnguge English Jpnese 3 Chinese 8 Setting the DP imge formt b This section describes the procedures to chnge the imge formt. Disply the [Setting] screen. Click [Generl] tb. 3 Click b, nd select from the list displyed. The selected imge formt displyed in the field on the right. You cn select from following imge formts. No. Imge Formt JPEG-HIGH JPEG-LOW 3 TIFF 49

54 9 Setting the clibrtion of DP/DP7 b If the difference in brightness or color between the right hlf nd the left hlf of the imge cquired using DP/DP7 is noticeble, you cn correct the imge to the norml sttus by clibrting DP/DP7. If DP/DP7 is not connected, this item is not displyed. 3 Disply the [Setting] screen. Click [Generl] tb. Click [DP Cmer] b. Clibrtion strts. For subsequent opertions, follow the screens displyed. 0 Displying About b This section describes the procedure to disply the informtion of this product. 3 Disply the [Setting] screen. Click [Generl] tb. Click [...] b. The screen to disply the informtion of this product ppers. Click [OK] to close the screen. The informtion displyed on this screen re described below. Item nme b c d e f b c d e f Product nme, version OS nme Informtion of the cmer connected FPGA informtion of the cmer Firmwre informtion of the cmer Version of the cmer driver 50

55 6-4 [Property] screen On the [Property] screen, you cn confirm the contents specified in the [Min] screen nd [Setting] screen. This section describes the items displyed on the [Property] screen. Clicking [Defult] will return the settings to the initil vlues. b The informtion displyed on this screen re described below. Item nme Select Imge c d e f g h i j k l m n b c d e f g h i j k l m n Count Confluency Number of Imges Are of Culture Vessel Objective Mgnifiction Adpter Mgnifiction Size of Imge Sensor Sve Result Working Folder File Nme DP Imge Formt Cell Scle Br Focus Indictor 5

56 6-5 Sving the settings You cn sve the items displyed on the [Property] screen s the setting file. This section describes the procedures. Disply the [Min] screen. Click to disply the screen to select the folder. Set the folder nd the file nme to sve settings nd click [Sve]. TIP The clibrtion results of [Count] or [Confluency] during mesurement re lso included in the items to be sved. 6-6 Loding the settings You cn lod the setting files sved nd chnge the settings. This section describes the procedures. Disply the [Min] screen. Click to disply the screen to select the folder. Select the folder nd the file nme to lod settings nd click [Open]. 5

57 7 Effective mesurement of cell count/confluency 7- Zoom re c This section describes the procedures to operte the zoom re. Item Description Click this icon to zoom out. The b d b c d Zoom View sme opertion cn be performed by pinching in on the tblet PC. Click this icon to zoom in. The sme opertion cn be performed by pinching out on the tblet PC. Displys the mgnifiction (selection from 3 levels: X, X nd 4X). Displys the ctul pixel. If the number of pixels of the imge is lrger thn the resolution of PC, this is not displyed. 7- Using the cell scle br effectively Displying the cell scle br llows you to know the mesurble cell size. Align the cell to the left line nd set the zoom mgnifiction so tht the cell size is between nd b. Exmple When the cell is smller thn the mesurble size b When the cell is lrger thn the mesurble size 53