1 00:00:05,01 --> 00:00:11,04 This is the third part of our video series on how to use the Well Navigator in cellSens software. 2 00:00:11,04 --> 00:00:17,19 In this video, I’ll explain how to group wells and assign individual observation conditions to each group. 3 00:00:17,19 --> 00:00:22,04 Refer to the first part of this video series for the necessary preparation steps 4 00:00:22,04 --> 00:00:26,22 and the second video for the basic well plate image acquisition procedure. 5 00:00:26,22 --> 00:00:30,10 First, I will show you how to group and organize wells. 6 00:00:30,10 --> 00:00:33,11 These are wells that were saved in the second video. 7 00:00:33,11 --> 00:00:36,21 Click “Add a comment to the selected well.” 8 00:00:36,21 --> 00:00:41,00 Left-click your mouse and drag it across the wells to select them. 9 00:00:41,00 --> 00:00:45,16 You can then type a comment, such as the name or concentration of the dye. 10 00:00:45,16 --> 00:00:49,14 Select the preferred highlight color, and then click “OK.” 11 00:00:49,14 --> 00:01:04,03 Repeat the same steps for the rest of the wells. 12 00:01:04,03 --> 00:01:09,23 It’s useful to group the wells according to the dye, type of cell, or cell condition. 13 00:01:09,23 --> 00:01:14,04 Then open the Experiment Manager pane, and click “New.” 14 00:01:14,04 --> 00:01:17,19 In the Experiment window, you can plan complex experiments 15 00:01:17,19 --> 00:01:20,16 simply by dragging and dropping commands. 16 00:01:20,16 --> 00:01:24,15 I will assign different observation conditions to each group. 17 00:01:24,15 --> 00:01:30,17 Select an observation method, for example “DAPI”from the “Image Acquisition” command list. 18 00:01:30,17 --> 00:01:33,23 And then click anywhere in the Experiment window. 19 00:01:33,23 --> 00:01:37,13 Draw a stage loop around the DAPI command. 20 00:01:37,13 --> 00:01:43,09 Then drag the stage to any well in the DAPI group in the Well Navigator. 21 00:01:43,09 --> 00:02:09,23 Click the DAPI command, and then click the “Live” button to adjust the focus and exposure time. 22 00:02:09,23 --> 00:02:14,18 Click “Get Settings” to save the exposure time in the Experiment Manager. 23 00:02:14,18 --> 00:02:18,14 Then click “Live” again to turn it off. 24 00:02:18,14 --> 00:02:23,22 Click the stage loop command, and then select DAPI from the “Position group” list 25 00:02:23,22 --> 00:02:30,00 so only the wells belonging to the DAPI group are acquired with the DAPI observation settings. 26 00:02:30,00 --> 00:02:42,12 Select and then copy and paste the commands in the Experiment window. 27 00:02:42,12 --> 00:02:53,09 Click the DAPI command, and then change the “Observation Method” to “RFP.” 28 00:02:53,09 --> 00:02:59,22 Click “Apply Settings,” and then click “Live.” 29 00:02:59,22 --> 00:03:03,21 Move the stage to any well in the RFP group. 30 00:03:03,21 --> 00:03:18,06 Adjust the exposure time, and then click “Get settings” to save it. 31 00:03:18,06 --> 00:03:20,20 Then click “Live” again. 32 00:03:20,20 --> 00:03:26,04 Click the stage loop command for RFP, and then change the “Position Group” to RFP 33 00:03:26,04 --> 00:03:32,08 so only the wells belonging to the RFP group are acquired with the RFP observation settings. 34 00:03:32,08 --> 00:03:39,06 Now we have assigned different observation conditions to different wells according to their group. 35 00:03:39,06 --> 00:03:42,19 This yellow warning symbol appears in the Experiment window 36 00:03:42,19 --> 00:03:47,03 to alert you when there is an error in the experiment settings. 37 00:03:47,03 --> 00:03:51,10 The error message is saying that these two commands need to be connected. 38 00:03:51,10 --> 00:03:55,01 When that has been done, the alert disappears. 39 00:03:55,01 --> 00:03:58,21 Next, we are going to define the Z coordinate. 40 00:03:58,21 --> 00:04:06,08 I'll use the TruFocus™ z-drift compensation. 41 00:04:06,08 --> 00:04:09,23 In this case, I'll set it to “Continuous ZDC mode,” 42 00:04:09,23 --> 00:04:14,22 which ensures that the distance from the vessel bottom to the observation plane remains constant. 43 00:04:14,22 --> 00:04:20,18 For more information on the ZDC, refer to the second video in this series. 44 00:04:20,18 --> 00:04:23,11 Click “Live” to start the focus adjustment, 45 00:04:23,11 --> 00:04:32,08 and then click “Find offset” to set distance of the ZDC offset. 46 00:04:32,08 --> 00:04:41,11 Click “Live” again, and then connect the ZDC command and DAPI command. 47 00:04:41,11 --> 00:04:45,11 To enable verification of the acquisition image during the experiment, 48 00:04:45,11 --> 00:04:58,17 select the “Online display” check box in the Experiment Manager for each acquisition command. 49 00:04:58,17 --> 00:05:03,18 Before starting the acquisition, let’s check the saved Acquisition Settings. 50 00:05:03,18 --> 00:05:09,14 It’s possible to set the file naming parameters so the group name is saved in each image file name. 51 00:05:09,14 --> 00:05:13,06 Under Document Name, select “All Options.” 52 00:05:13,06 --> 00:05:24,15 Then, add “Position Group” to the file name. 53 00:05:24,15 --> 00:05:39,09 Look at the preview to check the order of the properties. 54 00:05:39,09 --> 00:05:44,19 Then, under “Saving,” verify the location where files are stored. 55 00:05:44,19 --> 00:05:50,10 In this case, I’m going to create a subfolder in the “Pictures” directory named “Well B_Date,” 56 00:05:50,10 --> 00:06:04,20 which will automatically generate the year, the day, and the month of the acquisition. 57 00:06:04,20 --> 00:06:10,21 Click “Start” to start the acquisition. 58 00:06:10,21 --> 00:06:14,08 The TruFocus ZDC locates the focus position, 59 00:06:14,08 --> 00:06:20,12 and then the stage moves to well B4, which is the first well in the DAPI group. 60 00:06:20,12 --> 00:06:24,15 Then the system acquires a DAPI image. 61 00:06:24,15 --> 00:06:27,18 Once the image acquisition for the DAPI group is finished, 62 00:06:27,18 --> 00:06:32,14 the stage moves to the RFP group and continues the acquisition. 63 00:06:32,14 --> 00:06:36,15 All image acquisitions are complete. 64 00:06:36,15 --> 00:06:42,05 Now, let’s check the images. 65 00:06:42,05 --> 00:06:49,09 Here is the subfolder that I created earlier in the “Pictures” directory. 66 00:06:49,09 --> 00:06:54,03 There are 90 images available since I saved 9 positions in 10 wells. 67 00:06:54,03 --> 00:07:01,05 And, each file name contains either the “DAPI” or “RFP” group name. 68 00:07:01,05 --> 00:07:19,18 In this case, these images were properly acquired using the DAPI or RFP method, as per the group name. 69 00:07:19,18 --> 00:07:26,05 For more information on cellSens™ software, visit olympus-lifescience.com.