Change Details

= Tutorial= {F8105850} {F8105851} {F14084374} Outdated : F8105712 [Worflow and Data management](https://docs.google.com/presentation/d/1jfdDF_viFlqg3St1r2829cHj5HYXFfUfRe7wRWt3410) = Relocate Settings = {nav Settings > Relocate Settings} {F15082039} [[ https://c4science.ch/w/bioimaging_and_optics_platform_biop/image-processing/imagej_tools/perkinelmer-stitching/ | Using Fiji to process your Operetta images]] = Mind the Byte = **One full well from a 96-well plate **(4.5mm x 4.5mm field of view) Full resolution acquisition **20x air objective** **1Gb per plane **(Single channel, single slice, single timepoint) Example acquisition, 2 channels, 11 slices : **9.35 GB per well** = Write archive = It takes **~1 hour for 100 GB** = Harmony 4.8 installation = Start Autorun.exe \\svfas6.epfl.ch\biop\biop\MICROSCOPY\Operetta\2019-10-17 SW-Update Harmony 4.9 MDe Select: Office PC > install Warning : if the computer states : "you need to restart the computer", restarting the software will not do anything... To proceed with the installation : * Run Regedit.exe * Find the Windows Registry : HKEY_LOCAL_MACHINE\SYSTEM\CurrentControlSet\Control\Session Manager : PendingFileRnameOperations * Delete the PendingFileRnameOperations registry (right click > delete > ok) * You can now proceed to do the installation The **Region and Language Settings of the PC should be in English UK or US** otherwise the software crashes when you start it. Connect the Operetta database. Start Harmony 4.9. Go on: settings > data management > database settings Add a new database (click on **+**) Computer name: SV-01-366 Alias name: Operetta CLS = Plate reference = | **Brand**| **Ref Name** | **Ref Nbr** | **Qty** | **EUR**| **Remarque** | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/cellcarrier-96-ultra-lid-8x20b-6055300 | CellCarrier Ultra 96 ]] | 6055300|50 | 388.00 | works with 20x et 63x water| |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/cellcarrier-96-ultra-lid-8x20b-6055308 | CellCarrier Ultra 96 ]] | 6055308 |160 |1241.00 | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/viewplate-96-f-tc-10x5b-6005182 | ViewPlate-96F]] | 6005182 | 50 | 390.00 | Can't access the wells on the border with high mag. obj. + issue with finding focus using 40x & 63x | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/viewplate-96-f-tc-10x5b-6005225 | ViewPlate-96F]] | 6005225 | 5x10 | 372.00 | |Falcon | [[ https://ecatalog.corning.com/life-sciences/b2c/EUOther/en/Microplates/Assay-Microplates/96-Well-Microplates/Falcon%C2%AE-96-well-Polystyrene-Microplates/p/353219 | 96Well ImagingPlate ]] | FA-353219 | 4 x 8 | CHF 206-290 | Can't access the wells on the border with high mag. obj. + issue with finding focus using 63x | = Export flat-fielded images = It is possible to export the flat-fielded image by using the script located in \\svfas6.epfl.ch\biop\public\0-BIOP_Data\Scripts Operetta\Export_Flatfielded_Images inside the Harmony Software This is a small tool which can apply the flat-field correction. It is based on the image processing core of Harmony (acapella.exe). It runs on Harmony 4.8 and seems to work fine. Should you still have Harmony 4.6 you probably have to edit the batch file for the correct path to acapella.exe. Unfortunately I can’t test it on older versions, but I don’t really expect problems. - First you have to export measurements from Harmony. On the Operetta CLS this can even be exports with “Referenced Images”, i.e. you will just get an “index.ref.xml” file. Or if you prefer the normal export, the “index.idx.xml” is also fine. - Then you run the batch file, which just calls the acapella.exe interpreter with the script file as parameter. This script will then ask you for an index.xml-file. Select the file that was created during the Harmony export. - It will create a new folder and write all flatfield corrected images into that folder. It uses a similar naming convention as Harmony, just with the prefix “flex_”. It will also generate a new appropriate index.xml file, in case your system works with the xml files. These scripts can run on any Harmony installation that has a valid license dongle.

= Tutorial= {F8105850} {F8105851} {F14084374} Outdated : F8105712 [Worflow and Data management](https://docs.google.com/presentation/d/1jfdDF_viFlqg3St1r2829cHj5HYXFfUfRe7wRWt3410) = Relocate Settings = {nav Settings > Relocate Settings} {F15082039} [[ https://c4science.ch/w/bioimaging_and_optics_platform_biop/image-processing/imagej_tools/perkinelmer-stitching/ | Using Fiji to process your Operetta images]] = Mind the Byte = **One full well from a 96-well plate **(4.5mm x 4.5mm field of view) Full resolution acquisition **20x air objective** **1Gb per plane **(Single channel, single slice, single timepoint) Example acquisition, 2 channels, 11 slices : **9.35 GB per well** = Write archive = It takes **~1 hour for 100 GB** = Harmony 4.8 installation = Start Autorun.exe \\svfas6.epfl.ch\biop\biop\MICROSCOPY\Operetta\2019-10-17 SW-Update Harmony 4.9 MDe Select: Office PC > install Warning : if the computer states : "you need to restart the computer", restarting the software will not do anything... To proceed with the installation : * Run Regedit.exe * Find the Windows Registry : HKEY_LOCAL_MACHINE\SYSTEM\CurrentControlSet\Control\Session Manager : PendingFileRnameOperations * Delete the PendingFileRnameOperations registry (right click > delete > ok) * You can now proceed to do the installation The **Region and Language Settings of the PC should be in English UK or US** otherwise the software crashes when you start it. Connect the Operetta database. Start Harmony 4.9. Go on: settings > data management > database settings Add a new database (click on **+**) Computer name: SV-01-366 Alias name: Operetta CLS = Plate reference = | **Brand**| **Ref Name** | **Ref Nbr** | **Qty** | **EUR**| **Remarque** | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/cellcarrier-96-ultra-lid-8x20b-6055300 | CellCarrier Ultra 96 ]] | 6055300|50 | 388.00 | works with 20x et 63x water| |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/cellcarrier-96-ultra-lid-8x20b-6055308 | CellCarrier Ultra 96 ]] | 6055308 |160 |1241.00 | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/viewplate-96-f-tc-10x5b-6005182 | ViewPlate-96F]] | 6005182 | 50 | 390.00 | Can't access the wells on the border with high mag. obj. + issue with finding focus using 40x & 63x | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/viewplate-96-f-tc-10x5b-6005225 | ViewPlate-96F]] | 6005225 | 5x10 | 372.00 | |Falcon | [[ https://ecatalog.corning.com/life-sciences/b2c/EUOther/en/Microplates/Assay-Microplates/96-Well-Microplates/Falcon%C2%AE-96-well-Polystyrene-Microplates/p/353219 | 96Well ImagingPlate ]] | FA-353219 | 4 x 8 | CHF 206-290 | Can't access the wells on the border with high mag. obj. + issue with finding focus using 63x | = Export flat-fielded images = It is possible to export the flat-fielded image by using the script located in \\svfas6.epfl.ch\biop\public\0-BIOP_Data\Scripts Operetta\Export_Flatfielded_Images inside the Harmony Software This is a small tool which can apply the flat-field correction. It is based on the image processing core of Harmony (acapella.exe). It runs on Harmony 4.8 and seems to work fine. Should you still have Harmony 4.6 you probably have to edit the batch file for the correct path to acapella.exe. Unfortunately I can’t test it on older versions, but I don’t really expect problems. - First you have to export measurements from Harmony. On the Operetta CLS this can even be exports with “Referenced Images”, i.e. you will just get an “index.ref.xml” file. Or if you prefer the normal export, the “index.idx.xml” is also fine. - Then you run the batch file, which just calls the acapella.exe interpreter with the script file as parameter. This script will then ask you for an index.xml-file. Select the file that was created during the Harmony export. - It will create a new folder and write all flatfield corrected images into that folder. It uses a similar naming convention as Harmony, just with the prefix “flex_”. It will also generate a new appropriate index.xml file, in case your system works with the xml files. These scripts can run on any Harmony installation that has a valid license dongle. # Tips and Tricks ## Digital Phase Contrast (DPC) NOTE: When setting up the DPC parameters, find a field where there are no artifacts or floating dead cells as these will affect the optimization. 1. Find the focus position (offset) in brightfield where you see your cells the **least**. That is, the most in-focus plane. 2. Set your exposure time and laser power so that you are using a good part of the histogram (max values around 50000 gray levels 3. Set yourself on "High Contrast" Mode and hit "Snapshot DPC" 4. The system will acquire 50 images and find the ones with the highest contrast to use as top plane and lower plane 5. Set the DPC to "Manual" 6. Change the Filter and Speckle Filter in the following way": Speckle to 0 in order to keep mitotic cells, otherwise they get removed. Filter to 0.5 if your cells have holes in them, or larger than 1 if there is too much background around the cells.

= Tutorial= {F8105850} {F8105851} {F14084374} Outdated : F8105712 [Worflow and Data management](https://docs.google.com/presentation/d/1jfdDF_viFlqg3St1r2829cHj5HYXFfUfRe7wRWt3410) = Relocate Settings = {nav Settings > Relocate Settings} {F15082039} [[ https://c4science.ch/w/bioimaging_and_optics_platform_biop/image-processing/imagej_tools/perkinelmer-stitching/ | Using Fiji to process your Operetta images]] = Mind the Byte = **One full well from a 96-well plate **(4.5mm x 4.5mm field of view) Full resolution acquisition **20x air objective** **1Gb per plane **(Single channel, single slice, single timepoint) Example acquisition, 2 channels, 11 slices : **9.35 GB per well** = Write archive = It takes **~1 hour for 100 GB** = Harmony 4.8 installation = Start Autorun.exe \\svfas6.epfl.ch\biop\biop\MICROSCOPY\Operetta\2019-10-17 SW-Update Harmony 4.9 MDe Select: Office PC > install Warning : if the computer states : "you need to restart the computer", restarting the software will not do anything... To proceed with the installation : * Run Regedit.exe * Find the Windows Registry : HKEY_LOCAL_MACHINE\SYSTEM\CurrentControlSet\Control\Session Manager : PendingFileRnameOperations * Delete the PendingFileRnameOperations registry (right click > delete > ok) * You can now proceed to do the installation The **Region and Language Settings of the PC should be in English UK or US** otherwise the software crashes when you start it. Connect the Operetta database. Start Harmony 4.9. Go on: settings > data management > database settings Add a new database (click on **+**) Computer name: SV-01-366 Alias name: Operetta CLS = Plate reference = | **Brand**| **Ref Name** | **Ref Nbr** | **Qty** | **EUR**| **Remarque** | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/cellcarrier-96-ultra-lid-8x20b-6055300 | CellCarrier Ultra 96 ]] | 6055300|50 | 388.00 | works with 20x et 63x water| |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/cellcarrier-96-ultra-lid-8x20b-6055308 | CellCarrier Ultra 96 ]] | 6055308 |160 |1241.00 | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/viewplate-96-f-tc-10x5b-6005182 | ViewPlate-96F]] | 6005182 | 50 | 390.00 | Can't access the wells on the border with high mag. obj. + issue with finding focus using 40x & 63x | |PerkinElmer | [[ http://www.perkinelmer.com/fr/product/viewplate-96-f-tc-10x5b-6005225 | ViewPlate-96F]] | 6005225 | 5x10 | 372.00 | |Falcon | [[ https://ecatalog.corning.com/life-sciences/b2c/EUOther/en/Microplates/Assay-Microplates/96-Well-Microplates/Falcon%C2%AE-96-well-Polystyrene-Microplates/p/353219 | 96Well ImagingPlate ]] | FA-353219 | 4 x 8 | CHF 206-290 | Can't access the wells on the border with high mag. obj. + issue with finding focus using 63x | = Export flat-fielded images = It is possible to export the flat-fielded image by using the script located in \\svfas6.epfl.ch\biop\public\0-BIOP_Data\Scripts Operetta\Export_Flatfielded_Images inside the Harmony Software This is a small tool which can apply the flat-field correction. It is based on the image processing core of Harmony (acapella.exe). It runs on Harmony 4.8 and seems to work fine. Should you still have Harmony 4.6 you probably have to edit the batch file for the correct path to acapella.exe. Unfortunately I can’t test it on older versions, but I don’t really expect problems. - First you have to export measurements from Harmony. On the Operetta CLS this can even be exports with “Referenced Images”, i.e. you will just get an “index.ref.xml” file. Or if you prefer the normal export, the “index.idx.xml” is also fine. - Then you run the batch file, which just calls the acapella.exe interpreter with the script file as parameter. This script will then ask you for an index.xml-file. Select the file that was created during the Harmony export. - It will create a new folder and write all flatfield corrected images into that folder. It uses a similar naming convention as Harmony, just with the prefix “flex_”. It will also generate a new appropriate index.xml file, in case your system works with the xml files. These scripts can run on any Harmony installation that has a valid license dongle. # Tips and Tricks ## Digital Phase Contrast (DPC) NOTE: When setting up the DPC parameters, find a field where there are no artifacts or floating dead cells as these will affect the optimization. 1. Find the focus position (offset) in brightfield where you see your cells the **least**. That is, the most in-focus plane. 2. Set your exposure time and laser power so that you are using a good part of the histogram (max values around 50000 gray levels 3. Set yourself on "High Contrast" Mode and hit "Snapshot DPC" 4. The system will acquire 50 images and find the ones with the highest contrast to use as top plane and lower plane 5. Set the DPC to "Manual" 6. Change the Filter and Speckle Filter in the following way": Speckle to 0 in order to keep mitotic cells, otherwise they get removed. Filter to 0.5 if your cells have holes in them, or larger than 1 if there is too much background around the cells.