User manual LEICA WELL PLATE ACQUISITION WIZARD WIDEFIELD APPLICATION LETTER

[. . . ] · Step 1: selection of micro plate pattern, user defined patterns, activation and deactivation of wells · Step 2: alignment of micro plate, channel definition for best focus · Step 3: best focus routine searches for focus level in each well · Step 4: setup of acquisition parameters, start experiment · Step 5: merging tile scan images Step 1: Define Pattern in Step 1 the type of micro plate is selected and the wells to be visited during the experiment are activated. A predefined pattern for 96 well plates, for 24, 12, and 6 well plates can be selected in the template pull down menu. the predefined template 96 ­ 60 wells is a 96 well plate pattern with deactivated outer wells. this pattern is required when screening with high nA objectives with a short working distance. [. . . ] once the save button is pressed, the newly generated template will appear in the matrix definition window. 9 Note: each position of a sub array still can be combined with a tile scan matrix. each single element of a sub array can be deactivated separately. 6 definition of a new template with sub arrays 10 widefield Application letter 5 Step 2: Align in step 2 the micro plate is aligned and the acquisition parameters for the best focus function are defined. the best focus function will be used in the next step to search for the focus level in the wells automatically and generate a focus map of the micro plate. for micro plate alignment four active wells in the corners of the screening area are necessary. Note: the XY position of the first corner is already sufficient to identify the XY-position of the micro plate. the XY position of all other corners is not necessary to correct for, if the stage moves correctly to the wells in the remaining corners. in each corner it is important that the focus level is read in correctly. these focus levels will be used to generate a first approximation of focus level distribution of the micro plate and is required to generate the focus map in the next step of the wizard. 14 6 widefield Application letter w e l l p l At e A c q u i S i t i o n Workflow: 1. Go to the Acquisition (11) tab and define a channel (12) used for the best focus function. Additional channels can be defined, but best focus will use the first channel only for focus search. Best focus works with both, brightfield and fluorescence channels. please make sure the images are not saturated. otherwise the best focus algorithm might not find a focus level. 11 12 2. Go to the Align (13) tab and move the micro plate to the position marked with a red frame (14). find the focus within this well manually and press Read Position (15). the stage now will move automatically to the next corner of the matrix. find the focus within the well manually and press Read Position. this procedure will be repeated for the next two corners of the matrix. once all four corners are aligned, a message will indicate that alignment is completed. the user now can navigate within the micro plate by a left mouse click on the wells in the matrix definition window. the user cannot proceed to step 3 if alignment is not completed. Note: if any of the positions used for alignment are deactivated or if there is no specimen to focus at, please mark an adjacent active position by left mouse click and use this position for alignment. 13 15 widefield Application letter 7 Step 3: Focus Map in this step a focus map of the micro plate is generated. A best focus function automatically searches for the focus in each specified well. [. . . ] press Pause and Relocate after a cycle is finished and apply an offset to all micro plate positions by selecting All Positions (22) and pressing Continue (23). 22 23 10 widefield Application letter w e l l p l At e A c q u i S i t i o n Step 5: Post-Process this step is used to merge tile scan images. it only is needed if a tile scan area was defined in step 1. Workflow: 1. Go to a well position (24) within the experiment tree. the last two digits of the folder´s name define the position of the well within the micro plate. [. . . ]