Simplified Instructions: IXMicro High Throughput Microscope S1225

Transcription

1 Simplified Instructions: IXMicro High Throughput Microscope S1225 This is an Overview Only intended to refresh specific items learned in the Hands-On Training Use only in conjunction with your own much more detailed notes taken during your training

2 1, Reserve Time on Scheduler 2, Contact Fred if You Wish to Use Something Other Than Configuration B

3 3, Power-Up Turn on Lamp ( I Position)

4 3, Power-Up Turn on IXMicro ( I Position)

5 3, Power-Up Turn on Filter Wheel ( I Position)

6 3, Power-Up Turn on Climate Control ( I Position)

7 3, Power-Up Set Climate Control (right: cell chamber) (left: compound plates)

8 3, Power-Up Turn on Computer ( I Position)

9 4, Access Software Use Windows Log-In/Password provided to you at training

10 4, Access Software Open B Config

11 4, Access Software If not using Config B, open other Configs only after arranging different Config set-up with Fred

12 5, Software Opening Click Yes (relevant only for fluidics)

13 5, Software Opening Server (MDCStore01) Log-In/Password provided to you at training

14 5, Software Opening Highlight User

15 6, Set Instrument Controls Open Screening: Plate Acquisition Setup

16 6, Set Instrument Controls Step Through Menus for Plate Acquisition Setup

17 6, Set Instrument Controls Note: Once Set-Up, simply load existing settings file and jump to Acquire Plate plate after...

18 6, Set Instrument Controls Enter Your Name under Experiment Set (the Set is your folder) Enter Experiment Base Name (i.e. the name of specific study)

19 6, Set Instrument Controls If New Set-Up, Step Through: A, objective/camera settings

20 6, Set Instrument Controls B, Plate Type Highlight only from pull-down. Change nothing else. DO NOT MODIFY ANYTHING HERE EVER

21 6, Set Instrument Controls C, Wells in Plate to image Well at Wells to read

22 6, Set Instrument Controls D, Number and location of fields/well (Adaptive continues until >specified number of cells collected/well)

23 6, Set Instrument Controls E, Time points (for time lapse studies only)

24 6, Set Instrument Controls F, Fluidics (for adding compounds before run only)

25 6, Set Instrument Controls G, General Acquisition Controls Number of wavelengths collected/field Type of Autofocus (laser preferred) Flat-field shading corrections (Yes/No)

26 6, Set Instrument Controls H, Autofocus Locations Plate and Well Bottom recommended First well acquired recommended All sites recommended

27 6, Set Instrument Controls I, Laser Settings Best to Let Fred Work out for your settings Always activate Short Working Distance Always activate Bottom of Well settings

28 7, Set Collection Parameters Set initial collection parameters for each λ Select fluorophore from pull-down, see chart Select Exposure Time (ms) Select Offset initially as 0

29 7, Set Collection Parameters Open Screening: Plate Acquisition and Control Opens Box to Examine Collection Parameters

30 7, Set Collection Parameters Click Find Sample (uses initial settings) Activate Show Live Set Step Size (1 recommended) Count Steps Up or Down for best focus

31 7, Set Collection Parameters Modify collection parameters for each λ Modify Exposure Time (ms) for intensity Modify Offset for best focus

32 8, Finish Collection Set-Up (Most Users will do nothing more) Add in User-written macros (Journals) to modify run in specific ways (knowledgeable Users only)

33 8, Finish Collection Set-Up (Most Users will do nothing more) Activate Display...acquisition if wish to see Click Display Image and size/locate display for manual configuration of display

34 8, Finish Collection Set-Up (Most Users will do nothing more) Starts specified analyses while running (must have analysis parameters set & saved)

35 8, Finish Collection Set-Up (Most Users will do nothing more) Starts specified analyses while running If running on Offline Computers, their control must be activated

36 9, Start Run Acquire Plate under Summary Or (next page)

37 9, Start Run Acquire Plate under Plate Acquisition and Control

38 10, View Data Post-Run Open Screening: Review Plate Data

39 10, View Data Post-Run Select Plate to Review

40 10, View Data Post-Run Select Plate to Review Scroll to your folder Click Date collected Click Experiment Name, Open with Select (can also double-click)

41 10, View Data Post-Run Select Images to Review Check to open thumbnails for fluorophores you wish to view Montage : number of thumbnails to preview starting from highlighted well

42 10, View Data Post-Run Select Images to Review Click on specific thumbnail to open all fluorophore channels for that image Drag arrow to scale image for best viewing

43 11, Image Analysis Select Analysis protocol from pull-down

44 11, Image Analysis Click Configure Settings to open parameter interface for that protocol

45 11, Image Analysis Enter Object classification parameters almost always starts with setting most distinguishable objects ( Nuclei ) Click Preview

46 11, Image Analysis Objects Identified according to parameters are highlighted

47 11, Image Analysis Click indicated button to toggle between actual image and objects classified Modify Object classification parameters and repeat until objects best identified

48 11, Image Analysis Run Analyses for one of -all sites -selected wells highlighted in interface -single site