Start in Acquisition Mode (in Viewer: Split Channel / Activate Intensity Histogram)
Optimize detector settings: Reuse/Create a detection sequence from file metadata or saved setting file => CHECK before use (LabJournal notes – link)
Hardware: Objective, filter ranges, laser (l, power), HyD (type, mode, Gain, AI denoise),
Acq Settings: Pixel Size (Format & Zoom), Pix Dwell time (Speed-Galvo/Res, Bi-Dir), Pinhole, Acc/Avg, Z step, Z range, t interval-duration
Search Dye, select and drag it onto the preferred HyD Detector (or drag onto empty space for automated setting), repeat for every dye
Choose optimal HyD Type for Dye (order S1>X2>S3>X4>R5, start at shortest wavelength)
Choose sequential mode to record dye (sets)
<+> Add a detector group, Ms(R) to reset a group
Line recommended, Frame allows different Acc or Avg per group
<Uncheck> to temporarily skip its acquisition
Avoid Dye Assistant, it resets filter ranges and does not include the HyD-R detector of the system (use it to get dye combination hints, do not <Apply>)
Choose mode of the HyDs (analog/digital/photon count (FLIM)/reflection)
Set Emission Filter Slider range
Fit emission spectra (shallow => ¯ Crosstalk & ¯ Signal), tweak Yield/Crosstalk by shifting the range
Ms(Clk)2 on slider to type l-limits or Clk-n-Drag ends
No overlap with laser line (>8nm away) & AFC aborts when a filter ³750 nm
Set Excitation Laser l
Fit excitation spectra – reduce Crosstalk by shifting from optimal, use PanelBox or Ms(Clk)2 the line to type
Inspect bleed/Crosstalk by varying Laser Intensity for the ‘donor’ channel, Intensity of the ‘acceptor’ Channel should not change, best evaluated in Viewer with Split channel & Histogram activated
Interactive elements open different setting GUIs (see numbered items in figure 2)
Choose group that generates the Transmission channel (TransPMT >> <ON>, set its Gain/Offset)
Set LUTs or channel coloring and save the setting in your data folder
Recommend to choose color blind-friendly sets that work well for presentation via beamers etc.)
Figure 2: Interactive GUI elements in the Image Compass
Use CCI form (on desktop or CCI website) dedicated to structure documentation of settings, save as: Setting-UID in your data folder before filling the form. Setting comments, for example Z range, Z step, T Interval
<FAST Live> to scan quickly (fast setting tweakable, it scans only the activate -white marked- Detector Group)
<Live> scans all channels as set (starts by Panelbox <Right Button>)
<Capture> takes image channels and omits other dimensions<START> records all set dimensions
Compare images in <Viewer> by <+> to add another Project Image, linking synchs Zoom/Intensity changes, <X> to remove a parallel view
Acquisition Parameter setting
Laser Intensity (use the Panel Box <Smart Intensity> or Ms(Clk)2 line to type)
Scan Acquisition mode XY
Do not oversaturate: Bit type intensity maxima (8b>256, 16b> 65536), use Glow-OverLUT in viewer to evaluate
<Zoom in> prompts to make a ROI and Zoom into it
<Bidirectional X> scan @ double speed but striping/doubling artefacts can occur (changing with zoom, speed>700, format change etc.), it needs manual Phase-X correction
<Format> & <Zoom Factor> define XY Pixel Size (diffraction limit or not)<Speed> defines Pixel Dwell Time: slower => signal , the <Resonance Mode> locked high speed: needs higher laser powers, and avg/acc to improve S/N
<Pinhole> defines along with l Optical section & Z step size (choose Airy 1 for the most important channel wlem)
<Rotation> orientate sample to optimally fit field
Many of the acquisition GUI is available in the <Navigator> as well in grouped GUIs. Beam Path= image Compass, Scan Mode = XY scan parameters, Z stack, Time, positions GUIs are activated in the <Toolbar>. Generally, preparing imaging is easiest done in the acquisition tab while overview/position/tile/ROI functions are done in the navigator.
Ms(Clk)2 move to position and makes <Preview> image, Ms(Wheel) to Zoom.
<AFC> performs quite well in <Spiral Scan>, Ms(R) to select variants of the spiral scan
Avoid LUT pileup of all (tiles and) previews in the images listing by unchecking or deleting unnecessary previews/Tiles. Setting LUT for the active FOV is quite difficult otherwise.
<Split Channel View> for active position FOV, a GUI offers options to overlay. Switch back to <Stage Overview> to resume tile actions
<Task List> Check/Unchecking ROIs/wells can be per group as well. <START> will only record checked <Tasks>
<Save Overview…> saves the checked pre/over-views in <Projects>
<Mosaic Merge> generates a single large file in <Projects>
Figure 4: Navigator
Focus MAP
Make Spiral overview (low mag)
Select ROI with drawing tools
< Check > keep Drawing Tool active
<+F> to set focus XY position (remove by Select> Delete)
<+AF> to use in Timelapse / Stage (set <..on Demand> – everty xth in time / position)
<Focus Map…> to activate dialog and list XY
<Set Z> Manual / Autofocus for active or all point(s) => Values Z/AFC are shown
OPTION: Shift Entire MAP in Z by defined offset (workaround to do asymmetric stacking)
CONTEXT Menu: Subtract Tool / Group Scan Regions / Paste Regions in Chambers etc.
Choose optimal HyD Type for Dye (order S1>X2>S3>X4>R5, start at shortest wavelength)
