@@ -140,37 +140,97 @@ OME Tiff is recommended single-channel FRAP experiments. It stores FRAP regions
## Image Acquisition
### Choosing the right illumination
If you have any questions regarding the software, you can always go to the Visiview helpcenter and search for a topic:
In the illumination dropdown menu, you can choose the illumination that matches your hardware settings - meaning the selected SD & camera (W1 or X1, sCMOS or EM-CCD). Choose the laser line you need to excite your fluorophore (DAPI, GFP, mCherry & Cy5 - meaning 405, 488, 561 and 647 excitation lasers). For each illumination (in case of multi-color imaging) go to 'Show live' adapt the following parameters to achieve the desired image quality (in that order):
- the camera gain (only available for the EMCCD, a good starting value is 300)
- the illumination time (if you have a mobile sample, you might save time by reducing the illumination time)
- the laser power (be aware that higher laser powers and illumination times can result in irreversible photobleaching, which impairs e.g. brightness analysis).
In the 'illumination' dropdown menu, you can choose the illumination that matches your hardware settings - meaning the selected SD & camera (W1 or X1, sCMOS or EM-CCD). Choose the laser line you need to excite your fluorophore (DAPI, GFP, mCherry & Cy5 - meaning 405, 488, 561 and 647 excitation lasers). The 'Quadband' illuminations have a filter that works for all wavelengths (so your recordings will be faster in case of multicolor imaging), however you will potentially experience more crosstalk, which you can check under https://www.fpbase.org/spectra/. If you are not sure, ask the facility for their help in figuring out whether you can use the Quadband. For each illumination (in case of multi-color imaging) go to 'Show live' adapt the following parameters to achieve the desired image quality (in that order):
- the EMCCD Gain (only available for the EMCCD, a good starting value is 300), which only increases your signal on the detection side (no increase in photobleaching)
- the Exposure (if you have a mobile sample, you might save time by reducing the illumination time)
- the Laser power (be aware that higher laser powers and illumination times can result in irreversible photobleaching, which impairs e.g. brightness analysis). Do not use more than 10% of the laser power! If you are getting close to 10%, let the facility know and they will realign the lasers
When acquiring the brightfield channel, you only have to adapt the intensity of the Dialamp.
Always tick 'Save Sequence to Disk' and set a path under 'Directory' (ideally save it directly to the Hive or Fileshare). Only tick 'Separate Files' if you want to save each recording individually (meaning each color channel etc.). **Important:** Sometimes the box next to 'Save Sequence to Disk' or 'Separate Files' disappears (which is a bug). Solution: untick 'Time-lapse', 'Wavelength', 'Z-series' and 'Stage positions', then you should see it again. Tick the box next to 'Save Sequence to Disk' or 'Separate Files' and then tick again 'Time-lapse', 'Wavelength', 'Z-series' or 'Stage positions'. Even if the box disappears again now, it will remain active.
Tick Time-lapse and choose your number of time points, your time interval or your duration. You only have to choose two of them and the third one will adapt automatically. If you are only doing a time-lapse (no other wavelengths, z-series or stage positions), you can also tick 'streaming' for a faster recording. The time interval is only the waiting time between two time points, after which an image is recorded and needs to be read out from the camera chip (so there will always be a couple of 10-100ms added to your time interval, which you can read out from your metadata). If you set a time interval of 0 the recording will be done as fast as possible (meaning the time interval is then only the illumination time plus the readout time of the camera - multiplied by the number of wavelengths, z-planes and positions, depending on your experiment: also switching in between wavelengths or moving to different z-planes or positions takes time!). If you set a time interval >0 then it will automatically be increased, in case you have chosen it too short for your number of wavelengths, z-planes and/or positions. If you still want to further decrease your time interval, you have to reduce your number of wavelengths, z-planes and/or positions per time point.
Tick Time-lapse and choose your number of time points, your time interval or your duration. You only have to choose two of them and the third one will adapt automatically. If you are only doing a time-lapse (no other wavelengths, z-series or stage positions), you can also tick 'streaming' for a faster recording. The time interval is only the waiting time between two time points, after which an image is recorded and needs to be read out from the camera chip (so there will always be a couple of 10-100ms added to your time interval, which you can read out from your metadata). If you set a time interval of 0 the recording will be done as fast as possible (meaning the time interval is then only the Exposure time plus the readout time of the camera - multiplied by the number of wavelengths, z-planes and positions, depending on your experiment: also switching in between wavelengths or moving to different z-planes or positions takes time!). If you set a time interval >0 then it will automatically be increased, in case you have chosen it too short for your number of wavelengths, z-planes and/or positions. If you still want to further decrease your time interval, you have to reduce your number of wavelengths, z-planes and/or positions per time point.
In the Time-lapse tab you can also tick Autofocus and FRAP (for a detailed explanation see further below).
Tick Wavelength and choose your number of fluorophores (brightfield counts as an extra wavelength). For each slot in the 'Current' dropdown menu pick an illumination in the 'Illumination' dropdown menu (in the order in which you want to record them). Ticking 'same exposure/gain for all wavelengths' increases the speed of your recording, it it however not recommended, as different fluorophores usually exhibit different brightnesses, which you then have to compensate with the laser power only.
If you have also ticket the 'z-Series' tab, you will have the option in the 'Wavelength' tab to tick 'Z-Plane Series' for each illumination **individually** (meaning you could e.g. record a brighfield image without a z-Series and than 4 colors with a z-Series each). You can also apply a flatfield correction, in case you are doing quantitative brightness analysis (for a detailed explanation go to the Visiview helpcenter and search for flatfield correction)
Tick Z-Series. Go to 'show live', focus on your sample, then tick 'Centered Range'. Do not change to focus after that. You can now either stay with the ticked 'Centered Range' and choose a value for the 'Centered Range' (=size of your z-stack) or you can untick it and go to 'View top offset' and 'View bottom offset' and live-change the value next to it to change the top and the bottom of your z-Series. Be aware that the z-Series tab does not recognize a change in the focus (as e.g. the Zeiss Zen software does), so you can **only** adapt your range by changing the values for 'View top offset' and 'View bottom offset'.
You can now adapt the number of steps or the size of the steps. For optimal resolution in z, click the macro 'set opt z' in the right sidebar. If you experience a lot of photobleaching, you might consider trading resolution (larger steps) for less photobleaching.
#### Stage
##### Different stage positions
Tick 'Stage positions'. Go to your desired positions using the joystick, **focus properly(!)**, change the name of your position (e.g. 1,2,3,...) and click 'Add'. Importantly, also the z-value is saved with each position. If you want to update your positions (e.g. just the z-focus right before starting your experiment or after adding a substance to your probe), click on each of your positions, click 'Go to', change the focus, click 'Add' and confirm that the current position will be overwritten. You can also pick a 'Settle time' (meaning a waiting time after moving to a new position), in case your sample exhibits some motion after being moved.
a. Tick 'Stage positions' and then choose 'Scan slide' in the dropdown menu.
b. Choose the Fill Order 'Vertical' and click on the square symbol right below (it only shows when you hover over it with your mouse) - the Scan Slide Detail View should open
c. Click on 'Setup' in the right upper corner, then close just the Scan Slide Detail View
d. Put on a sample, go to 'Show Live' and find a central, significant, solitary object. Choose an illumination time <100ms, put your joystick's/controller's speed to S (slow) and draw a ROI (not larger than 100x100 pixels) around your object.
f. If the calibration is sucessful confirm by clicking 'Yes'. If it has failed repeat with a different object, different laserpower or a different ROI. If it does not work after a couple of tries, you have to restart the software/PC and potentially the microscopes hardware and try again (unfortunately it is sometimes buggy).
h. Click 'clear'. Then go to 'Show Live' and use the joystick/controller to move around your sample and define the area you want to scan. If you have moved to far, go to the side that you want to reduce (corners do not work), click inside the Scan Slide Detail View window & press Control + Shift while moving the joystick to reduce the area.
i. Record an overview image.
#### Custom
Here you can set up a completely customized experiment. In order to do that use the Visiview helpcenter or contact the facility.
