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andor_faq [2016/04/17 01:36] (current)
bioimaging created
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 +====== Andor Revolution FAQ ======
 +== Q. Why do I see a striped pattern in my image? ==
 +**A. ** If you see strips in your image then the spinning disk rotation may not be synchronized with the camera exposure time. Mismatches between the camera exposure time and spinning disk rotation speed are usually not a problem for long exposure times (> 100 ms) but they can seriously impair image quality when exposure times are down to less than 100 ms. A mismatch occurs whenever the exposure time is not an integral multiple of the time necessary to sweep one scan frame, in which case scanning will continue into the next frame and produce a striped pattern. iQ doesn'​t synchronize spinning disk rotation with camera exposure times but you can check this [[http://​​tutorials/​spinningdisk/​synchronization/​index.html|online interactive flash tutorial]] to check which spinning disk rotation speed you can set in the Yokogawa tab on iQ for a given exposure time.
 +== Q. I keep hetting this error message '​WriteTIF. Error writing to file <​filename>​. Check disk space?'​ when I try to save my data from the ImageList. How can I save my files? ==
 +**A.** Large files, i.e. multiposition zstacks can actually be saved. ​ First, open the original image, which is, say, a 30GB file, with zstacks in time at 5 XY positions. ​ Go to the "​Edit"​ menu, and select the "​Selector"​ option. ​ This is the master menu for making image subsets within the IQ software. ​ To split each of the 5 XY positions to into a single file (for saving later), click the "​XY"​ tab within the selector ​ window, and set both the "​min"​ and max" to 0.  This corresponds to XY position 1.  Leave all the other sliders in Z, time, and channel as they are, and give the file a name, in the "​filename"​ box.  If your original file was named "​test",​ the new file will be, by default, named "​test-selector"​ unless you change it.  (in case you see anything different in the box, it hasn't actually loaded the file properly---quit the software, and load it again!). ​ Click on "​create image",​ then refresh the image list, and your new file should be at the top, and be 6GB in size (again, if it's not...quit, and try again). ​ Now you can select the file from the image list, and and save it---it will automatically split it into the appropriate number of files based on size, in the case of a 6GB file you should end up with 3 files. ​
 +== Q. How do I use the Perfect Focus System (PFS) for z-stacks and multiple positions? ==
 +**A.** It depends on how you define the z-scan, but you will __always configure the PFS for the first position only__ and you do it in the scan configuration wizard, __after you define the z-stack parameters__. If you are setting up the z-scan by defining a middle position for the z-stacks (e.g. you are looking at adherent cells where you focus on the cell nuclei and want to image say 20 um above and below) then you just activate PFS for the middle plane of the first position after the z-stack parameters screen (click on the PFS button on the microscope and adjust the offset to bring the middle plane into focus, then turn off the PFS and go to step 6 below). If you setup the z-scan by defining where to start and where to end instead, then do the following:
 +1) On the Scan Setup, choose multi-field and Z series
 +2) On the Z Series options, unselect "focus on series centre for all fields"​ and define the z series start and end positions (let's say start at 60 and end at 50, 1 um distance between slices -> 11 planes)
 +3) On the next screen, where you define the different XY(Z start) positions, the piezo goes to the middle position (55); make sure that the grayed value on (start) is also 55 (move the piezo up and down to set the grayed value)
 +4) Activate the PFS on the microscope. The plane being imaged changes according to the PFS offset and now you have to adjust this offset to get back the image that corresponds to the middle position of the Z series. This can be tricky if you have no particular reference or way to know where exactly is the middle position (as opposed to adherent cells where we know that the middle corresponds to the cell nuclei). Try to adjust the PFS offset to get an image that looks like what you were seeing before you turned PFS on.
 +5) __Turn off the PFS__.
 +6) Adjust the piezo for the start position again (i.e. the grayed value must correspond to the start position, 60) and move to the next XY positions to set the different Z start positions
 +7) After saving the scan, create a protocol with Time, Multi-channel and Scan
 +8) In the protocol, under '​repeat XY' insert an Autofocus instruction
 +9) Start the protocol. The PFS will adjust focus for the first position, then it turns off to acquire all positions and Z series in the first time point and then it turns on again to wait for the second time point, always adjusting the focus before the first XY.
 +[[andor_revolution|Back to the Andor Revolution page]]
andor_faq.txt ยท Last modified: 2016/04/17 01:36 by bioimaging