Greetings.
I am currently building an laser scan confocal microscope to be used in my Master’s project. Optical elements such as galvo scan mirrors, lenses, laser, filters and PMT are assembled on a breadboard and the system is controlled by a National Instruments DAQ card using LabVIEW and a fairly known program, GPScan.
The main problem is that all images seem to be tainted by black stripes resembling the raster scan pattern of the laser on the sample, as you can see in the following images. Both are fluorescence imaging of Rhodamine B stained paper, with resolutions of 1000x1000 pixels and dwell times of 1 and 10us, respectively.
I have searched some posts in the forum, and other users have reported that the lack of an optical table might be to blame for this issue. The setup is not atop one, as all in my laboratory are currently in use, but am also not 100% sure this could be the issue, as the patterns on those posts do not seem to match the ones I am experiencing.
So, with that in mind:
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I will try to use the setup on an optical table ASAP, but was wondering if anyone has experienced this and has any alternative insight or could point to other possible causes of the black stripes.
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I also suspect foul on the GPScan program, since it contains some problems when it comes to image distortion at high mirror speed (notice the left side of the images is a duplicate of the right side). Therefore, I have been thinking about switching to MicroManager as my primary control software. Would that be possible, considering I use a NI DAQ card and Thorlabs PMT and Galvo Scan Mirrors? If anyone has other suggestions, they are also welcome.
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For higher acquisition times (dwell time >100 us), a wave pattern appears over the image, as shown in the next picture. would that be aliasing caused by the large difference between acquisition and scan speeds (Nyquist Theorem)?
Thanks in advance.
Perhaps you are seeing (periodic) power fluctuations in the excitation laser? Or your PMT is picking up background from some other source like room lights or a computer monitor?
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It’s a possibility. The setup is in a place where I can turn off the lights, but the rest of the laboratory remains lit. There is an optical curtain I can pull to minimize the brightness and I also cover the entire setup with optical cloth while I am working, but my current situation doesn’t allow me to work in perfect dark, so there is always the possibility that some light punches through.
I’ll try moving the setup to the optical table as soon as possible, where we usually work in the dark.
However, I don’t think that explains how the stripes are continuous. If light interference was indeed the cause, I’d imagine each individual stripe to present different intensities along their length.
This kind of pattern is symptomatic of something oscillating relative to the oscillation of the mirrors. I’ve seen it in two instances: (1) a thin sample on a broken piezo Z stage that was oscillating at a set frequency such that the fluorescent sample was moving in and out of the focus plane and (2) flickering of fluorescent room light in the transmitted light image.
Your draping and curtains make room light an unlikely cause, though do check there isn’t an LED somewhere inside your rig.
Your images could be consistent with a thin sample and a table oscillating at the building frequency (typically the fans for the fume hoods and building air handling). Have you tried a thick sample like the Chroma plastic slide? If you don’t have those, a double stick tape chamber filled with 1:10 highligher fluid in water works well, too. The Koch lab is a great protocol for creating the doubestick tape chambers.
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Thank you! Will give it a try.
I also talked to one of our senior researchers, and his hypothesis is that, since the scan pattern is raster rather than zigzag, the PMT continues to acquire signal when the laser makes its way from the end of one line to the beginning of the next one, causing the stripes.
I will see how I could implement your solution as well as attempting to change the scan pattern to a zigzag one.