Hello,
I encountered differences in the PSF FWHM between pre-mounted commercial TetraSpeck beads slides and self-made custom beads slides using TetraSpeck beads in suspension (same actual bead diameter / microscope / PSF width estimation algorithm). The PSF width is larger for the self-made custom beads slide. I tested to mount the coverslip with and without mounting media, and the PSF width with mounting media is closer to the one of the commercial slide.
- I was wondering whether anybody observed such a difference before as well?
- Does anybody have ideas about possible reasons for the observed difference in the PSF width or suggestions how to circumvent it?
- Could it be that the hardening mounting media slightly compresses the beads leading to a smaller bead diameter?
Thanks a lot for any input and ideas.
Here is some more background information:
For the evaluation of the imaging performance of a standard fluorescence microscope I used the pre-mounted TetraSpeck fluorescent beads slides from Thermofisher (“TetraSpeck Fluorescent Microspheres Size Kit”, fluorescent microspheres mounted on microscope slide, using the region with 500nm beads, ). As I need to evaluate the imaging performance in another fluorescence channel, I now prepare my own custom microscope beads slides. For this I use the TetraSpeck fluorescent beads in suspension (“TetraSpeck™ Microspheres, 0.5 μm, fluorescent blue/green/orange/dark red”, in theory the same beads that are used for the commercial pre-mounted beads slide) and add another type of fluorescent beads. However, I encountered quite some difference in the size of the estimated PSF FWHM when using the commercial slide and custom slides on the same microscope and using the same algorithm to detect the beads and estimate the PSF. The PSF width is larger for the custom slide, especially pronounced for FITC and TRITC. For the preparation of the slides I follow the proposed protocol from Thermofisher. In short, just diluting the beads in suspension in DIW, pipetting the solution onto a microscope slide and letting it dry. I mounted the coverslip on top of the microscope slide in two different ways, just by cluing the coverslip with nail-polish without mounting media, and by using mounting media (“SlowFade Diamond Antifade Mountant”) to attach the coverslip. For the slides with mounting media, the PSF width is closer to the one when using the commercial slides, but still significantly larger for FITC. I performed some experiments to investigate the difference, but none were conclusive.
Hi Esther,
Without seeing images, it is hard to say for certain, but what you are observing is most likely due to your choice of mounting medium, specifically the RI of that medium. I believe the precast Tetraspeck slides use Thermo’s Prolong GLASS media, which cures to a 1.52 refractive index. Slowfade has a 1.42 RI so that mismatch (assuming you are using an oil immersion objective) will increase spherical aberration and thus your PSF measurements.
Coverslip choice also impacts these measurements as most objectives are designed for 170um of glass thickness. Use a high precision coverslip (170um +/- 5um) for best results.
Edit: keep in mind that in order to properly measure the optical characteristics of a system you want to minimize aberrations as much as possible. This means matching the refractive index of the specimen as closely as possible to crown glass (what the objective lenses are made from) - which is 1.52. If you deviate from this - any measurements you make will suffer in some pretty interesting ways!
If you can share lateral and axial images of your beads this would help diagnose things more accurately.
Cheers,
-Jason
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Hello Jason,
Thank you a lot for your response and your assessment with the additional information.
I am using a dry objective without immersion media and high precision 170um coverslips.
I have ordered the Prolong GLASS media from Thermofisher to reduce the refractive index mismatch to check whether this leads to PSF widths closer to the commercial beads slides.
Best,
Esther
Hi Esther.
Firstly, your beads should be dried onto the coverslip- in this situation, the RI of the mounting medium does not impact on the PSF size at all, and you are imaging at the distance the microscope system is expecting to image at (moving away from the coverslip greatly impacts PSF shape). Secondly, the coverslip thickness. The pre-made slide does not state the coverslip thickness used (I assume you have T14792), and I don’t know where you can buy 1.5H coverslips of that size. This makes me suspicious about the thickness they use and this may impact the apparent resolution. Thirdly, the bead size you are using is probably very very close to the limit you can use (I don’t know as you don’t state the NA or the microscope type), and if you have a good NA, it is definitely too large. eg, NA0.75 for an air lens will give FWHM resolution of 357nm on a widefield at 525nm emission, and a 0.5NA would be 536nm.
I am part of a consortium of volunteers that try to standardise this sort of thing, so we have done a lot of these tests (including testing mounting RI). We have a sample prep protocol and an imaging protocol (admittedly aimed at confocal and high NA lenses, but easily adaptable for your needs) that might help, Monitoring the point spread function for quality control of confocal microscopes .
So my guess with the info provided is that you are comparing beads on the slide (yours) to beads on the coverslip and that is giving the difference. There is the fact that they are different beads too, and it is worth checking the lot certificates- they do differ from batch to batch.
Lastly, as part of our comparisons, we’ve played with mounting medium options. I doubt the pre-made slide is prolong glass (again, they give no info). We find the sample doesn’t keep as long with Prolong glass- the background increases noticeably compared to Gold, and a good signal to background ratio is essential for the software to accurately determine the FWHM as it impacts the Gaussian fits.
Lastly lastly, if you’re interested, feel free to join the community of volunteers at quarep.org : working group 5 does this work. (Sorry, shameless plug).
Glyn