Recommendations for fluorescent nano-beads for PSF confocal

Probes & Prep
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Hi,

We have an INCELL 6000 analyzer, which has confocal capabilities, using a line scanning confocal, which appears to be somewhere between wide field and confocal.

Images are quite blurry, especially in the z-plane. We’ve had some success using deconvolution with a theoretical PSF, but would like to try capturing a more accurate PSF using fluorescent nano-beads.

Does anyone have any recommendations on which products to use? I believe we want beads around the 175nm, and but I’m not sure.

Also any tips for imaging would be appreciated.

Thanks in advance

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Any particular wavelength?
Bang Laboratories is one source.
I have also used TetraSpeck beads.

Sub 200nm beads should be fine for this purpose.

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Hi @NachoDave

I also posted your question on Twitter. There are some answers that may be useful. Let me know when you get a bead image and I can help you extract a good PSF for deconvolution.

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Hi Brian, thanks for this.

I’m not actually collecting the images myself, but is actually found the marker pen solution poster by searching older forum posts. I relayed it to the guy in the lab who is going to take the images and he’s going to try the zebra pens and also the commercial beads they use for bench marking. I’ll let you know when we have images.

Thanks again

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Hi @Hazen_Babcock

I shall check out these beads. Thank you

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“…1, 0.5 and 0.2 μm beads are TetraSpeck Microspheres from Life Technologies…”
from A line scanning confocal fluorescent microscope using a CMOS rolling shutter as an adjustable aperture
https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2818.2012.03642.x

Some years ago (2013 or 2014), the author visited my previous company to give a talk. In my previous company we would use ~100nm beads for a more conventional confocal. They are using 200nm beads as the smallest. Recently (2017) in my current lab, I put together something similar on an optics table, using generic beads found on Taobao or beads made by another lab in the material science department. Unlike other “slit” or “line” scanning setups, it is a variable “slit.” Normally, a “slit” or a pair (excitation/detection) would be fixed and terminology such as “line spread function” would be employed to characterize resolution (derive the MTF) as a cylindrical lens is often used (with the intensity unevenly distributed, with better resolution in the middle and worse at the edges, similar to some light sheet) Alternatively a “line” of fixed pinholes may be used as in “swept-field” confocal. Usually in a “swept-field” they have several sets of “lines” of pinholes, with each set of different diameters, for excitation and detection, which can be quickly changed using a piezo.

Lateral and axial resolution is not really that great in any of these systems compared to more conventional confocal approaches, however you do get some optical sectioning ability. Apologize for the quotation marks, as if you have read a lot of the different papers about these slit/line approaches, the terminology/jargon gets a bit confusing. Likewise, the different papers have different (sometimes dubious) methods to characterize resolution.

In the INCELL it will depend on the settings in the software which will determine the width of the “aperture” (the width of the rolling shutter). Did you call Cytiva (Molecular Devices) to ask them? The service engineer who installed or maintains it could perhaps explain to you how they QC it on-site to verify its performing properly. Something such as that is probably not in the user manual, but the engineer has to have a standard sample or SOP to prepare one. Its was quite a pricey piece of equipment so I would expect they may be able to help if you ask.

Also, some of the machines Thermo sells employ a similar mechanism as the INCELL, using the same patent under license.

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Hi @ShawnPatrickCasey

Thank you for help. The paper you reference is really useful.

Yes, we are in contact with service engineers at Cytiva, I will ask them about their QC protocols. We are able to adjust the (virtual) aperture size, however even at the lowest setting we are still getting significant amounts of out of focus light in each image plane, making segmentation difficult.

With help from @bnorthan we have performed deconvolution on our images, using a theoretical wide field PSF, which definitely improves our segmentation. We want to try and measure the PSF to improve the deconvolution results further.

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Sorry, that paper is not exactly that great (more of a whitepaper).

By “segmentation” you refer to some image processing, or are sectioning?

The main problem is that the “PSF” is variable with “slit” width? There is some other image processing besides a simple subtraction operation going on in their software.