Detection versus resolution

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crb5
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Joined: Sat Sep 12, 2020 11:55 pm

Detection versus resolution

#1 Post by crb5 » Sat Oct 30, 2021 8:16 pm

I acquired a Zero Mode Waveguide (ZMW) chip from the lab where we use them to record fluorescence from single molecules and I wondered what I would see under my "hobby-grade" Amscope 120 microscope. Zero mode waveguides are made by depositing a 100 nm layer of metal on a coverslip and etching thousands of 70 nm diameter holes using an electron beam. These nano-wells restrict the volume of excitation in a fluorescence microscope, so reducing background signal. The technique is used for high-throughput DNA sequencing https://www.pacb.com/videos/video-intro ... equencing/.

I illuminated the ZMW chip with a laser pointer held at an angle of about 45 degrees while recording the image through a 10x 0.25 NA objective lens. A pattern of dots spaced at 10 um intervals was easily resolved but the diameter of the dots was around 1 um, limited by the resolution of a 0.25 NA lens. So, even with a cheap microscope we can see (detect) the holes even though we cannot resolve their true diameter. Most of the laser beam was cleanly reflected from the chip surface (where it gave a nice diffraction pattern on the wall) and the microscope is detecting the small amount of scattered light from each hole. I think the ghost image of dots arises from a secondary reflection from the glass coverslip.
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PeteM
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Location: N. California

Re: Detection versus resolution

#2 Post by PeteM » Sat Oct 30, 2021 8:52 pm

Darn you crb5. I was perfectly happy before viewing your post. Looks very cool. Now, I want one of these Zero Mode Waveguides.

Any idea of where/how to get single instances?

MichaelG.
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Location: North Wales

Re: Detection versus resolution

#3 Post by MichaelG. » Sat Oct 30, 2021 8:54 pm

This is all very new to me … and intriguing: So I searched for some information and now I’m really confused
ZMWs are arrayed at a pitch slightly above the Abbe resolution limit for a high numerical aperture microscope objective. At this density, approximately 6.25 million ZMWs can be placed in an area of one square millimeter.
Ref. https://www.researchgate.net/figure/Col ... _228349791

This does not seem consistent with your description, or your image
… so what am I missing ?

MichaelG.
Too many 'projects'

crb5
Posts: 101
Joined: Sat Sep 12, 2020 11:55 pm

Re: Detection versus resolution

#4 Post by crb5 » Sat Oct 30, 2021 9:02 pm

Sorry, Pete. This was used ZMW chip handed down by my colleagues at Stanford. You could try contacting PacBio https://www.pacb.com/smrt-science/smrt-sequencing/ - they may give out samples or used chips.

crb5
Posts: 101
Joined: Sat Sep 12, 2020 11:55 pm

Re: Detection versus resolution

#5 Post by crb5 » Sat Oct 30, 2021 9:30 pm

This does not seem consistent with your description, or your image
… so what am I missing ?

MichaelG.
The pitch can be varied. In the article you reference they state "The NEMS process can be used to readily manufacture large arrays of regular ZMWs. Uniform arrays with inter ZMW spacing ranging from 300 nm to several micrometers were manufactured [400 nm pitch shown in Fig. 4b]." The PacBio chip we used had 150,000 holes in about a 5 mm diameter array, which gives about a 10 um spacing. The paper you reference may be an improved version or PacBio stuck with their pitch as they use fluorophores which include those in the far red and they want to avoid any possible overlap in signal between the holes.

MichaelG.
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Joined: Mon Apr 10, 2017 8:24 am
Location: North Wales

Re: Detection versus resolution

#6 Post by MichaelG. » Sat Oct 30, 2021 9:57 pm

Thanks for the clarification

I may catch-up eventually :oops:

MichaelG.
Too many 'projects'

PeteM
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Joined: Tue Dec 06, 2016 6:22 am
Location: N. California

Re: Detection versus resolution

#7 Post by PeteM » Sun Oct 31, 2021 12:47 am

crb5 wrote:
Sat Oct 30, 2021 9:02 pm
. . . You could try contacting PacBio https://www.pacb.com/smrt-science/smrt-sequencing/ - they may give out samples or used chips.
Thanks for the lead. I should check if Nanopore - a local sequencing startup - has something as well.

apochronaut
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Joined: Fri May 15, 2015 12:15 am

Re: Detection versus resolution

#8 Post by apochronaut » Thu Aug 18, 2022 8:49 am

This seems like it is a phenomenon of light against a black background . Diffraction in pinholes serves to blur and thus enlarge the image. of each hole. It plays on our psychology, like specks if light on a bright day, peeking through a tree canopy. You can't really see the gap due to optical artifacts until the light becomes oblique and defines the leaf edges.
If the pattern were reversed ; dark spots in BF, diffraction bands would increase the size of the specks increasing the dark to light ratio. There is no real resolution, just a contrasted pattern. The same phenomenon gives cheap achromat lenses a kind of artificial contrast, by increasing the dark to light ratio in the image. Diffraction creates a detectable image but nothing of consequence.

In older microscope objectives made prior to the advent of coating, in comparing lower N.A. achromats to higher N.A. apochromats of the same magnification: achromats are more forgiving of a mis-adjusted condenser and diaphragm, yielding images with better apparent contrast under such conditions than apochromats. However, under scrutiny and with the substage components more critically adjusted, it is obvious that in the achromat, contrast due to diffraction replaces resolution in the apochromat. This is in addition to the more conventionally understood chromatism based relationship between the two.

crb5
Posts: 101
Joined: Sat Sep 12, 2020 11:55 pm

Re: Detection versus resolution

#9 Post by crb5 » Thu Aug 18, 2022 10:40 pm

This seems like it is a phenomenon of light against a black background
I think we are saying the same thing. The holes in the zero mode waveguide are being detected but are only "resolved" by a cheap objective lens because they are 10 um apart. Some forms of super-resolution microscopy use the same trick. The position of a single fluorophore in the image is determined from the center of the diffraction limited blurr - and by photo-activating and then bleaching individual fluorophores, a high-resolution image is built-up one molecule at a time.

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