Posted by
Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deep-tissue-imaging-with-spinning-disc-comparison-with-other-confocal-techniques-tp7561610p7567106.html
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Hi Jan,
Comparisons between systems have to be done very carefully to be really meaningful, since there are so many variables between instruments. Even between similar types of instruments, there can be substantial differences, the more so as the instruments get more complex. A slightly misaligned pinhole can send your confocal performance and image quality down the drain, and even 1 Airy unit may mean different things on different microscopes. Just yesterday I was reading a brochure about one of the major vendor's new confocal, where they describe their hexagonal pinhole, which at 1 Airy units gives (according to the brochure) 30% more transmission than a square pinhole. The term pinhole tends to evoque a little circle in my mind, so I often forget that pinholes in confocals can be square, circular, or hexagonal. In the example above, clearly, the manufacturer was comparing the light throughput of an hexagonal pinhole with a square pinhole where the Airy unit would correspond to the diagonal. However, they don't say that their hexagonal pinhole would transmit less light than a circular pinhole, or in the case of square pinholes, 1 Airy Unit may be defined as the length of the square, in which case, the square pinhole still wins (with the hexagonal pinhole the Airy unit is defined as the largest diameter, i.e. the 1 Airy hexagon fits inside a 1 Airy circle). Anyway, this is just to illustrate that a trivial difference such as the geometry of the pinhole can result in a 20-30% difference in efficiency between two systems, all other things being equal (which they rarely are). Another trivial example, with one of our confocals, using to alternate (and theoretically functionally similar) dichroics can give as much as 20-30% difference in image intensity.
Back to the paper anyway, If you look at Figure 1, you'll see that the starting image (40 microns deep) is about 2.5 times brighter with Nipkow than with conventional confocal (you can measure it with imagej, for what it is worth on a reproduction). If you crank up the contrast, you will see signal in the conventional confocal images down to 120-140 microns. How would those compare to the Nipkow system if the confocal image had been optimized, and initial brightness of the various systems had been equalized? who knows....
As you point out, I am also concerned by the 5 mW laser power used for all instruments. Besides the fact that we do not know where this power was measured, it is clear that this is way too much power to use on a conventional confocal (we typically use around 0.1 mW, definitely less than 0.5 mW, and I don't know too many samples that could take much imaging at 5 mW). On the other hand, it is probably a reasonable amount of power for a spinning disk (or widefield), and maybe on the low end for two-photon, but can't really say. In any event, if I were to collect a 200 micron stack on our point scanning confocal at 5 mW laser power, I would proably see massive bleaching after the first few sections. This could be what the authors are observing. Again, hard to know.
You may also notice that the fields of view shown in Fig 1 for all three systems are identical. Would the successive imaging of the same region on different microscopes not bleach the sample, which would obviously put the first instrument at an advantage? In this case, which images were acquired first, and which were acquired last?
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
http://www.fhcrc.org----- Original Message -----
From: "Jan Pala" <
[hidden email]>
To:
[hidden email]
Sent: Friday, May 18, 2012 3:19:08 AM
Subject: Re: Deep tissue imaging with spinning disc - comparison with other confocal techniques
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Dear Sebastian,
I agree with George and you. I still do not understand the excitation
comparison - based on your experience, is there somehow comparable the
excitation laser power in between different confocal principles (5 mW) as
presented in the paper?
For sure, I am also very interested in some deep explanation and analysis
of observed and presented results.
Thank you.
Best regards,
Jan
On Fri, May 18, 2012 at 9:10 AM, Sebastian Rhode <
[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hi Jan,
>
> since our company manufactures both kind of systems, spinning disc confocal
> and 2-Photon systems, just my opinion on this paper.
>
> What I have seen so far during our tests, shows a clear advantage of 2PM if
> depth penetration is your primary(!) goal. If it comes to speed, spinning
> disc confocal (SDC) systems have a clear advantage. And in terms of cell
> viability, 2PM and SDC are better than a conventional LSM microscope.
>
> What puzzles me a bit, is the comparison between the penetration depth of
> the SDC and LSM. There are several points (some of them already mentioned
> by
> George here), which makes it difficult for me to really compare the
> results.
>
> But nevertheless the findings are quite interesting, even if there are open
> questions to answer. But I would be really interested in the explanation
> about the observed results.
>
> Dr. Sebastian Rhode
> Project Manager Research & Development
>
> TILL Photonics GmbH
> an FEI Company
>
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