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Here's a link to a recent article on the Argolight test slides:

http://argolight.com/wp-content/uploads/2013/09/2013_Royon_Imaging-and-Microscopy.pdf

I'd echo Glenn's question: Has anyone out there tried these slides yet? Very curious to know how well they meet the claims of the article. How do they prevent photobleaching I wonder, and for how long?

John



On 2014-03-15, at 11:43 PM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Well, that's very interesting. This is exactly the kind of test specimen I had in mind for fluorescence microscopy. I had not heard of this company before now. Thank you Glenn for pointing them out. I think I'll have to try using one of their test slides. What I find most interesting here is Argolight's ability to manufacture complex 3D structures with the fluorescent brush technology. The question then becomes - what's a good/challenging test specimen 3D pattern for confocal microscopy that everyone could all agree on? 
> 
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research Inc.
> A Division of Andor Technology
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
> 
> 
> On 2014-03-15, at 4:56 PM, Glenn Merrill-Skoloff wrote:
> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> There’s a commercially available product that claims to meet most of the requirements John lists, below. Has anyone used this slide?
>> 
>> http://argolight.com/product-micro/
>> 
>> My Best,
>> — Glenn
>> 
>> Glenn Merrill-Skoloff
>> Division of Hemostasis and Thrombosis
>> Director, Intravital Microscopy Core
>> 
>> 617-735-4040 (Office)
>> 617-735-4007 (Lab)
>> 617-735-4000 (Fax)
>> 
>> 
>> 
>> From: John Oreopoulos <[log in to unmask]<mailto:[log in to unmask]>>
>> Reply-To: Confocal Microscopy List <[log in to unmask]<mailto:[log in to unmask]>>
>> Date: Saturday, March 15, 2014 at 12:35 AM
>> To: "[log in to unmask]<mailto:[log in to unmask]>" <[log in to unmask]<mailto:[log in to unmask]>>
>> Subject: Re: Confocal NEMA?
>> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Kurt,
>> 
>> I'm not familiar with NEMA, but the general topic of testing/standardizing confocal sensitivity, resolution, and image quality is of great interest to me. If you search the confocal listserver archive, you'll find several postings about this. In theory, you could develop a standard test for confocal sensitivity, resolution, and image quality, but what should the test specimen be? Others have worked on this to some extent, and a few names that come to mind are Fred Brakenhoff, Claire Brown, Robert Zucker, and Mike Model. I recommend looking up their papers to see what sorts of test specimens have been proposed and tried in the past. Unfortunately, I don't think there is one single test specimen that can be used to determine all instrument parameters of interest (sensitivity, resolution - spatial, temporal, spectral-, and image quality, etc.).
>> 
>> In my mind, an ideal test specimen for confocal fluorescence imaging would have the following properties:
>> 
>> 1. Be cheap, readily available, and reproducible to a high degree
>> 2. Able to absorb and emit light over broad range of wavelengths
>> 3. Be portable and emit constant intensity (no photobleaching) perhaps even with a known number of photons under certain conditions
>> 4. Does not saturate easily, and has a linear response to excitation light power
>> 5. Has a known 3D structure of specific shapes/patterns/sizes with a high level of precision determined by some other imaging technique (electron microscopy for example).
>> 6. Has a refractive index close to water or glass
>> 
>> Again, I don't think such a sample exists, but if you can think of one, let me know! I think one of the other problems here is: how would you define and quantify things like "image quality"? I'm curious how NEMA does this for PET. Spatial resolution is a bit more straightforward to quantify, although here too there are different ways to define resolution and ways to measure it. Sensitivity measurements are complicated by the need to keep many other imaging parameters constant when examining day-to-day performance/sensitivity of one instrument, or comparing the performance of two different instruments - see Jim Pawley's "39 steps".
>> 
>> Your question also brings to mind a quote from another very well-written article on the topic which I never forget:
>> 
>> "This complex relationship between qualitative and quantitative content of fluorescence images also expresses itself in the way that commercial instruments are assessed. On the one hand it is unavoidable that users initially are strongly influenced by the visual quality of the images presented by the instrument. On the other hand the longer term scientific value of the instrument depends crucially both on the quality of the visual information and the effectiveness with which it can be reduced for quantitative analysis... The increased complexity of a fluorescent image (in terms of the number of dimensions that can be measured), and the variability of sample preparation, makes it less easy to assess quantitatively the quality of the image. This may seem a surprising, or at least somewhat bleak, assessment. But consider this: In the 20 years or more since introduction of laser scanning confocal microscopes it has not been feasible, from published data, to assess how these systems compare (in terms of absolute units associated with measurements). No one can assert with confidence that instrument A in use in 1990 has better or worse sensitivity than instrument B operating in 2006. There is, therefore, a paramount need for standardized test samples and procedures for their use."
>> 
>> -taken from:
>> Dixon, A., T. Heinlein, and R. Wolleschensky, Need for standardization of fluorescence measurements from the instrument manufacturer's view standardization and quality assurance in fluorescence measurements ii. 2008. 6: p. 3-24.
>> 
>> I find the second last statement above quite frustrating, especially from the point of view of someone who works in the industry. I can tell you that the experience of demoing a microscope system to a potential customer often goes something like this: The potential customer prepares a typical biological specimen that they are familiar with from experience and then images of this specimen are captured with the demo system. These images are then compared to a similar set of images that were produced on another competing instrument (maybe - sometimes comparison images are not available). Usually there are statements made to the effect of "yes/no, the images on this instrument look better/worse, therefore I should buy/decline instrument A/B". I think anyone with a good imagination can figure out that this is a very subjective way to compare instrument performance and that there are a lot of potential pitfalls involved that might bias the assessment one way or another. I'm not saying this isn't a good method to judge an microscope's general imaging performance. Indeed, you should always take a prospective microscope system out for a "test drive", but a more quantitative test that doesn't solely depend on the opinion of how "good" an image looks would be better for everyone in my opinion.
>> 
>> And Kurt, supposing a good set of text specimens and protocols were developed - how could we all come to agree on them? Who would set the standards and govern them for microscopy? Surely the industry players would have much to say (or dispute!) about that. Again, as stated above, there is a paramount need in microscopy for standardized test samples and procedures. It's a cause I certainly would be willing to devote time to were there an organized and concerted effort to make it happen.
>> 
>> Curious to hear other people's thoughts on this.
>> 
>> 
>> John Oreopoulos
>> Staff Scientist
>> Spectral Applied Research Inc.
>> A Division of Andor Technology
>> Richmond Hill, Ontario
>> Canada
>> www.spectral.ca
>> 
>> 
>> 
>> On 2014-03-14, at 1:14 PM, Kurt Anderson wrote:
>> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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>> *****
>> hi all,
>> anyone who has worked in clinical or pre-clinical imaging will be familiar with the National Electrical Manufacturer’s Association, or NEMA.
>> they are like a north american DIN, in that they set standards for the specification and performance of electrical equipment ranging from conduit to clinical PET scanners.
>> see here<http://www.nema.org/Standards/Pages/All-Standards-by-Product.aspx> for an interesting list of standards.
>> recently i’ve been involved in using NEMA protocol NU4-2008 to characterise a pre-clinical PET scanner.
>> among other things, the protocol specifies the samples and methods to be used for quantification of the instrument sensitivity, resolution, and image quality.
>> my naive question for a friday afternoon is this: why dont we have a NEMA standard for confocal microscopes?
>> is anyone aware of this issue ever having been discussed by NEMA?
>> cheers
>> kurt
>> .
>> Prof. Kurt I. Anderson
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