Re: simple test to compare confocals (semi-commercial response)

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cosmic-ray-particles-centenary-The-Chase-2012-tp7578803p7578821.html

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Arvydas,

This question has been posed to the listserver several times over the years. Head to head comparisons of various instruments, while not impossible, are extremely non-trivial. To be absolutely fair, the tests have to take into account many instrument parameters; for example, read through Pawley's "39 steps":

http://www.google.ca/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CEQQFjAA&url=http%3A%2F%2Flabs.pbrc.edu%2Fcellbiology%2Fdocuments%2F39steps.pdf&ei=01wpUODWIqq36gH484GYCg&usg=AFQjCNHwENzXTObG9pAP5cVHyzUkybIErA

Unfortunately, there are no standard samples for fluorescence microscopy, but as you mentioned, there have been several researchers who have tried to come up with something that works well. I would also recommend taking a look at the following publications:

Zwier, J.M., et al., Image calibration in fluorescence microscopy. Journal of Microscopy-Oxford, 2004. 216: p. 15-24.

Zwier, J.M., et al., Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and sipchart-based calibration procedures. Journal of Microscopy-Oxford, 2008. 231(1): p. 59-69.

Murray, J.M., et al., Evaluating performance in three-dimensional fluorescence microscopy. Journal of Microscopy-Oxford, 2007. 228(3): p. 390-405.


Personally, I am a big fan of Mike Model's "concentrated dye solutions" which, like the thin polymer films described in the publications by Zwier et al., offer a simple means to assess field illumination uniformity, (axial) resolution, and intensity across the visible spectrum (3 diagnostics from one type of sample):

Model, M.A. and J.K. Burkhardt, A standard for calibration and shading correction of a fluorescence microscope. Cytometry, 2001. 44(4): p. 309-316.

Model, M.A. and J.L. Blank, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope. Journal of Microscopy-Oxford, 2008. 229(1): p. 12-16.

These dye solutions are cheap and very easy to make, not requiring any special equipment (something very important for any kind of microscope imaging "standard"). We used these types of solutions a year ago for characterizing the illumination uniformity in spinning disk confocal microscopes. More information can be found on the Spectral Applied Research Website here:

http://www.spectral.ca/_files/file.php?fileid=fileChRCVXJwJl&filename=file_2_Borealis_Uniformity_Protocol.pdf
http://www.youtube.com/watch?v=Fn8Q5AYusOI
http://www.spectral.ca/Downloads/index.html

Your question also brings to mind a quote from another very well-written article on the topic:

"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.

Cheers,

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2012-08-13, at 3:36 PM, Arvydas Matiukas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear List,
>
> I am looking for  simple and quick test (and sample) to compare
> acquisition across different confocals. Specifically, to our LSM510
> there was recently added Nikon C2 , and users want to know how
> they compare by few practical parameters, e.g. signal sensitivity,
> spectral bleedthrough, bleaching, and  maybe resolution  .
>
> I am aware of papers by Zucker, Pawley, Cole that describe detail
> evaluation of confocal performance, and even recently measured
> some PSFs. However, I am looking for just simple and quick
> test that would allow direct visual comparison of images (simple
> analysis like getting intensity histogram is fine) acquired on different
> confocals.
>
> Please share your thoughts and/or experience.
>
> Thank you in advance,
> Arvydas
> ***************************
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> Department of Pharmacology
> SUNY Upstate Medical University
> 766 Irving Ave., WH 3167
> Syracuse, NY 13210
> tel.: [phone]
> fax: [phone]
> email: [hidden email]