Posted by
Emmanuel Gustin on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cosmic-ray-particles-centenary-The-Chase-2012-tp7578803p7578836.html
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Hello,
It is not exactly the same problem, but to track performance of our
confocal over time, I used to try to keep the characteristics of the
resulting image stable, instead of the settings. The images were from
the fluorescent bead slides that are sold by Invitrogen, and we always
adjusted settings so that (with standardized choice of objectives,
pinhole, laser power, scan speed and zoom factors) the image spanned the
range of the A/D convertor. Then I wrote down the PMT settings to see
the evolution of the system over time. The results were reasonably
consistent and indicative of the state of the instrument.
Across different systems you cannot directly compare the numbers, but
you can get an impression of where the settings are in the "usable
range" of PMT and laser power settings for a particular system.
We also use their reference slide that contains muntjac skin fibroblasts
with 3-color staining. A convallaria slide is useful because it is a
fairly thick, bright and stable sample that is very easy to image. The
fibroblast slide takes a bit more time to bring into focus and adjust
(which perhaps gives a more relevant impression of a system's
user-friendliness), but its F-actin staining usually gives a better idea
of a system's resolution than convallaria. It does bleach easier than
convallaria.
Best Regards,
Emmanuel
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]]
On Behalf Of Arvydas Matiukas
Sent: Tuesday, 14 August, 2012 22:24
To:
[hidden email]
Subject: Re: simple test to compare confocals
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Dear list and George,
Thanks for all the suggestions regarding simple confocal test.
I meant a test that allows to quickly verify that confocal is
functioning normally and stable. One of typical situations is when I
need to prove to a customer that confocal is OK, and it is the bad
sample that produces poor image.
I agree that in detail testing/evaluation mentioned in some replies is
useful and interesting to perform for a new system or after a major
overhaul. I would not be very enthusiastic to do it often (e.g. weekly).
My special thanks to George whose Convallaria slide based simple test is
the best aligned with my own line of thought (which I did not present
initially to avoid any bias and get fresh ideas). I have been routinely
doing similar test on our LSM510 to verify normal and stable performance
(green/red fluorescence at FITC and Texas Red settings).
Now based on George's experience I will use the test to quickly compare
confocals. I 100% agree that it is very important to keep identical
imaging parameters. However, one of caveats may be different emission
filters (in terms of bandwidth and transmission).
I assume this simple and quick test while not being a substitute to a
strict/detail comparison answers the concerns of the Core user who has
to switch between confocals.
Best wishes,
Arvydas
>>> George McNamara <
[hidden email]> 8/13/2012 9:36 PM >>>
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Convallaria (lily of the valley) cross section slide - simplest to
borrow from your local Zeiss sales rep or service engineer. Basically,
if an Zeiss LSM or Leica SP# gives a decent image, it passes as far as a
Zeiss or Leica service engineer is concerned (unless you are looking
over their shoulder or better driving the scope and see any issues).
I've not seen Nikon service work on a confocal, so this test might be
confocal vendor standard. If you do not have a slide already; bummer
(might be some lily growing outside that you could harvest). The
www.carolina.com web site search engine is so incredibly bad, I was
unable to get a hit just now (amazon.com was no better). You can
probably find it from other prepared slide companies on the Internet.
Practical parameters:
gain 600 (not that all vendors "600' are the same, or even two PMTs in
the same scanhead).
offset so that all pixel values are above zero (i.e. no laser light)
12-bit data mode low laser power (ND filter or AOTF control level ...
need to run the Argon laser in the "good"power range) NO AVERAGING
(averaging is cheating) Fastest scan speed available [shortest pixel
dwell time] (ideally these will be identical on both instruments) ... my
thanks to Jonathan Boyd of Leica for demonstrating that "faster is
better" (SP5, standard scan speeds vs resonant scanner, sum images when
needed to get same total dwell time for each mode).
Same size image format for all instruments, i.e. 2048x2048 pixels
highest performance objective lens available, i.e. plan apo 63x/1.4 NA
oil, full resolution - by my math 60 nm pixel size for 1.4 NA
(explanation in previous messages at the listserv).
Note: Zeiss and Nikon oil do not play well together. Need to remove the
oil from the coverglass, clean the coverglass with 70% ethanol, before
oiling for the other scope.
George
p.s. I have not acquired any of my Convallaria slides for Sebastian
Munck's et al PiMP super-resolution calculation method (J Cell Sci 2012,
PubMed 22357945), but am looking forward to doing so in the future. I
have been getting very nice results with 30 nm pixel size on both our
LSM710 and Leica SP5 with 63x/1.4NA, relatively bright initially
specimens, filter size 1.625 (instead of default of 1.1 which is for
somewhat larger pixel size), 16-bit output (so I don't have to remember
where the 32-bit to 16-bit command is located), ~1% photobleaching per
time point. I find it most useful to have one channel time series per
LIF or LSM file. My thanks to Sebastian and Glen M for sending me the
ImageJ plugin.
On 8/13/2012 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.: 315-464-7997
> fax: 315-464-8014
> email:
[hidden email]
>
>