Posted by
Peter Gabriel Pitrone on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cosmic-ray-particles-centenary-The-Chase-2012-tp7578803p7578832.html
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Hello folks,
I don't know how much it will help, but you can look through my TechRMS
thesis on a similar topic:
http://goo.gl/qmf2OHave fun!!
Pete
On Wed, August 15, 2012 2:39 am, George McNamara wrote:
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|
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|
| Hi Arvydas,
|
| You're welcome. To test instrument stability, run the Convallaria
| overnight or over the weekend with the most important laser lines (hint:
| all are important, but Zeiss and possibly other some other vendors
| software only lets you go to four tracks ... I've done 20 scan tracks on
| the Leica SP5 - let me check different AOTF power settings). To avoid
| photobleaching the Convallaria slide (too much), use low laser power. My
| main test (explained in archived posts) is to send to transmitted light
| detector, no averaging, same low gain, condenser field aperture shrunk
| down to be in the field and adjusted to be in focus. Use an edge of the
| Convallaria slide so you have some blank area. If you are (un)lucky the
| building temperature will change during the test (I don't have access to
| an USB thermometer data recorder - would not be surprised if hidden in
| the instrument logs are occasional temperature readings from inside the
| laser enclosures).
|
| If any of the laser lines do fluctuate, don't assume it is the laser
| itself (or themselves) - Bob Zucker has suggested the AOTF(s) could be
| temperamental by way of temperature fluctuations. Of course at very low
| laser output (AOTF at <5%, certainly at 0.5%) there could be unavoidable
| fluctuations.
|
|
| Enjoy,
|
| George
|
|
|
| On 8/14/2012 4:23 PM, Arvydas Matiukas wrote:
|> *****
|> To join, leave or search the confocal microscopy listserv, go to:
|>
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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>>>
|>>>>
|> *****
|> To join, leave or search the confocal microscopy listserv, go to:
|>
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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]
|>>
|>>
|>>
|>
|
--
Peter Gabriel Pitrone - TechRMS
Microscopy/Imaging Specialist
Prof. Dr. Pavel Tomancak group
Max Planck Institute for
Molecular Biology and Genetics
Pfotenhauerstr. 108
01307 Dresden
"If a straight line fit is required, obtain only two data points." - Anon.