Re: simple test to compare confocals

Posted by George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cosmic-ray-particles-centenary-The-Chase-2012-tp7578803p7578831.html

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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:

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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.: [phone]
>> fax: [phone]
>> email: [hidden email]
>>
>>
>>      
>