Laser stability over time

[Milan Esner]

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Dear colleagues,

we have new Zeiss LSM880 confocal microscope in our facility and performing measurements of laser stability over time.
I am using slide with mirror and measuring reflection on internal detectors.
However the results shows quite high fluctuation for all lasers.
I would like to ask you if you are measuring the stability in time of lasers on your microscopes and what is the correct method for this measurement and also how big fluctuation you observe on your system?

Thanks a lot for sharing your experience. Best regards,

Milan

[George McNamara]

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Hi Milan,

It could be the specimen focus changes over time. This can -- and should
-- be evaluated by X-Z scan.

I recommend:

* continuing with your reflection slide, though consider sometimes
testing a fluorescent slide like Chroma's plastic slides (or a piece of
a fluorescent plastic clipboard). If you can afford it, ArgoLight's
calibration targets are supposed to be non-bleaching.

* imaging 50:50 edge of slide and air.

* turning on the transmitted light detector, operating that at modest
gain, helps to have a neutral density filter between the condenser
(which you can parfocalize by shrinking the condenser numerical aperture
to get it in focus) and detector.

* Each laser in its own scan track ... extremely low laser power (under
2% may be unstable because of the AOTF intensity control, not the lasers
themselves). I typically operate the lasers to the ChD transmitted image
is approximately the same brightness in all tracks (keep ChD gain constant).

* "no moving parts" (other than the scanners) in the light path.

* 1 minute interval overnight ... since now Friday, can do over the weekend.

* and sure, do this in X-Z scan (field rotated to accommodate edge).

* Do every night (room temperature and other variables may be different)
and over weekends.

Have low expectations!

I encourage you to post online your results and interpretation.

When i moved to UMiami in 2007 our LSM510 had huge fluctuations. One of
the Zeiss field service engineers found 6 (or more?) years of dust in
the trap under the RTC (real time computer), which was sitting on the
floor. After the dust trap was cleaned, the 510's fluctuations were
greatly diminished (good), though not eliminated (sigh). Too bad Zeiss
did not do thorough preventive maintenances every time (i.e. every 6
months while under warranty and service contract).

enjoy,


George

On 12/15/2017 5:34 AM, Milan Esner wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear colleagues,
>
> we have new Zeiss LSM880 confocal microscope in our facility and performing measurements of laser stability over time.
> I am using slide with mirror and measuring reflection on internal detectors.
> However the results shows quite high fluctuation for all lasers.
> I would like to ask you if you are measuring the stability in time of lasers on your microscopes and what is the correct method for this measurement and also how big fluctuation you observe on your system?
>
> Thanks a lot for sharing your experience. Best regards,
>
> Milan
>
>
>  

--


George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu

July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract
supporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023
figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures

[Valeria Berno-4]

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Dear all,

I don't have very much experience on FLIM-FRET technique and up to now I
had to "use" a sample with already a fluorochrome in it.

Now I have 2 users who would like to start an experiment from scratch
and I want to start with the best conditions.

Which are the best pair of recombinant protein (for
transfection/infection) and the best pair of dyes (for direct
conjugation) to start with for a FLIM FRET experiment?

We have a single photon confocal equipped with a white laser ( so pretty
much open to all wavelength) and the FLIM system (Picoquant).

thanks in advance for all your useful reply

have a nice day

Valeria


--
ALEMBIC
Advanced Light and Electron Microscopy BioImaging Center
San Raffaele Scientific Institute
DIBIT 1
via Olgettina 58, 20132 - Milano - Italy

Tel [phone]
Fax [phone]
e-mail: [hidden email]
home page: www.hsr.it/research/alembic



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[Zdenek Svindrych-2]

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Hi Milan,
I did some quick measurement to determine short-term fluctuations of the
lasers on our LSM880, basically just transmitted light detector at fairly
low gain (but the PMT response should still be linear), a quick series of
about 100 images, 'stdev' stack projection in ImageJ.

The 633 HeNe and the 561 DPSS lasers were very stable (0.5 % RMS noise, I
did not checked how big contribution comes from the detector noise), but the
Ar-ion laser fluctuates much more, around 1 % RMS at 488 nm, around 1.5 %
RMS at 458 nm. The 514 line was the least stable, with RMS of 2 % at full
power and 3 % at standby. You can compare it with your numbers. A 3 % RMS
noise would become visible when you get 1000 (or more) photons per pixel...

I did not look at the spectral properties of the noise, but it looks like
most of it is within microsecond to millisecond range.
If you are interested in long-term fluctuations (hours, days), you first
need to make sure you have very stable detector (maybe a photodiode, or a
PMT in photon-counting mode?), or very stable light source to compare with.
You can also compare two different lasers to see where the fluctuations are
coming from.

The way you did your measurements (with a mirror in focus, confocal
detection) the main contribution will be the mechanical stability (z drift)
and a bunch of other things...

Good luck!

Best, zdenek
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
email: [hidden email]

---------- Původní e-mail ----------
Od: Milan Esner <[hidden email]>
Komu: [hidden email]
Datum: 15. 12. 2017 10:46:16
Předmět: Laser stability over time
"*****
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*****

Dear colleagues,

we have new Zeiss LSM880 confocal microscope in our facility and performing
measurements of laser stability over time.
I am using slide with mirror and measuring reflection on internal detectors.

However the results shows quite high fluctuation for all lasers.
I would like to ask you if you are measuring the stability in time of lasers
on your microscopes and what is the correct method for this measurement and
also how big fluctuation you observe on your system?

Thanks a lot for sharing your experience. Best regards,

Milan



"

[Periasamy, Ammasi (ap3t)-2]

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Hello Valeria,
Many people uses Cerulean-Venus or Teal(mTFP)-Venus for FLIM-FRET.
You can also use mTurquoise2-Venus. Teal and mTurquoise are more photostable than Cerulean.
Or,
You can attend our annual workshop to get trained on FLIM-FRET. If you are interested contact me off the list.
http://www.kcci.virginia.edu/workshop-2018 
Hope this helps.
Happy Holidays!
Ammasi

Dr. Ammasi Periasamy
Professor & Center Director,
WM Keck Center for Cellular Imaging,
Departments of Biology and Biomedical Engineering,
Univeristy of Virginia,
409 McCormick Rd., Charlottesville, VA 22903, USA.

http://www.kcci.virginia.edu/people/profile/ap3t
Phone: [phone] or 982-4869
Fax: [phone]
E-mail: [hidden email]

FRET/FLIM Workshop-March 5-9, 2018: http://www.kcci.virginia.edu/workshop 



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Valeria Berno
Sent: Monday, December 18, 2017 4:05 AM
To: [hidden email]
Subject: FLIM-FRET dye pair

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Dear all,

I don't have very much experience on FLIM-FRET technique and up to now I had to "use" a sample with already a fluorochrome in it.

Now I have 2 users who would like to start an experiment from scratch and I want to start with the best conditions.

Which are the best pair of recombinant protein (for
transfection/infection) and the best pair of dyes (for direct
conjugation) to start with for a FLIM FRET experiment?

We have a single photon confocal equipped with a white laser ( so pretty much open to all wavelength) and the FLIM system (Picoquant).

thanks in advance for all your useful reply

have a nice day

Valeria


--
ALEMBIC
Advanced Light and Electron Microscopy BioImaging Center San Raffaele Scientific Institute DIBIT 1 via Olgettina 58, 20132 - Milano - Italy

Tel [phone]
Fax [phone]
e-mail: [hidden email]
home page: www.hsr.it/research/alembic



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