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Testing Confocal Reproducibility

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Cameron Nowell

Testing Confocal Reproducibility

Hi List,

I want to test how reproducible the intensity collected by our Olympus FV1000 system is. I am planning on doing the following:

- Set up a Chroma slide for each wavelength (405, 473, 559 and 635)

- Capture a single plane from the brightest depth of the slide every 30 seconds for an hour or so.

- Repeat this on subsequent days by loading the acquisition parameters, so the only thing that has changed is the day of the week.

I have already tested stability over a 5 hour period and there is only 0.9% variation over that time. The question is though that while the system is stable for any given run, are the same results (intensity) achievable on subsequent days using the same settings.

Does what i propose seem reasonable? Any other suggestions to try?

Cheers

Cam

Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104

Facility Website

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

Stephen Cody-2

Re: Testing Confocal Reproducibility

Dear Cameron,

Your test sounds reasonable. A reproducible depth of focus will be
crucial. I'm not sure how you will ensure this is controlled
accurately from day to day. Maybe comparing vertical sections may be
safer. You will need to ensure the PMT's and lasers are given the same
amount of time each day to warm up. You may need to set the PMT's to a
set voltage during the warm up period. Although difficult, it is
possible to bleach the plastic test slides with high laser powers.
Using reasonable laser power will probably reduce bleaching to
negligible levels. See papers by Robert (Bob) Zucker, I'm sure these
will will help.

Cheers
Steve Cody

2009/12/7 Cameron Nowell <[hidden email]>:

> Hi List,
>
>
>
> I want to test how reproducible the intensity collected by our Olympus
> FV1000 system is. I am planning on doing the following:
>
>
>
> -          Set up a Chroma slide for each wavelength (405, 473, 559 and 635)
>
> -          Capture a single plane from the brightest depth of the slide
> every 30 seconds for an hour or so.
>
> -          Repeat this on subsequent days by loading the acquisition
> parameters, so the only thing that has changed is the day of the week.
>
>
>
> I have already tested stability over a 5 hour period and there is only 0.9%
> variation over that time. The question is though that while the system is
> stable for any given run, are the same results (intensity) achievable on
> subsequent days using the same settings.
>
>
>
> Does what i propose seem reasonable? Any other suggestions to try?
>
>
>
>
>
> Cheers
>
>
>
> Cam
>
>
>
>
>
>
>
> Cameron J. Nowell
> Microscopy Manager
> Centre for Advanced Microscopy
> Ludwig Institute for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>
> Office: +61 3 9341 3155
> Mobile: +61422882700
> Fax: +61 3 9341 3104
>
> Facility Website
>
>
>
> ________________________________
> This communication is intended only for the named recipient and may contain
> information that is confidential, legally privileged or subject to
> copyright; the Ludwig Institute for Cancer Research Ltd does not waive any
> rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do not
> necessarily reflect the views of the Ludwig Institute for Cancer Research
> Ltd.
>

--
Stephen H. Cody

Cameron Nowell

Re: Testing Confocal Reproducibility

Hi Stephen,

Yes focus has proved a problem in the first run. So i am now capturing a z series instead. So far the results show exactly what i thought they would. No fluctuation in the system.

I have also made sure i am in the linear response range for the PMTs on the FV1000 system (500-750V). Initial tests at 350V were to sensitive to very minor fluctuations in the system.

Cheers

Cam

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody
Sent: Wednesday, 9 December 2009 11:16 AM
To: [hidden email]
Subject: Re: Testing Confocal Reproducibility

Dear Cameron,

Your test sounds reasonable. A reproducible depth of focus will be
crucial. I'm not sure how you will ensure this is controlled
accurately from day to day. Maybe comparing vertical sections may be
safer. You will need to ensure the PMT's and lasers are given the same
amount of time each day to warm up. You may need to set the PMT's to a
set voltage during the warm up period. Although difficult, it is
possible to bleach the plastic test slides with high laser powers.
Using reasonable laser power will probably reduce bleaching to
negligible levels. See papers by Robert (Bob) Zucker, I'm sure these
will will help.

Cheers
Steve Cody

2009/12/7 Cameron Nowell <[hidden email]>:

--
Stephen H. Cody

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This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.