[ome-users] CZI dimension

Thu Mar 6 18:25:33 GMT 2014

Hi Paul

1) You can use a ‘perfect’ or delta-function irf, in fact this is the default if you don’t import an irf explicitly.
This is placed by default at the first data point (with a perfect detector you will see no light, except background before the excitation).
As you have guessed you therefore need to exclude points before the peak to get this to work.

2) You can do this using the 'Time Min' control on the Data tab.
You can also move the irf in time using the 'IRF shift ‘ control on the IRF tab.

3) Re ROIs : Top left  Just under ‘File’ are three shapes. You can use these to draw an ROI on the intensity image & fit this region
as a single summed decay.

You can also use the ‘Integrated Min’ control on the data tab to threshold dim pixels out of the fit. Recommended!

4) If you fit only the tail after the peak (as described) My guess is that you will get a v. similar answer to other software
as it’s a fairly simple task. Once you add in irfs & multi-exponentials we’re in 40 page thesis territory I’m afraid.

Please note that although, currently, FLIMfit can’t open .msr files directly from disc you can open these images from an OMERO 5.0.0 server.
In addition we hope to release a version with this capability (via bio-formats) very shortly.

Regards

ian

On 6 Mar 2014, at 17:17, Paul Thomas (SCI) <P.Thomas at uea.ac.uk<mailto:P.Thomas at uea.ac.uk>> wrote:

Hi Ian,

I’ve messed around with FLIMfit a little this afternoon, and can import data files (*.ome.tif) and my IRF data. I was able to “shift” the IRF and use the program to generate lifetimes, but what I would like to do to start with is to compare ImSpectorPro with FLIMfit, and then to see the effect of the IRF. So, I have some basic questions on FLIMfit:

1) Can I fit data without the IRF? – I’ve tried this but I’m obviously not doing it correctly; probably related to my second question.
2) Can I set limits on the data so that I make a fit from the “peak” to the end of the recording (see example from ImSpectorPro attached)?
3) Is it possible to choose an ROI to fit (again, see example from ImSpectorPro attached) rather than a point?
4) I’m afraid I don’t know exactly how ImSpector fits the curve (I can probably find out), but I’d like to use as similar conditions as I can with FLIMfit to get a reasonable comparison. I’m not sure there’s a simple answer to this one, but some guidelines as to what options to choose (or avoid) might help? – This may require a tutorial!

Thanks,
Paul.
____________________
Dr. Paul Thomas, Manager,
The Henry Wellcome Laboratory for Cell Imaging,
Faculty of Science,
University of East Anglia,
Norwich Research Park,
Norwich,
NR4 7TJ,
United Kingdom.

e-mail: p.thomas at uea.ac.uk<mailto:p.thomas at uea.ac.uk>
Tel: +44-1603-592196
Fax: +44-1603-592250
Imaging web-site: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab
Personal web-page: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab/people

From: Munro, Ian [mailto:i.munro at imperial.ac.uk]
Sent: 06 March 2014 13:47
To: Paul Thomas (SCI)
Subject: Re: [ome-users] CZI dimension

Hi Paul

You’re already aware of FLIMfit then. Good!
 Feel free to ask if you need help with using it. We are aware that we need to get some user guides out.
FLIMfit is capable of using an image as an irf i.e. if you take a picture of something with a known lifetime (or an extremely short one like a scatterer)
you can then import that image as a irf.
Regards

Ian

On 6 Mar 2014, at 13:39, Paul Thomas (SCI) <P.Thomas at uea.ac.uk<mailto:P.Thomas at uea.ac.uk>> wrote:

Hi Ian,

Yes, I have a TCSPC detector from LaVision, and do generate FLIM files. I can do some analysis with ImSpectorPro, but have had trouble correcting for the IRF (not available in ImSpectorPro). I’ve never been able to analyse my data with the plug-in in Fiji, but have downloaded FLIMfit – although I haven’t learned how to use it properly, yet.

Regards,
Paul.
____________________
Dr. Paul Thomas, Manager,
The Henry Wellcome Laboratory for Cell Imaging,
Faculty of Science,
University of East Anglia,
Norwich Research Park,
Norwich,
NR4 7TJ,
United Kingdom.

e-mail: p.thomas at uea.ac.uk<mailto:p.thomas at uea.ac.uk>
Tel: +44-1603-592196
Fax: +44-1603-592250
Imaging web-site: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab
Personal web-page: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab/people

From: Munro, Ian [mailto:i.munro at imperial.ac.uk]
Sent: 06 March 2014 13:05
To: Paul Thomas (SCI)
Subject: Re: [ome-users] CZI dimension

Dear Paul

Given your interest in .msr files cam I ask if you are interested in FLIM ?

Ian

On 6 Mar 2014, at 08:56, Paul Thomas (SCI) <P.Thomas at uea.ac.uk<mailto:P.Thomas at uea.ac.uk>> wrote:

Hi Curtis,

Thanks. In addition, I should also thank the team for including support for ImSpectorPro (*.msr) files. I’ve had no problems (yet!) opening these files since upgrading to Bioformats 5.

Paul.
____________________
Dr. Paul Thomas, Manager,
The Henry Wellcome Laboratory for Cell Imaging,
Faculty of Science,
University of East Anglia,
Norwich Research Park,
Norwich,
NR4 7TJ,
United Kingdom.

e-mail: p.thomas at uea.ac.uk<mailto:p.thomas at uea.ac.uk>
Tel: +44-1603-592196
Fax: +44-1603-592250
Imaging web-site: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab
Personal web-page: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab/people

From: ctrueden.wisc at gmail.com<mailto:ctrueden.wisc at gmail.com> [mailto:ctrueden.wisc at gmail.com] On Behalf Of Curtis Rueden
Sent: 05 March 2014 20:08
To: Paul Thomas (SCI)
Cc: melissa at glencoesoftware.com<mailto:melissa at glencoesoftware.com>; vbindokas; ome-users at lists.openmicroscopy.org.uk<mailto:ome-users at lists.openmicroscopy.org.uk>
Subject: Re: [ome-users] CZI dimension

Hi Paul,

> I had to do a complete reinstallation of Fiji to update Bioformats to version 5.

I completed the update of Fiji to use Bio-Formats 5 earlier today. Simply updating Fiji should now give you a working Bio-Formats 5.0.0. And enabling the Bio-Formats update site (http://fiji.sc/Bio-Formats#Daily_builds) should give you the latest development builds.

Regards,
Curtis

On Thu, Feb 27, 2014 at 10:11 AM, Paul Thomas (SCI) <P.Thomas at uea.ac.uk<mailto:P.Thomas at uea.ac.uk>> wrote:
This didn't work for me, I had to do a complete reinstallation of Fiji to update Bioformats to version 5.
Paul.

____________________
Dr. Paul Thomas, Manager,
The Henry Wellcome Laboratory for Cell Imaging,
Faculty of Science,
University of East Anglia,
Norwich Research Park,
Norwich,
NR4 7TJ,
United Kingdom.

e-mail: p.thomas at uea.ac.uk<mailto:p.thomas at uea.ac.uk>
Tel: +44-1603-592196<tel:%2B44-1603-592196>
Fax: +44-1603-592250<tel:%2B44-1603-592250>
Imaging web-site: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab
Personal web-page: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab/people

> -----Original Message-----
> From: ome-users-bounces at lists.openmicroscopy.org.uk<mailto:ome-users-bounces at lists.openmicroscopy.org.uk> [mailto:ome-users-<mailto:ome-users->
> bounces at lists.openmicroscopy.org.uk<mailto:bounces at lists.openmicroscopy.org.uk>] On Behalf Of Melissa Linkert
> Sent: 27 February 2014 01:23
> To: vbindokas
> Cc: ome-users at lists.openmicroscopy.org.uk<mailto:ome-users at lists.openmicroscopy.org.uk>
> Subject: Re: [ome-users] CZI dimension
>
> Hi Vytas,
>
> > Might there be instructions on how to install the rc?  I did the
> > drag/drop in FIJI with bio-formats and/or loci_plugins jars [one at a
> > time], but when i ask help/about Loci, it still reports vers
> > 4.4.10-DEV. I take it I'm not running 5.0.
> > I've deleted all bio-formats and loci jars I found in fiji, except
> for
> > the installed rc.
> > What am I doing wrong?
>
> The best way to use 5.0.0 with Fiji is by following the instructions
> here:
>
> http://fiji.sc/Bio-Formats#Daily_builds
>
> Downloading the files by hand and copying them into the plugins folder
> is not recommended when using Fiji, as it is very easy to accidentally
> have an older version of the plugin still installed.
>
> If "Help > About Plugins > ..." shows something other than 5.0.0 after
> following those instructions, please let us know.
>
> Regards,
> -Melissa
>
> On Tue, Feb 25, 2014 at 09:27:04AM -0600, vbindokas wrote:
> > Dear Melissa,
> > Thanks for the response.
> > Might there be instructions on how to install the rc?  I did the
> > drag/drop in FIJI with bio-formats and/or loci_plugins jars [one at a
> > time], but when i ask help/about Loci, it still reports vers
> > 4.4.10-DEV. I take it I'm not running 5.0.
> > I've deleted all bio-formats and loci jars I found in fiji, except
> for
> > the installed rc.
> > What am I doing wrong?
> > thanks!
> >
> > On 2/24/2014 6:45 PM, Melissa Linkert wrote:
> > >Hi Vytas,
> > >
> > >>>It appears bioformats importer does not recognize the CZI fields
> > >>>created by the recent Lightsheet microscope system.  The
> lightsheet
> > >>>stored angular rotation "views" within the structure in addition
> to
> > >>>CH, T, Z. The importer does open the first 'view', but not beyond
> that.
> > >>>Can I request that tag/data be read for CZI?
> > >Which version of Bio-Formats are you currently using?  If you
> haven't
> > >already, I would suggest updating to 5.0.0-rc1:
> > >
> > >http://downloads.openmicroscopy.org/bio-formats/5.0.0-rc1
> > >
> > >and/or the very, very soon-to-be-released 5.0.0.  5.0.0-rc1 contains
> > >many improvements to .czi support, and I would expect it to open all
> > >rotations in a lightsheet dataset.
> > >
> > >>>The Zeiss software export is not very friendly [outputs views in
> > >>>stacks per Z and time], meaning that output must be then split and
> > >>>resorted to get data suitable for openSPIM processing.
> > >>>These are hugely big.
> > >>>If the czi could be opened as a virtual stack and then written out
> > >>>with the usual sequential tif naming, then it at least saves one
> > >>>step and much time/space.
> > >>>[I can try to upload a smaller czi file (5GBs), if that helps]
> > >A sample dataset would be very helpful if you find that there are
> > >still problems with 5.0.0-rc1/5.0.0.<http://5.0.0./>  If you are interested in
> > >uploading a file, please let me know and I will send the current FTP
> > >server information off-list.
> > >
> > >Regards,
> > >-Melissa
> > >
> > >On Fri, Feb 21, 2014 at 10:23:43AM -0600, Curtis Rueden wrote:
> > >>Hi Vytas,
> > >>
> > >>>It appears bioformats importer does not recognize the CZI fields
> > >>>created by the recent Lightsheet microscope system.
> > >>I am replying with the OME-users mailing list in CC, which is the
> > >>best place to report Bio-Formats issues.
> > >>
> > >>Regards,
> > >>Curtis
> > >>
> > >>
> > >>On Wed, Feb 19, 2014 at 8:45 AM, vbindokas
> <vbindoka at bsd.uchicago.edu<mailto:vbindoka at bsd.uchicago.edu>>wrote:
> > >>
> > >>>Dear Curtis,
> > >>>It appears bioformats importer does not recognize the CZI fields
> > >>>created by the recent Lightsheet microscope system.  The
> lightsheet
> > >>>stored angular rotation "views" within the structure in addition
> to
> > >>>CH, T, Z. The importer does open the first 'view', but not beyond
> that.
> > >>>Can I request that tag/data be read for CZI?
> > >>>The Zeiss software export is not very friendly [outputs views in
> > >>>stacks per Z and time], meaning that output must be then split and
> > >>>resorted to get data suitable for openSPIM processing.
> > >>>These are hugely big.
> > >>>If the czi could be opened as a virtual stack and then written out
> > >>>with the usual sequential tif naming, then it at least saves one
> > >>>step and much time/space.
> > >>>[I can try to upload a smaller czi file (5GBs), if that helps]
> best
> > >>>regards,
> > >>>
> > >>>--
> > >>>__
> > >>>
> > >>>Vytas Bindokas, Ph.D.
> > >>>Research Assoc. / Assoc. Prof.,
> > >>>Director, BSD Light Microscopy Core Facility
> > >>>phone: 773-702-4875<tel:773-702-4875>
> > >>>
> > >>>      [address for letters ONLY (see shipping addr below):] Dept
> > >>>Pharmacol Physiol Sci MC0926
> > >>>947 E 58th Street
> > >>>The University of Chicago
> > >>>Chicago IL 60637
> > >>>Room Abbott 129
> > >>>
> > >>>
> > >>>shipping address (main KCBD site):
> > >>>V. Bindokas
> > >>>900 E 57th Street
> > >>>KCBD room 1250, Microscopy Core
> > >>>The University of Chicago
> > >>>Chicago IL 60637
> > >>>
> > >>>
> > >>>email vbindoka at bsd.uchicago.edu<mailto:vbindoka at bsd.uchicago.edu>
> > >>>web site for LMCF:
> > >>>http://digital.uchicago.edu/index.html
> > >>>
> > >>>
> > >>_______________________________________________
> > >>ome-users mailing list
> > >>ome-users at lists.openmicroscopy.org.uk<mailto:ome-users at lists.openmicroscopy.org.uk>
> > >>http://lists.openmicroscopy.org.uk/mailman/listinfo/ome-users
> >
> > --
> > __
> >
> > Vytas Bindokas, Ph.D.
> > Research Assoc. / Assoc. Prof.,
> > Director, BSD Light Microscopy Core Facility
> > phone: 773-702-4875<tel:773-702-4875>
> >
> >      [address for letters ONLY (see shipping addr below):] Dept
> > Pharmacol Physiol Sci MC0926
> > 947 E 58th Street
> > The University of Chicago
> > Chicago IL 60637
> > Room Abbott 129
> >
> >
> > shipping address (main KCBD site):
> > V. Bindokas
> > 900 E 57th Street
> > KCBD room 1250, Microscopy Core
> > The University of Chicago
> > Chicago IL 60637
> >
> >
> > email vbindoka at bsd.uchicago.edu<mailto:vbindoka at bsd.uchicago.edu>
> > web site for LMCF:
> > http://digital.uchicago.edu/index.html
> >
> _______________________________________________
> ome-users mailing list
> ome-users at lists.openmicroscopy.org.uk<mailto:ome-users at lists.openmicroscopy.org.uk>
> http://lists.openmicroscopy.org.uk/mailman/listinfo/ome-users
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