How to make a spectrum graph?

[Avi Jacob]

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Hey all,

We have a novel stain that I did a lambda-lambda scan on with our Leica
SP8. I have the images and the data from the report that the acquisition
program exports.
How do I make a spectrum graph from this? i.e. The ex-em graph every
fluorophore has. I suppose I can create one with excel by plotting the
intensity from the images from scan, but there must be a more correct way.
(Please don't tell me to use Matlab...)

Cheers,
Avi

--
Avi Jacob, Ph.D.
Head of The Kanbar Light Microscopy Unit
The Mina & Everard Goodman Faculty of Life Sciences
Bar-Ilan University, Ramat-Gan 5290002, Israel
Cell: 052-5802544 (call here first), Desk: 972-3-5317647
http://tinyurl.com/BIU-Microscopy

[Benjamin Smith]

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ImageJ can also make pretty nice 2D fluorescence plots for free.  In Excel,
make a 2D matrix of the normalized intensity for each ex/em pair.  Save
this as a text file, then import the text file into ImageJ using
File->Import->Text Image.  Then go to Image->Lookup Tables->Physics,
and voilà you'll have a 2D fluorescence plot just like figure C in this
paper:
http://imagebank.osa.org/getImage.xqy?img=dTcqLmZ1bGwsb2UtMTktUzYtQTExODQtZzAwMg&article=oe-19-S6-A1184-g002


On Mon, May 6, 2019 at 12:24 AM Avi Jacob <[hidden email]> 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.
> *****
>
> Hey all,
>
> We have a novel stain that I did a lambda-lambda scan on with our Leica
> SP8. I have the images and the data from the report that the acquisition
> program exports.
> How do I make a spectrum graph from this? i.e. The ex-em graph every
> fluorophore has. I suppose I can create one with excel by plotting the
> intensity from the images from scan, but there must be a more correct way.
> (Please don't tell me to use Matlab...)
>
> Cheers,
> Avi
>
> --
> Avi Jacob, Ph.D.
> Head of The Kanbar Light Microscopy Unit
> The Mina & Everard Goodman Faculty of Life Sciences
> Bar-Ilan University, Ramat-Gan 5290002, Israel
> Cell: 052-5802544 (call here first), Desk: 972-3-5317647
> http://tinyurl.com/BIU-Microscopy
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[daj1u06]

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Hi, the function to generate the curve in the system's dye database is built into the SP8 software - having done the scan, go into the quantify tab and do a stack profile on a bright (but not saturated) ROI / area of the signal within the image. It will draw the graph for you. Right click on the graph and a drop down menu will give you the option to export to the dye database. The entry is generated automatically under the user's profile dye set - you can then move it to the all user Leica (master) dye set folder using windows explorer and it will appear there

[daj1u06]

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in LAS-X, spectral database for user lives here:
 C:\Users\....yourusername...\AppData\Roaming\Leica Microsystems\LAS X\SpectralDatabase

master set (leica) here:
C:\Program Files\Leica Microsystems CMS GmbH\LAS X\SpectralDatabase

search for .lsf files in windows explorer

can freely copy between these 2 folders

can also copy the files between SP5 and SP8 systems.

Cheers, DAJ

[Roy Edward-2]

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Dear Avi,
it might be useful to know that we only ever receive requests for our products’ spectra in Excel file format, perhaps just because of the ubiquity of the s/w.

We embedded a dual Ex/Em graph in the first one and simply update the input data subsequently.
You can crib from ours if you want but you might prefer something more elegant..
However, you can download one example here showing 4 overlaid emission profiles emanating from common excitation wavelengths:
DRAQ7™ EX and EM SPECTRA Raw Data (XLSX)<http://www.biostatus.com/site/biostatus/documents/DRAQ7.SPECTRA%20002%20180117.xlsx>

I like the idea of taking that through to ImageJ (as suggested by Ben Smith, UCB).
Best regards,
Roy

PS loved your Matlab comment - my 24 year-old geoscientist son has already developed a Matlab aversion.

Roy Edward
BioStatus Limited
56a Charnwood Road, Shepshed, Leicestershire LE12 9NP
T [phone] | F [phone] | W www.biostatus.com<http://www.biostatus.com>


Date:    Mon, 6 May 2019 10:21:50 +0300
From:    Avi Jacob <[hidden email]<mailto:[hidden email]>>
Subject: How to make a spectrum graph?

Hey all,

We have a novel stain that I did a lambda-lambda scan on with our Leica
SP8. I have the images and the data from the report that the acquisition
program exports.
How do I make a spectrum graph from this? i.e. The ex-em graph every
fluorophore has. I suppose I can create one with excel by plotting the
intensity from the images from scan, but there must be a more correct way.
(Please don't tell me to use Matlab...)

Cheers,
Avi

--
Avi Jacob, Ph.D.
Head of The Kanbar Light Microscopy Unit
The Mina & Everard Goodman Faculty of Life Sciences
Bar-Ilan University, Ramat-Gan 5290002, Israel
Cell: 052-5802544 (call here first), Desk: 972-3-5317647
http://tinyurl.com/BIU-Microscopy

[Craig Brideau]

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Once again the University of Calgary (Alberta, Canada) is hosting the
Canadian Light Microscopy Course. This is a week-long workshop/crash course
in various microscopy techniques. This course it pitched towards mid-level
researchers with some experience who are looking to branch out into new
techniques and improve their abilities with analysis and quantitative
imaging. The course includes seminars and hands-on labs with our wide
variety of microscope systems. I recommend this for anyone looking to get
more out of their microscopes!

Topics this year:

-Quantitative microscopy and analysis (Getting actual numbers from your
images, deconvolution, etc)
-Machine Learning (the analysis kind)
-FRAP and friends (aka poking your sample with light to see what happens)
-TIRF (really tiny light sheet)
-Light Sheet (regular light sheet)
-Tissue Clearing (fun with solvents!)
-3D printing for microscopy (how to put that weird custom
chamber/filter/camera on your microscope)
-Super resolution (cheating Abbe)
-Live cell (how to keep it that way)
-AND MORE!

If you are interested in attending, please contact the CLMC committee to
reserve a spot ([hidden email]).
Website and registration details: https://www.ucalgary.ca/clmc/
The hosts: https://snyder.ucalgary.ca/imaging/
LinkedIn:
https://www.linkedin.com/feed/update/urn:li:activity:6531532624161316864/

Craig