Posted by
Jacqueline Ross on
May 08, 2007; 10:40pm
URL: http://imagej.273.s1.nabble.com/Please-Divide-an-Image-with-ImageJ-tp3699513p3699518.html
Dear Don,
Just a quick question regarding your image capture.
I'm just wondering why you are using an LP665 for emission collection of
your Alexa568.
You should have another more suitable barrier filter such as an OG590 or
something around 600nm.
If you are using an LP665, you will be missing out on a huge proportion
of your photons from the 568nm label.
Do you have any other filters in your system?
Cheers,
Jacqui.
Jacqueline Ross
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND
Tel: 64 9 373 7599 Ext 87438
Fax: 64 9 373 7484
http://www.health.auckland.ac.nz/biru/
-----Original Message-----
From: ImageJ Interest Group [mailto:
[hidden email]] On Behalf Of
Liu, Dongfang (NIH/NIAID) [F]
Sent: 09 May 2007 04:30
To:
[hidden email]
Subject: Image correction help!
Dear all,
Recently, I came across a problem with alexa568 and alexa647 on our TIRF
system. I have to label my two proteins using the two different dyes
(alexa568 and alexa647)simultaneously(because I have to use four dyes in
one chamber, alxa488, alexa555, alexa568 and alexa647).
Problem: When I used 568nm laser to excite the alexa568 dyes on lipid
bilayer(the emission filter is LP665), I got the fluorescence of
alexa647 in alexa568 emission channel(because 568 laser can
partially-10% excite the alexa567 laser.).
The protocol for acquiring image alexa568 and alexa647:
1, Acquire the alxa568 and alxa647 image in sequence.
2, Excitation laser for alexa568 is 568nm, emission for alexa568 is
LP665
Excitation laser for alexa568 is 647nm, emission for alexa647 is
LP665
From the spectrum properties, we can know the fluorescence of Alexa647
can not breakthrough into alexa568 channel(even I used the same emission
filter to acquiring the fluorescence of alexa568 and alexa647, because
647nm laser cannot excite the alexa568 dye)
What I want to do: I want to remove the fluorescence of alexa647 from
alexa568 channel. How to do it? Any comments would be greatly
appreciated!
Thank you very much! Have a nice day!
Best regards,
Don