From owner-biophys@hgmp.mrc.ac.uk Wed May 10 13:44:26 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 2393517A4E; Wed, 10 May 2000 13:44:25 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by hgmp.mrc.ac.uk (8.9.3/8.9.3) with ESMTP id NAA02041 for ; Wed, 10 May 2000 13:44:22 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id NAA10682 for biophys-list@hgmp.mrc.ac.uk; Wed, 10 May 2000 13:44:20 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Piers Mahon X-Newsgroups: bionet.biophysics Subject: DUAl RATIO IMAGING Date: Wed, 10 May 2000 13:50:48 +0100 Organization: University of Cambridge, England Lines: 21 Message-ID: <39195B28.F69BD275@mole.bio.cam.ac.uk> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.03 [en] (WinNT; I) To: biophys@hgmp.mrc.ac.uk Sender: owner-biophys@hgmp.mrc.ac.uk Precedence: bulk I am about to try quantitative ratio imaging on a TCS using CYAN and YELLOW fluorescent proteins with different sub-cellular location signals. I am expecting any positive results to be very subtle images differences (i.e. not blue and yellow blobs in different parts of the cell, but a cyan/yellow signal ratio that varies in sub-sections of the same blob). If you have any experience of CYAN FP/ YELLOW FP imaging, or related dual imaging, please get in touch. I'm particularly interested in advice in three areas: 1) selecting the right filter ranges to obtain the best bleed through / sensitivity compromise, 2) Indications of the random noise levels I can expect in my negative controls of dual transformants containing CYAN and YELLOW with the same location signals, 3) Suggestions for statistical tests that I can perform to see if ratio images with different signals on the two reporters are more variable than images captured using the same signal. Many thanks in advance, Piers Mahon .