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Dear Christophe,

You might like to look at an applications note in
www.clontech.com/archive/JUL01UPD/pdf/FourColor.pdf by Hawlwy et al.
which seems to do just what you want (but at the flow cytometry level)
using only optical filter-based systems.
Whether the filters can be incorporated into your TCS-NT is of course
another matter, which can only be answered by your Leica
representatives.

You may, however, find this problem more easily soluble by using the
TCS-SP2 which Leica designed specifically for the type of problem you
seem to be addressing here. Nevertheless I always like to see someone
trying to extract the best from older technology which is still
basically excellent in its design.
Good luck.

Jim


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Christophe Leterrier
Sent: Thursday, November 25, 2004 4:47 PM
To: [log in to unmask]
Subject: TCS-NT with eCFP and eYFP

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Hi everybody,

Just two questions about developement of a quite old Leica TCS-NT
Confocal, the one that uses conventionnal filters in the detection part
of the scanhead.

1) We would like to image eCFP and eYFP, not necessarily simultaneously.
We have an Ar/Kr and we currently have the 458, 488,
568 and 647 nm lines working (our AOTF can support up to 4 lines). For
eYFP eGFP settings are quite OK (488 excitation, RSP500 dichroic, BP
525/50 emission). I tried to image eCFP using 458 nm excitation with the
same dichroic and emission filters but I got nothing. I was wondering if
I could get a dichroic with <500nm in order to detect eCFP fluorescence
at lower wavelength. For simultaneous detection, is there any DD458/488
? or should we get the 514nm laser line working for eYFP and use a
DD458/514 ? I tried to have a look at the literature but authors rarely
go in such optical details in the material & methods...

2) We just got a nice XY automated stage, but the only Leica LCS macro I
got can memorize XY position but not the motorized Z position. Do anyone
have a macro that memorizes XYZ position for multiple time-lapse
experiments ? More general, do anyone know where I can find a reference
for all objects used by the Leica TCS VBA macro programming ?

Thanks,


-- Christophe LETERRIER -------------------

Ph D Student
Neuronal Receptors Team
Neurobiology Laboratory
ESPCI-CNRS UMR 7637
10, rue Vauquelin 75231 Paris Cedex 05

Tel 01 40 79 51 84
Fax 01 40 79 47 67
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