Shuko

A confocal microscope is the wrong technology for
luminescence imaging.  You need to use a low light
camera detector (e.g. photon counting camera or
cooled CCD) for this purpose.

Nick


Date sent:              Wed, 21 Feb 2001 14:49:36 -0500
Send reply to:          Confocal Microscopy List <[log in to unmask]>
From:                   "Shuko Harada, M.D." <[log in to unmask]>
Organization:           University of Pennsylvania School of Medicine
Subject:                Re: Transcriptional activity using Luciferase
To:                     [log in to unmask]

> Dear Confocalist
>
> I would like to visualize real-time transcriptional
> activity using the promomter fused upstream of
> luciferase cDNA (plasmid pGL3 basic; Promega) in living
> cells using photon counting imaging after transiently
> transfected with plasmids. We have BioRad MRC 1024
> (both inverted and upright, Kr/Ar and Ti:Sapphire
> laser). The reference I have does not explain detail
> and some of key references are missing from the
> library, unfortunately. Has anyone used this technique?
> Could anyone give me some suggestions, considerations,
> protocols? Especially the methods regarding to how to
> collect pGL firefly luciferase signal (transcriptional
> activity) and Renilla reniformis luciferase signal (as
> control) separately in living condition, how to expose
> to luciferin (incubate with DMSO?, how long stable?),
> and what filter to use to collect image etc. (Since I
> am collecting luminescence, I guess I do not have to
> excite, right?) Thank you for your help!!
>
> Shuko
>
> --
> Shuko Harada, M.D.
> Assistant Professor, Research
> Dept. of Pathology and Lab Medicine
> University of Pennsylvania School of Medicine
> D101 Richards 37th Hamilton Walk
> Philadelphia, PA 19104
> Phone: 215-898-6738
> Fax: 215-573-2350


Dr Nick D. Read
Fungal Cell Biology Group
Institute of Cell and Molecular Biology
Rutherford Building
University of Edinburgh
Edinburgh EH9 3JH
UK

Email: [log in to unmask]
Tel: 0131-650-5335
Mobile: 0776-013-6521
FAX: 0131-650-5392