I think the 'triplet state' argument used for ground state d
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Hi Colin,
I think the 'triplet state' argument used for ground state depletion is a little
confusing/misleading as triplet states are usually not long lived enough
(typically a few us) to make a suitable dark state for super-resolution. A more
convincing description of the photo-physics/chemistry lies in the partially
reduced states described by the Sauer group (under the dSTORM acronym -
Heilemann et Al 2008, van de Linde et Al 2008, and several others) or in the
case of cyanine dyes, direct thiol conjugation. More recent publications from
the Hell group are starting to recognise this and usually say 'the triplet or
another dark state' or something to that effect. A high intersystem crossing
rate is thus not necessarily what you are after (this will also increase
photobleaching and reduce the photon count from each blink). We started out
using A488, but have since switched to near IR fluorophores (Alexa 647, 680 etc
...) in a dSTORM type buffer which seem to behave much better. If you want to
stick with A488 I'd suggest a viscous buffer (eg glycerol), as this prolongs the
dark state ( Baddeley et Al, 2009), improving contrast. If you really do want to
go down the route of enhancing isc, try some of the older dyes such as
fluorescein or TRITC - a lot of modern dye development has been directed at
reducing inter system crossing.
cheers,
David
----- Original Message ----
From: Colin Rickman <
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To:
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Sent: Wed, 27 April, 2011 8:02:48 AM
Subject: Fluorophores for ground state depletion
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Hi
Can anybody recommend a fluorophore for use in ground state depletion
microscopy. I have been using Alexa 488 with some success and am able to acquire
data at the single molecule level for localisation. However, I would like to
know if there are better options within the Alexa range or in other organic dyes
with enhanced propensity to enter triplet states, longer duration in the triplet
state, etc. that make them better candidates.
Hope someone can help.
Regards
Colin
Dr Colin Rickman
Life Science Interface
Department of Chemistry (WP 2.03)
School of Engineering and Physical Sciences
Heriot-Watt University
Edinburgh
EH14 4AS
Tel: +44 131 4514193 (Office)
http://www.lifescienceinterface.hw.ac.ukhttp://www.eps.hw.ac.uk/departments/chemistry/cr.htm
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