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June 2011

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From:
Dan Metcalf <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Thu, 23 Jun 2011 06:48:38 -0700
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I've been using an in-house built dSTORM microscope and so I don't have any
direct experience with commercial super-resolution microscopes. Here are
some thoughts though:

*dSTORM * (also called ground state depletion microscopy by Stefan Hell and
Leica). This will get you up to approximately 20 nm resolution. You can use
Alexa, Cy and Atto dyes so long as they're matched up with the relevant
lasers and filters on the microscopes. The key for us to getting good
resolution is to have a nicely labelled sample with nice bright structures
and low background. Having all the fluorophores in a similar plane of focus
also seems to help. For both of these reasons you'll see that these
techniques are combined with TIRF or a 'partial' TIRF. The only real
down-side for me so far is that you need to acquire multiple frames over
several minutes. This means it's better suited to a fixed sample and a
microscope that is in a thermally stable and vibration-free environment.

The fluorophore blinking behaviour is achieved with a combination of
reducing buffer and a high laser power. This is in constrast to nSTORM which
has coupled activator-receptor fluorophores with normal laser powers. I'd
recommend thinking about dSTORM as well as nSTORM.

I believe Zeiss will be launching a dSTORM system soon and Leica are
starting to demonstrate their version. 


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