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Zeiss Elyra PALM system and Nikon N-storm system - experiences?

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Michelle Peckham

Zeiss Elyra PALM system and Nikon N-storm system - experiences?

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Just wondering if anyone has had either of these two systems demonstrated? Or has even bought one? And what their experiences with these systems is – do they really give good 'super-resolution' results?

Dan Metcalf

Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

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.

Schoenle, Andreas

Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

In my humble opinion, your statement should read *GSDIM*[+] (also called
dSTORM).

In the paper introducing the name 'dSTORM', Heilemann et al.[3] do not point
out that (unpaired!) Cy5 molecules had been used a year earlier by Bock et
al. [2] for a stochastic single molecule switching type of nanoscopy. In
simple words, they coined the acronym dSTORM for something that had been
published already.[#]
GSDIM [4] introduces the mechanism of pumping molecules out of the ground
state to an arbitrary dark state. Explicitely, it states that this concepts
should be applicable to virtually any dye under the right conditions. In
their second paper[5], published after the GSDIM paper, Heilemann et al. then
analyze in detail the switching mechanism of this concept but decided to
ignore the acronym GSDIM. Rather they rebranded their acronym 'dSTORM' (which
was originally coined just for Cy5 without Cy3) to other 'conventional' dyes
such that it also contained the GSDIM method, which was shown already earlier
by Fölling.
Moreover, Fölling et al. pointed out the generality of the ground state
depletion switching mechanism.

[1] Rust et al. (2006) Nature Meth.
(http://www.nature.com/nmeth/journal/v3/n10/full/nmeth929.html)
[2] Bock et al. (2007) Appl. Phys. B
(http://www.nanoscopy.de/publications/pdf/Appl._Phys._B_88_161-165.pdf)
[3] Heilemann et al. (2008) Angew. Chem.
(http://onlinelibrary.wiley.com/doi/10.1002/ange.200802376/full)
[4] Fölling et al. (2008) Nature Meth.
(http://www.nanoscopy.de/publications/pdf/Nature_Meth._5_943-945.pdf)
[5] Heilemann et al. (2009) Angew. Chem.
(http://onlinelibrary.wiley.com/doi/10.1002/ange.200902073/full)

[+]
*GSD* refers to the general mechanism of switching a dye by ground state
depletion, which had been introduced as early as 1995 for STED-type
nanoscopies. This very same mechanism of switching a dye to a metastable dark
state is then used in *GSDIM* (aka dSTORM) to stochastically switch single
molecules.
[#]
The fluorophores (Cy5/Alexa647) and off-switch used in what was originally
termed dSTORM are identical to those in the original STORM paper[1], which
BTW also states that the concept is applicable to any on-switchable dye. The
possibility of directly switching on was both known from earlier publications
and, more importantly, demonstrated in the context of nanoscopy in [2], where
two-color single molecule switching nanoscopy using the fluorescent protein
rsFastLime and Cy5 is presented.

--
Andreas Schoenle, Dr.
Department of NanoBiophotonics
Max-Planck-Institute for Biophysical Chemistry D-37070 Gottingen, Germany

phone:+49 551 201 2611
fax:+49 551 201 2505
http://www.mpibpc.gwdg.de/abteilungen/200/
mailto:[hidden email]

PGP-Key: http://www.mpibpc.gwdg.de/abteilungen/200/personals/aschoen.asc

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Dan Metcalf
Sent: Thursday, June 23, 2011 3:49 PM
To: [hidden email]
Subject: Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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.

--
View this message in context:
http://confocal-microscopy-list.588098.n2.nabble.com/Zeiss-Elyra-PALM-system-
and-Nikon-N-storm-system-experiences-tp6503765p6508337.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

Jose Moranosa

Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

He asks about experiences with available commercial instruments. This
information would be useful for all. Please, share your experiences if you
have them.
*
*Also, really there are any differences between
GSDIM/STORM/dSTORM/PALM/FPALM/PALMIRA/iPALM/etc? At the heart, all these
techniques are the same thing, yes? Please tell me what distinguishes
GSDIM/STORM/PALM. To me, it looks Leica/Nikon/Zeiss have claimed the same
thing with different names.

Ciao,

Jose*

From: *"Schoenle, Andreas" <[hidden email]>

> *Date: *June 25, 2011 10:47:06 AM EDT
> *To: *[hidden email]
> *Subject: **Re: Zeiss Elyra PALM system and Nikon N-storm system -
> experiences?*
> *Reply-To: *Confocal Microscopy List <[hidden email]>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> In my humble opinion, your statement should read *GSDIM*[+] (also called
> dSTORM).
>
> In the paper introducing the name 'dSTORM', Heilemann et al.[3] do not
> point
> out that (unpaired!) Cy5 molecules had been used a year earlier by Bock et
> al. [2] for a stochastic single molecule switching type of nanoscopy. In
> simple words, they coined the acronym dSTORM for something that had been
> published already.[#]
> GSDIM [4] introduces the mechanism of pumping molecules out of the ground
> state to an arbitrary dark state. Explicitely, it states that this concepts
> should be applicable to virtually any dye under the right conditions. In
> their second paper[5], published after the GSDIM paper, Heilemann et al.
> then
> analyze in detail the switching mechanism of this concept but decided to
> ignore the acronym GSDIM. Rather they rebranded their acronym 'dSTORM'
> (which
> was originally coined just for Cy5 without Cy3) to other 'conventional'
> dyes
> such that it also contained the GSDIM method, which was shown already
> earlier
> by Fölling.
> Moreover, Fölling et al. pointed out the generality of the ground state
> depletion switching mechanism.
>
> [1] Rust et al. (2006) Nature Meth.
> (http://www.nature.com/nmeth/journal/v3/n10/full/nmeth929.html)
> [2] Bock et al. (2007) Appl. Phys. B
> (http://www.nanoscopy.de/publications/pdf/Appl._Phys._B_88_161-165.pdf)
> [3] Heilemann et al. (2008) Angew. Chem.
> (http://onlinelibrary.wiley.com/doi/10.1002/ange.200802376/full)
> [4] Fölling et al. (2008) Nature Meth.
> (http://www.nanoscopy.de/publications/pdf/Nature_Meth._5_943-945.pdf)
> [5] Heilemann et al. (2009) Angew. Chem.
> (http://onlinelibrary.wiley.com/doi/10.1002/ange.200902073/full)
>
> [+]
> *GSD* refers to the general mechanism of switching a dye by ground state
> depletion, which had been introduced as early as 1995 for STED-type
> nanoscopies. This very same mechanism of switching a dye to a metastable
> dark
> state is then used in *GSDIM* (aka dSTORM) to stochastically switch single
> molecules.
> [#]
> The fluorophores (Cy5/Alexa647) and off-switch used in what was originally
> termed dSTORM are identical to those in the original STORM paper[1], which
> BTW also states that the concept is applicable to any on-switchable dye.
> The
> possibility of directly switching on was both known from earlier
> publications
> and, more importantly, demonstrated in the context of nanoscopy in [2],
> where
> two-color single molecule switching nanoscopy using the fluorescent protein
> rsFastLime and Cy5 is presented.
>
> --
> Andreas Schoenle, Dr.
> Department of NanoBiophotonics
> Max-Planck-Institute for Biophysical Chemistry D-37070 Gottingen, Germany
>
> phone:+49 551 201 2611
> fax:+49 551 201 2505
> http://www.mpibpc.gwdg.de/abteilungen/200/
> mailto:[hidden email]
>
> PGP-Key: http://www.mpibpc.gwdg.de/abteilungen/200/personals/aschoen.asc
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On
> Behalf Of Dan Metcalf
> Sent: Thursday, June 23, 2011 3:49 PM
> To: [hidden email]
> Subject: Re: Zeiss Elyra PALM system and Nikon N-storm system -
> experiences?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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.
>
>
> --
> View this message in context:
>
> http://confocal-microscopy-list.588098.n2.nabble.com/Zeiss-Elyra-PALM-system-
> and-Nikon-N-storm-system-experiences-tp6503765p6508337.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
>

David Baddeley

Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The short (and somewhat simplified) answer is that anything with PALM in it uses
fluorescent proteins and/or caged compounds, whereas the rest use different
subsets of organic dyes which must first be shelved into a dark state. The
equipment and data analysis algorithms required are largely identical and the
proliferation of names has more to do with turf wars, marketing, and
intellectual property circumvention than anything else.

This acronym proliferation has not been aided by the fact that many of the key
discoveries have happened more or less simultaneously in a number of different
labs (PALM, STORM, and fPALM were all in press at the same time. With GSDIM,
dSTORM and RPM [Baddeley et Al, 2009], it was even closer, with all papers
having been submitted to the same journal at the same time - unfortunately the
editors chose to only publish the GSDIM paper and the other two had to be
resubmitted elsewhere, resulting in somewhat delayed publication).

Whilst I empathise with Andreas's frustration, I believe that there is a lot of
'prior art' in the field and that one also could construct a similar argument
for the use of other acronyms such as RPM or SPDM (eg. Reymann et Al 2008 -
first use of non carboxycyanine organic dyes for localisation microscopy,
Kaufman 1971, Wick 1975, Rundquist 1976 and others - observation of 'reversible
photobleaching' in ensemble populations, Song et Al 1996 and others -
spectroscopic characterisation of the mechanisms behind reversible
photobleaching and the effect of MEA - notably they came up with the same long
lived dark states which have more recently been described by Heilemann et Al).
In the end, however, this leads to a whole pile of confusion for the end user
and I believe that dSTORM is not a particularly bad acronym, due to the
relatively minimal embellishment over, and clear linkage to traditional STORM.

Both GSD and SPDM have the disadvantage of having previously
been associated with different techniques: a STED-like point scanning approach
and a spectrally separated position measurement method respectively, not helping
the confusion. GSD also has the disadvantage of being strongly linked to the
triplet as the long lived dark state - although the original paper does raise
the possibility of other dark states. Typical triplet lifetimes are usually
orders of magnitude too short to constitute the long lived dark state
(particularly in the presence of triplet quenchers such as MEA) and a partially
reduced radical state seems much more likely.

Cheers,
David

----- Original Message ----
From: Jose Moranosa <[hidden email]>
To: [hidden email]
Sent: Sun, 26 June, 2011 8:24:19 AM
Subject: Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

He asks about experiences with available commercial instruments. This
information would be useful for all. Please, share your experiences if you
have them.
*
*Also, really there are any differences between
GSDIM/STORM/dSTORM/PALM/FPALM/PALMIRA/iPALM/etc? At the heart, all these
techniques are the same thing, yes? Please tell me what distinguishes
GSDIM/STORM/PALM. To me, it looks Leica/Nikon/Zeiss have claimed the same
thing with different names.

Ciao,

Jose*

From: *"Schoenle, Andreas" <[hidden email]>

Guy Cox-2

Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Yes, well, I was reduced to coining this acronym: Arbitrary Conventions for Refining Original Names for Your Microscope.

OK, more seriously, I don't have any axe to grind in this one. My only connection was being a referee for one of the papers David mentions (and no, I didn't reject it and force it to be delayed!). However, the term STORM (Stochastic Optical Resolution Microscopy) does actually cover all the methods described here, so to my mind inventing a new name has no merit other than staking a claim. Hence, to me, dSTORM seems sensible, and acknowledges what has been done before. GSDim and RPM are unnecessary coinages. They are all varieties of the pre-existing technique STORM - in fact they are all essentially the same variety of that technique.

Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Baddeley
Sent: Sunday, 26 June 2011 11:35 AM
To: [hidden email]
Subject: Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The short (and somewhat simplified) answer is that anything with PALM in it uses
fluorescent proteins and/or caged compounds, whereas the rest use different
subsets of organic dyes which must first be shelved into a dark state. The
equipment and data analysis algorithms required are largely identical and the
proliferation of names has more to do with turf wars, marketing, and
intellectual property circumvention than anything else.

This acronym proliferation has not been aided by the fact that many of the key
discoveries have happened more or less simultaneously in a number of different
labs (PALM, STORM, and fPALM were all in press at the same time. With GSDIM,
dSTORM and RPM [Baddeley et Al, 2009], it was even closer, with all papers
having been submitted to the same journal at the same time - unfortunately the
editors chose to only publish the GSDIM paper and the other two had to be
resubmitted elsewhere, resulting in somewhat delayed publication).

Whilst I empathise with Andreas's frustration, I believe that there is a lot of
'prior art' in the field and that one also could construct a similar argument
for the use of other acronyms such as RPM or SPDM (eg. Reymann et Al 2008 -
first use of non carboxycyanine organic dyes for localisation microscopy,
Kaufman 1971, Wick 1975, Rundquist 1976 and others - observation of 'reversible
photobleaching' in ensemble populations, Song et Al 1996 and others -
spectroscopic characterisation of the mechanisms behind reversible
photobleaching and the effect of MEA - notably they came up with the same long
lived dark states which have more recently been described by Heilemann et Al).
In the end, however, this leads to a whole pile of confusion for the end user
and I believe that dSTORM is not a particularly bad acronym, due to the
relatively minimal embellishment over, and clear linkage to traditional STORM.

Both GSD and SPDM have the disadvantage of having previously
been associated with different techniques: a STED-like point scanning approach
and a spectrally separated position measurement method respectively, not helping
the confusion. GSD also has the disadvantage of being strongly linked to the
triplet as the long lived dark state - although the original paper does raise
the possibility of other dark states. Typical triplet lifetimes are usually
orders of magnitude too short to constitute the long lived dark state
(particularly in the presence of triplet quenchers such as MEA) and a partially
reduced radical state seems much more likely.

Cheers,
David

----- Original Message ----
From: Jose Moranosa <[hidden email]>
To: [hidden email]
Sent: Sun, 26 June, 2011 8:24:19 AM
Subject: Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

He asks about experiences with available commercial instruments. This
information would be useful for all. Please, share your experiences if you
have them.
*
*Also, really there are any differences between
GSDIM/STORM/dSTORM/PALM/FPALM/PALMIRA/iPALM/etc? At the heart, all these
techniques are the same thing, yes? Please tell me what distinguishes
GSDIM/STORM/PALM. To me, it looks Leica/Nikon/Zeiss have claimed the same
thing with different names.

Ciao,

Jose*

From: *"Schoenle, Andreas" <[hidden email]>

James Pawley

Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

STORM in a teacup?

As I may have noted to this list before, the
technique of centroid-finding as a method of
improving the resolution of an optical system
(but admittedly not of combining this with a
system for turning point emitters on and off)
goes back to a least to the 1970s:

An earlier digital system for locating and
storing the centroid of a single-photoelectron
event in an electronic image acquisition system
is described in

Herrmann, Krahl Rust and Ulrichs, Optik, 44,(1976)-4, pp 393-412 (see figure 7)

Herrmann, Krahl and Rust, Ultramicroscopy, 3 (1978) pp 227-235.

Herrmann, and Krahl, J. Microscopy, 127-1 (1982) pp 17-28

and many other places

The system was for viewing very low dose images
in an electron microscope and used an intensified
vidicon camera coupled to a phosphor conversion
screen. Each electron in the electron microscope
made a flash on the screen and this was then
amplified and detected by the intensified
vidicon. Using this procedure, the spot produced
by each microscope electron was quite large (4-5
tv lines wide) and consequently the resulting
image was very blurred. To overcome this, the
authors built (!) a 256x256 video-rate digital
image memory and then developed software (or more
likely hardware) that ran in real time to
calculate the centroid of each flash, slowly
accumulating an image in the digital memory in
which each flash was recorded as a single unit
event located where the centroid of the original
flash had been.

Of course, in this case, the source of the
blurring to be removed was the low resolution of
the intensified vidicon, not an imperfection in
the electron microscope itself. However, if you
consider the microscope+vidicon as a single
functional unit, the parallel is quite close: it
was made to "see and record" an electron landing
place; the newer LM analogs is made to "see and
record" the location of a fluorescent molecule.

I saw the Rust system operate in the early 1980s.
It was set to accumulate in an exponential
(running?) average manner (stored pixel value
added to new value and the sum divided by a
constant to create new stored value) suitable for
averaging about 120 frames (4 second decay). One
could watch the image of a single biological
molecule first begin to appear out of the
background and then turn into a featureless blob
as radiation damage took its toll.

This general system was later commercialized by
Hamamatsu in a system that they called PIAS
(Photon-counting image acquisition system, See:
http://www.sao.ru/drabek/CCDP/Hamamatsu/CAMERAS/PiasE.htm
for updated version.). Some PIAS system simply
gated the single-photoelectron flashes to a
standard height but a more advanced system was
also offered in which a computer again computed
the centroid of each flash and stored a single
count at its location. I have a catalog
describing the system dated May 1985.

Like many productive new developments, PALM and
STORM and the other variant are actually
improvements or recombinations of earlier
techniques although often the new "inventors"
were unaware of the earlier work. You can make
the case that the confocal was "invented" at
least 7 times, and Guy makes a good case for a
much longer list.

Priority is important. But it is not
all-important. Putting the last piece in the
puzzle may produce something that works, but this
doesn't change the fact that without many of the
earlier pieces, the puzzle would still remain
incomplete.

All that stated, my problem wit all of these
techniques is how they display the data. Usually
they place a blob into the image memory at the
location of the centroid and the narrowness and
brightness of this blob is often related to the
accuracy of the fit fit of the "Gaussian" to the
detected blob. But this says nothing about the
statistics of the dye molecules themselves. How
many were actually detected? Why are some shown
brighter and smaller when the main variable may
simply be how many times these they emitted
before being bleached (or exactly where they are
located in the evanescent field) rather than the
significance of the labeled molecule to the
function of the cell.

I worry that the proclivity of our visual system
to look for "edges" and then interpret these
collections of blob as though they were some sort
of "tapioca" pudding may lead us draw unsupported
interpretations from these images.

We may have solved "The Diffraction" problem but
"The Statistics" are still unclear because it has
stopped being the (nice, understandable Poisson)
statistics related to the difficulty in counting
a very small number of quantum events and become
the statistics that determine which individual
fluorophors have been recorded and what factors
in their local microenviroment might have been
important in this selection, as well as the
(Poisson-like?) errors associated with observing
very small numbers of molecules.

Regards,

Jim Pawley
--

>
>Yes, well, I was reduced to coining this
>acronym: Arbitrary Conventions for Refining
>Original Names for Your Microscope.
>
>OK, more seriously, I don't have any axe to
>grind in this one. My only connection was being
>a referee for one of the papers David mentions
>(and no, I didn't reject it and force it to be
>delayed!). However, the term STORM (Stochastic
>Optical Resolution Microscopy) does actually
>cover all the methods described here, so to my
>mind inventing a new name has no merit other
>than staking a claim. Hence, to me, dSTORM
>seems sensible, and acknowledges what has been
>done before. GSDim and RPM are unnecessary
>coinages. They are all varieties of the
>pre-existing technique STORM - in fact they are
>all essentially the same variety of that
>technique.
>
> Guy
>
>Optical Imaging Techniques in Cell Biology
>by Guy Cox CRC Press / Taylor & Francis
> http://www.guycox.com/optical.htm
>______________________________________________
>Associate Professor Guy Cox, MA, DPhil(Oxon)
>Australian Centre for Microscopy & Microanalysis,
>Madsen Building F09, University of Sydney, NSW 2006
>
>Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
>______________________________________________
> http://www.guycox.net
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On
>Behalf Of David Baddeley
>Sent: Sunday, 26 June 2011 11:35 AM
>To: [hidden email]
>Subject: Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?
>
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>
>The short (and somewhat simplified) answer is
>that anything with PALM in it uses
>fluorescent proteins and/or caged compounds, whereas the rest use different
>subsets of organic dyes which must first be shelved into a dark state. The
>equipment and data analysis algorithms required are largely identical and the
>proliferation of names has more to do with turf wars, marketing, and
>intellectual property circumvention than anything else.
>
>This acronym proliferation has not been aided by the fact that many of the key
>discoveries have happened more or less simultaneously in a number of different
>labs (PALM, STORM, and fPALM were all in press at the same time. With GSDIM,
>dSTORM and RPM [Baddeley et Al, 2009], it was even closer, with all papers
>having been submitted to the same journal at the same time - unfortunately the
>editors chose to only publish the GSDIM paper and the other two had to be
>resubmitted elsewhere, resulting in somewhat delayed publication).
>
>Whilst I empathise with Andreas's frustration, I
>believe that there is a lot of
>'prior art' in the field and that one also could construct a similar argument
>for the use of other acronyms such as RPM or SPDM (eg. Reymann et Al 2008 -
>first use of non carboxycyanine organic dyes for localisation microscopy,
>Kaufman 1971, Wick 1975, Rundquist 1976 and
>others - observation of 'reversible
>photobleaching' in ensemble populations, Song et Al 1996 and others -
>spectroscopic characterisation of the mechanisms behind reversible
>photobleaching and the effect of MEA - notably they came up with the same long
>lived dark states which have more recently been described by Heilemann et Al).
>In the end, however, this leads to a whole pile of confusion for the end user
>and I believe that dSTORM is not a particularly bad acronym, due to the
>relatively minimal embellishment over, and clear linkage to traditional STORM.
>
>Both GSD and SPDM have the disadvantage of having previously
>been associated with different techniques: a STED-like point scanning approach
>and a spectrally separated position measurement
>method respectively, not helping
>the confusion. GSD also has the disadvantage of being strongly linked to the
>triplet as the long lived dark state - although the original paper does raise
>the possibility of other dark states. Typical triplet lifetimes are usually
>orders of magnitude too short to constitute the long lived dark state
>(particularly in the presence of triplet
>quenchers such as MEA) and a partially
>reduced radical state seems much more likely.
>
>Cheers,
>David
>
>
>----- Original Message ----
>From: Jose Moranosa <[hidden email]>
>To: [hidden email]
>Sent: Sun, 26 June, 2011 8:24:19 AM
>Subject: Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?
>
>*****
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>
>He asks about experiences with available commercial instruments. This
>information would be useful for all. Please, share your experiences if you
>have them.
>*
>*Also, really there are any differences between
>GSDIM/STORM/dSTORM/PALM/FPALM/PALMIRA/iPALM/etc? At the heart, all these
>techniques are the same thing, yes? Please tell me what distinguishes
>GSDIM/STORM/PALM. To me, it looks Leica/Nikon/Zeiss have claimed the same
>thing with different names.
>
>Ciao,
>
>Jose*
>
>
>From: *"Schoenle, Andreas" <[hidden email]>
>
>> *Date: *June 25, 2011 10:47:06 AM EDT
>> *To: *[hidden email]
>> *Subject: **Re: Zeiss Elyra PALM system and Nikon N-storm system -
>> experiences?*
>> *Reply-To: *Confocal Microscopy List <[hidden email]>
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> In my humble opinion, your statement should read *GSDIM*[+] (also called
>> dSTORM).
>>
>> In the paper introducing the name 'dSTORM', Heilemann et al.[3] do not
>> point
>> out that (unpaired!) Cy5 molecules had been used a year earlier by Bock et
>> al. [2] for a stochastic single molecule switching type of nanoscopy. In
>> simple words, they coined the acronym dSTORM for something that had been
>> published already.[#]
>> GSDIM [4] introduces the mechanism of pumping molecules out of the ground
>> state to an arbitrary dark state. Explicitely, it states that this concepts
>> should be applicable to virtually any dye under the right conditions. In
>> their second paper[5], published after the GSDIM paper, Heilemann et al.
>> then
>> analyze in detail the switching mechanism of this concept but decided to
>> ignore the acronym GSDIM. Rather they rebranded their acronym 'dSTORM'
>> (which
>> was originally coined just for Cy5 without Cy3) to other 'conventional'
>> dyes
>> such that it also contained the GSDIM method, which was shown already
>> earlier
>> by Fölling.
>> Moreover, Fölling et al. pointed out the generality of the ground state
>> depletion switching mechanism.
>>
>> [1] Rust et al. (2006) Nature Meth.
>> (http://www.nature.com/nmeth/journal/v3/n10/full/nmeth929.html)
>> [2] Bock et al. (2007) Appl. Phys. B
>> (http://www.nanoscopy.de/publications/pdf/Appl._Phys._B_88_161-165.pdf)
> > [3] Heilemann et al. (2008) Angew. Chem.
>> (http://onlinelibrary.wiley.com/doi/10.1002/ange.200802376/full)
>> [4] Fölling et al. (2008) Nature Meth.
>> (http://www.nanoscopy.de/publications/pdf/Nature_Meth._5_943-945.pdf)
>> [5] Heilemann et al. (2009) Angew. Chem.
>> (http://onlinelibrary.wiley.com/doi/10.1002/ange.200902073/full)
>>
>> [+]
>> *GSD* refers to the general mechanism of switching a dye by ground state
>> depletion, which had been introduced as early as 1995 for STED-type
>> nanoscopies. This very same mechanism of switching a dye to a metastable
> > dark
>> state is then used in *GSDIM* (aka dSTORM) to stochastically switch single
>> molecules.
>> [#]
>> The fluorophores (Cy5/Alexa647) and off-switch used in what was originally
>> termed dSTORM are identical to those in the original STORM paper[1], which
>> BTW also states that the concept is applicable to any on-switchable dye.
>> The
>> possibility of directly switching on was both known from earlier
>> publications
>> and, more importantly, demonstrated in the context of nanoscopy in [2],
>> where
>> two-color single molecule switching nanoscopy using the fluorescent protein
>> rsFastLime and Cy5 is presented.
>>
>> --
>> Andreas Schoenle, Dr.
>> Department of NanoBiophotonics
>> Max-Planck-Institute for Biophysical Chemistry D-37070 Gottingen, Germany
>>
>> phone:+49 551 201 2611
>> fax:+49 551 201 2505
>> http://www.mpibpc.gwdg.de/abteilungen/200/
>> mailto:[hidden email]
>>
>> PGP-Key: http://www.mpibpc.gwdg.de/abteilungen/200/personals/aschoen.asc
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On
>> Behalf Of Dan Metcalf
>> Sent: Thursday, June 23, 2011 3:49 PM
>> To: [hidden email]
>> Subject: Re: Zeiss Elyra PALM system and Nikon N-storm system -
>> experiences?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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.
>>
>>
>> --
>> View this message in context:
>>
>>
>>http://confocal-microscopy-list.588098.n2.nabble.com/Zeiss-Elyra-PALM-system-
>> and-Nikon-N-storm-system-experiences-tp6503765p6508337.html
>> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>>
>>
>>

--
****************************************
Prof. James B. Pawley, Phone: 604-822-7801
3D Microscopy of Living Cells: Summer Course CELL: 778-919-3176
Info at: http://www.3dcourse.ubc.ca
"If it isn't diffraction, it is statistics":Microscopist's complaint, Anon.

Mark Bates

Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

hi all,

Thanks Jim for your synopsis of the related work in the EM field - I wasn't aware of that. Just to touch on a couple of the points raised, I agree with Jim regarding the statistics of switchable molecules - this can result in the STORM image becoming non-linear with respect to how the intensity in the image corresponds to the local concentration of fluorophores on the sample. This all depends on the nature of the switchable fluorophore of course, whether it activates once and then bleaches, or if it's reversibly switchable, etc. In principle it would be very useful to treat these images as quantitative maps of fluorophore position and concentration, and this is where one needs to be careful in considering the switching statistics of the fluorophore.

Regarding the name debate, PALM, STORM, FPALM etc all refer to precisely the same concept. PALM should not be associated strictly with proteins, nor should STORM be associated with one type of fluorescent probe. We made it clear in our original paper that the concept is applicable to any switchable fluorophore. I don't see the need for the acronyms which have been subsequently coined, except where there is new science involved (such as GSDIM, which extends the concept to any photophysical dark state).

best,
Mark Bates, Ph.D.
Max Planck Institute for Biophysical Chemistry
Göttingen 37077
Germany

Csúcs Gábor

Re: Zeiss Elyra PALM system and Nikon N-storm system - experiences?

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To join, leave or search the confocal microscopy listserv, go to:
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Leaving the discussion aside, I've seen both systems demonstrated and
recently took a look also at the Leica system. For me the main difference
(if I neglect all the licensing/patent issues) was that in the GSDIM
(Leica) they use considerably stronger lasers ( around 600 mW).

Greetings Gabor

Light Microscopy Centre
ETH Zurich,
Schafmattstrasse 18
CH-8093, Zurich
Switzerland

Phone: +41 44 633 6221

On 6/27/11 2:48 PM, "Mark Bates" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>hi all,
>
>Thanks Jim for your synopsis of the related work in the EM field - I
>wasn't
>aware of that. Just to touch on a couple of the points raised, I agree
>with
>Jim regarding the statistics of switchable molecules - this can result in
>the STORM image becoming non-linear with respect to how the intensity in
>the
>image corresponds to the local concentration of fluorophores on the
>sample.
>This all depends on the nature of the switchable fluorophore of course,
>whether it activates once and then bleaches, or if it's reversibly
>switchable, etc. In principle it would be very useful to treat these
>images
>as quantitative maps of fluorophore position and concentration, and this
>is
>where one needs to be careful in considering the switching statistics of
>the
>fluorophore.
>
>Regarding the name debate, PALM, STORM, FPALM etc all refer to precisely
>the
>same concept. PALM should not be associated strictly with proteins, nor
>should STORM be associated with one type of fluorescent probe. We made it
>clear in our original paper that the concept is applicable to any
>switchable
>fluorophore. I don't see the need for the acronyms which have been
>subsequently coined, except where there is new science involved (such as
>GSDIM, which extends the concept to any photophysical dark state).
>
>best,
>Mark Bates, Ph.D.
>Max Planck Institute for Biophysical Chemistry
>Göttingen 37077
>Germany
>
>--
>View this message in context:
>http://confocal-microscopy-list.588098.n2.nabble.com/Zeiss-Elyra-PALM-syst
>em-and-Nikon-N-storm-system-experiences-tp6503765p6520262.html
>Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

Clements, Ian

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