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Dear List members,

I would like to respond to Andreas Schoenle’s post. Even if Christoph Cremer was not Hell’s thesis advisor, he was a professor in Hell’s department during most of Hell's years as a graduate student, and their common interest in superresolution microscopy was an important connection. Furthermore, two decades earlier, Cremer and his brother had filed a patent application on the basic concept of the 4Pi microscope, which was to illuminate a point coherently from all directions, rather from just the one side as in previous microscopes, and then to scan that illuminated point in a raster pattern, detect the light emitted from the scanned point, and form an image with the detected light. In their patent application, the Cremer brothers even named their instrument the 4Pi microscope. That application is available for download from the German Patent Office.

Cremer has been gracious enough to credit Hell with improving his original concept by combining it with the confocal microscope concept, though needless to say, a patent examiner might call such a combination obvious. Hell’s ’09 Nature Methods review article, “Microscopy and its focal switch”, included a discussion of the history of the 4Pi microscope. By the time he wrote that article, Hell was obviously aware that the Cremer brothers were the original inventors of that instrument. Yet of the four references on the 4Pi microscope that Hell cited in that paper, the earliest was his own 1990 patent application, implying that Hell’s was the original inventor of that instrument. In that paper, Hell’s only acknowledgement of the Cremer’s work was, “based on inadequate assumptions about the nature of diffraction and hence did not lead to valid conclusions.” This comment speaks for itself.

Andreas is correct that this year’s Chemistry Nobel prize was awarded for the breakthrough in resolution, enabling not just an improvement over the Abbe diffraction limit, such as the 40% improvement afforded by Minsky's confocal microscope, but for the complete elimination of that limit, in principal enabling resolution at molecular dimensions. That breakthrough improvement is embodied in the “STED resolution equation,”

d = λ/2n sin α √ (1 + a Imax/Is)
which shows that as the intensity Imax of the STED beam increases without limit, the minimum distance d between just-resolvable points goes to zero without limit.

Hell claims that the first microscope embodying that breakthrough resolution performance was described in his 1994 paper with Wichmann. That is simply untrue, as anyone can easily see looking at the paper. The microscope described in the Hell and Wichmann paper is limited to a resolution improvement of about fourfold. At its maximum resolution, the fluorescent image is quenched out of existence, so further increases in the intensity of the STED beam make no difference. I urge members of this discussion group to download the 1994 Hell and Wichmann paper from Hell’s web site, and confirm this for themselves.

The key innovation that allowed a STED microscope to break free of the diffraction limit was to give the STED beam a “zero-point,” which is to say a point of zero intensity surrounded by points of non-zero intensity, so that at any distance from the zero-point, however close, the intensity is greater than zero, and as such can be increased to an arbitrary level by raising the mean intensity of the STED beam. Even at such a high mean intensity, the central point of the STED beam will remain at zero intensity, so the central point of the fluorescent spot remains at its initial level, regardless of how much the spot’s width is reduced.

The first STED microscope to contain the requisite zero-point, thus completely transcending the diffraction limit, was the STED microscope I invented independently of Hell and Wichmann and described in the patent application for US Pat. 5,866,911, filed the same year they submitted their original paper. I am extremely grateful to Guy for including a reference to that patent in his new book, and to Christoph Cremer, Barry Masters, Rainer Heintzmann, and the late Mats Gustafsson for citing that that patent in review papers. 

Steve Baer

On Oct 10, 2014, at 4:24 AM, Andreas Schönle <[log in to unmask]> wrote:

> *****
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> *****
> 
> Dear Guy, Nuno, dear list members!
> 
> Surely this discussion will now go on for a while but I would like to set a
> few facts straight, so it can be put on solid ground.
> 
> 1)
> Stefan Hell was never a student of Chirstoph Cremer. His PhD advisor was
> Prof. S. Hunklinger and both 4pi and STED microscopy were conceived by him
> independently. The principle of STED is completely unrelated to 4pi and 4pi
> has nothing to do with what the Nobel prize was awarded for (see below).
> 2)
> 4pi microscopy is not a super-resolution method (it is diffraction limited).
> Christoph Cremer's original proposal to use light coming from all directions
> was aimed at increasing lateral resolution and was based on the wrong
> assumption that inverting the light-field of a point source <<lambda by
> means of a hologram could beat the diffraction limit. 
> (this neglects the fact that the near field decays exponentially and that
> this cannot be inverted in conventional materials) 
> 3)
> Localization is not resolution. Impressive work was done localizing single
> or several light sources separable by spectral properties, stepwise
> bleaching etc. - usually with the goal of measuring distances. But dense
> images can (as of now) only be obtained by time-sequentially switching
> molecules on and off.
> This was first realized for ensembles in STED and molecule-by-molecule in
> PALM/STORM/FPALM. 
> 
> Again, this is not to diminish anybody's work in the field. Over the years I
> have read many articles describing important and inspiring work. And I
> wholeheartedly agree that the field would not be where it is without these
> contributions.
> But when discussing who should be honored for finding the key to circumvent
> the diffraction limit in optical imaging, it is important to identify those
> people that had the right idea, recognized its importance and proved this by
> actually putting it to work. 
> 
> Best regards,
> Andreas
> 
> --
> Andreas Schoenle, Dr.
> Abberior Instruments GmbH, D-37077 Goettingen, Germany
>  
> phone: +49 (551) 30724170
> fax: +49 (551) 30724171
> http://www.abberior-instruments.com
> mailto:[log in to unmask] 
> 
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On
> Behalf Of Nuno Moreno
> Sent: Freitag, 10. Oktober 2014 00:19
> To: [log in to unmask]
> Subject: Re: congratulations to Eric, Stefan and W . E. for Nob el P rize in
> Chemistry, ³ for t he development of super-reso lved fluores cenc e mi
> croscopy²
> 
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> *****
> 
> Dear Guy
> 
> You're absolutely right. I actually sent an internal email (for the
> institute) a few hours after the announcement with, among other stuff, this:
> 
> "As always there are many others that should be on the laureates list but
> one needed the final click. In my opinion Cremer from Heidelberg university
> should be one of them ...."
> 
> Nuno Moreno
> 
> 
> 
> 
> 
> On 09 Oct 2014, at 02:16, Guy Cox <[log in to unmask]> wrote:
> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>> 
>> The three names is always a problem - especially when the prize is awarded
> essentially for two different techniques.  Christoph Cremer (Stefan's
> supervisor) could well also feel he'd been passed over.
>> 
>>                                                           Guy
>>