History lesson

[Andrew York]

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I recently stumbled across a Wikipedia article that caught my eye:
http://en.wikipedia.org/wiki/Christoph_Cremer

I'm not familiar with the work described in this article, but it seems
astounding.
In particular:
Widefield localization microscopy of whole cells in two minutes, with 10 nm
resolution
Structured illumination microscopy with 40 nm resolution.

Have I been negligent in my citations? Why haven't I heard of this before?

[John Oreopoulos]

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Andrew,

See these publications:

Cremer, C., et al., Superresolution imaging of biological nanostructures by spectral precision distance microscopy. Biotechnology Journal, 2011. 6(9): p. 1037-1051.

Huber, O., et al., Localization microscopy (spdm) reveals clustered formations of p-glycoprotein in a human blood-brain barrier model. Plos One, 2012. 7(9).

Kaufmann, R., et al., Visualization and quantitative analysis of reconstituted tight junctions using localization microscopy. Plos One, 2012. 7(2).

My understanding is that this is yet another form of super-resolution localization microscopy. Here's a clipping from the first paper:

"Originally [10], in the scheme to explain the SPDM principle, the different spectral signatures were indicated using color. In later publications [22, 24], the color assignment was deliberately omitted to make it clear that differences in the fluorescence emission spectrum (called color) are just one of the many ways to realize a spectral signature difference useful for superresolution. In the original definition of ‘spectral signature’ [10, 14], any photophysical property of the fluorescent point emitters was included that allowed the independent registration (‘optical isolation’) of the respective diffraction patterns. In particular, it was recognized that the SPDM principle as described was applicable to various kinds of far-field fluorescence microscopy, including in particular focused, structured, and homogeneous illumination."

Cheers,

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2012-12-04, at 8:20 AM, Andrew York wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I recently stumbled across a Wikipedia article that caught my eye:
> http://en.wikipedia.org/wiki/Christoph_Cremer
>
> I'm not familiar with the work described in this article, but it seems
> astounding.
> In particular:
> Widefield localization microscopy of whole cells in two minutes, with 10 nm
> resolution
> Structured illumination microscopy with 40 nm resolution.
>
> Have I been negligent in my citations? Why haven't I heard of this before?

[David Baddeley]

having done my PhD in Christoph's lab I can confirm that he
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Hi Andrew,

having done my PhD in Christoph's lab I can confirm that he has a considerable pedigree in the fields of high and super- resolution microscopy and is probably not cited as much as he deserves. I can also confirm that all the claims on the wikipedia page will have some basis in measurement. That said, the wiki page is somewhat confusing and arguably conflates results from multiple different microscopy techniques (or fails to draw some of the distinctions/ offer some of the explanation that would be required to fully understand what is being claimed). Part of the reason for this is probably that the page was prepared by a member of the universities intellectual property team (judging by the edit history) who might not have fully appreciated some of the subtleties. Christoph himself can arguably also sometimes be guilty of taking a slightly optimistic view whilst trying to paint a broad picture. 

Addressing the two topics you have picked up on:
- 30-40 nm structured illumination resolution. This is size resolution and/or position resolution, not spatial resolution. The SMI microscope generates an axial standing wave between two microscope objectives, resulting in a structured illumination pattern with ~ 160 nm period. When an object is moved through this pattern, the resulting signal will have a modulation, the magnitude of which depends on it's size (infinitely small objects will dim completely in the troughs, whereas objects > ~ 200 nm show very little modulation. Based on the modulation observed, an estimate of the real size can be obtained. On objects with a well characterised shape (such as beads) it is possible to get an accuracy better than ~10 nm, but the method becomes less accurate when assumptions need to be made about the underlying object. 30-40 nm is probably a reasonable estimate of the precision with which sizes of biological structures can be determined. When determining
 positions, the illumination pattern can be used as a 'ruler' to improve axial localisation (not too dissimilar to an iPALM like approach). For bright objects (not single fluorphores), I regularly got 1-2 nm localisation precisions.

The references in the SMI section are not particularly helpful, in that they refer to two separate techniques - some (9, 12) are conventional lateral structured illumination, whereas 10 & 11 refer to the nano-sizing technique.

- 10 nm resolution in 2 minutes. It is not completely clear whether this is referring to a PALM/STORM type of approach, or an SMI type approach. SMI only needs about 20 frames to scan the object through the focus, so this is easily achievable. I suspect that it however refers to PALM/STORM type imaging and that the 10 nm is a localisation precision rather than a nyquist sampled end resolution, in which case it could possibly be considered oversold. It might well be a conflation of both.


One source of confusion is probably the re-purposing of acronyms (in this he's following the lead of the the Hell group who have, e.g., changed GSD from meaning a STED like point-scanning microscopy to a PALM/STORM variant). Maybe the following (somewhat subjective) disambiguation might help:

SMI - axial structured illumination used for size and/or position measurements with sub-diffraction (few nm on synthetic objects) accuracy. Also sometimes used to mean conventional SIM/PIM with a lateral illumination pattern and a 2-fold resolution improvement. It should be noted that Christoph was involved in some of the first lateral SIM experiments (Heintzmann and Cremer, 1999).

Vertico-SMI - an SMI microscope in which the two objectives are orientated vertically, and with an incubation chamber used for live cell imaging (the initial SMI prototypes were built on a breadboard with horizontal objectives). Being a microscope equipped with both a sensitive camera and laser illumination it was/is also used for PALM/STORM type measurements.

SPDM - a method of sub-resolution structure analysis based on measuring the relative positions of objects in different spectral channels (e.g. looking at the relative position of different parts of a gene locus - Esa et Al, JoM, 2000). Severely limited by the number of available spectral channels, but conceptually similar to PALM/STORM, and arguably a fore-runner. Also used / repurposed to mean PALM/STORM

SPDM-phymod - PALM/STORM, following the rational that switching is conceptually equivalent a spectral signature. Sometimes used to mean PALM/STORM with conventional fluorophores (we were the first to publish localisation microscopy images based on conventional fluorophore switching in Reymann et al, Chromosome research, 2008 - they were included as an afterthought so you need to dig through the article to find them, and were by all admissions pretty unconvincing with the switching chemistries etc ... not yet worked out).

LIMON - catch all acronym to refer to the Cremer group's super-resolution efforts. Similar in concept to RESOLFT.

cheers,
David


________________________________
 From: Andrew York <[hidden email]>
To: [hidden email]
Sent: Wednesday, 5 December 2012 2:20 AM
Subject: History lesson
 
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To join, leave or search the confocal microscopy listserv, go to:
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I recently stumbled across a Wikipedia article that caught my eye:
http://en.wikipedia.org/wiki/Christoph_Cremer

I'm not familiar with the work described in this article, but it seems
astounding.
In particular:
Widefield localization microscopy of whole cells in two minutes, with 10 nm
resolution
Structured illumination microscopy with 40 nm resolution.

Have I been negligent in my citations? Why haven't I heard of this before?