Navigation Links
New law for resolution allows unprecedented sharpness in fluorescence microscopy

Editor : Detailed Information with Figures (pdf download)

Max Planck researchers have succeeded in overcoming the law postulated by Ernst Abbe in 1873 for diffraction limited resolution in light microscopes. Stefan Hell and his co-workers have established a new law that promises unlimited resolution in fluorescence microscopy. Future applications range from the imaging of cell interiors to the measuring of lithographic structures in microchip manufacturing, and substantial improvements in the quantification of the reaction kinetics of organic molecules (Phys. Rev. Lett., April 15 and Phys. Rev. Lett. May 6).

Ever since its invention in the 17th century, the light microscope has served as a key instrument to the advancement of knowledge. For biology, this statement is true even today, since focused light is the only way to examine living cells non-invasively. However, because of the wave nature of light, focused light is subject to diffraction. As early as 1873, Ernst Abbe recognized that this fact imposes an absolute limit on resolution in microscopy. Abbe, whose 100th year death anniversary was in February, captured this limit in a formula, which states that objects at distances that are smaller than a certain limit cannot be separated in a light microscope. This resolution limit can be largely ascribed to the fact that light cannot be focused to a smaller spot due to diffraction. It seemed impossible to image details smaller than a distance where delta d is equal to 200nm using conventional lenses and visible light.

For a long time, Abbe's law was regarded as being insurmountable. For higher resolution, the textbook knowledge was that a complicated and costly electron or scanning tunneling microscope would be required. However, over the past few years, researchers from the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, have developed wi th Stimulated Emission Depletion (STED) microscopy, a physically consistent concept for breaking the diffraction limited resolution limit in fluorescence microscopy, and then verified it in experiments. Unlike in the light microscopes conceivable so far, in a STED microscope, the relevant focal fluorescence spot can, in principle, be reduced in size to the size of a molecule (2-5 nm). This is due to the fact that the spot size is no longer subject to Abbe's formula, but to a new law that differs from Abbe's original formula in a crucial factor - a square root term [1,2]:

In one of their latest publications [2], the Göttingen-based research team verifies this new law with their experiments. They show that even with conventional objectives and focused light, resolutions of 16 nm are feasible. Therefore for the first time ever, it has been demonstrated experimentally that even with focused-light optics, resolutions at the nano-level are possible in fluorescing samples. In an additional publication [3], the researchers show that STED microscopy allows one to image lithographic structures (which had been previously dyed with a fluorescent dye) of width as small as 40-80 nm, a size previously reserved for electron microscopy. This may be of importance for the lithographic manufacturing of microchips.

The reduction in size of the diameter of the effective fluorescence focus also has fundamental implications for methods that use fluorescence fluctuations to study the reaction kinetics of molecules in solutions. The smaller the focal volumes, the more effective and sensitive are these methods. Fluorescence fluctuation methods were also limited by diffraction as a result. Therefore, in a fourth study [4], the research team from Göttingen shows that using STED, measurement volumes notably below the resolution limit can be produced without confining the solution itself mechanically. This method may substantially improve the analysis of pharmaceutical active ingr edients and protein interactions in cells in future.

Original work:

[1]Hell, S. W.
Strategy for far-field optical imaging and writing without diffraction limit.
Phys. Lett. A 326(1-2): 140-145 (2004)

[2] Westphal, V., and S. W. Hell
Nanoscale Resolution in the Focal Plane of an Optical Microscope.
Phys. Rev. Lett. 94: 143903 (2005)

[3] Westphal, V., J. Seeger, T. Salditt and S. W. Hell
Stimulated Emission Depletion Microscopy on Lithographic Nanostructures.
J. Phys. B: At. Mol. Opt. Phys. 38: S695 - S705 (2005)

[4] Kastrup, L., H. Blom, C. Eggeling, S. W. Hell
Fluorescence Fluctuation Spectroscopy in Subdiffraction Focal Volumes.
Phys. Rev. Lett. 94: 178104 (2005)


'"/>

Source:Max-Planck-Gesellschaft


Related biology news :

1. High resolution snapshots detail dynamics of a cocaine antibody
2. High-resolution light microscope reveals the fundamental mechanisms of nerve communication
3. Recombination protein dynamics observed with single monomer resolution
4. With record resolution and sensitivity, tool images how life organizes in a cell membrane
5. Researchers find how protein allows insects to detect and respond to pheromones
6. Enzyme allows B cells to resist death, leading to leukemia
7. Tiny scaffolding allows stem cells to become working fat cells
8. Looks matter to female barn swallows
9. Stolen gene allows insect virus to enter cells
10. Researchers identify molecular anchor that allows bacterial invasion of central nervous system
11. Algal protein in worm neurons allows remote control of behavior by light
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:3/31/2016)... , March 31, 2016   ... ("LegacyXChange" or the "Company") LegacyXChange is excited ... of its soon to be launched online site for ... https://www.youtube.com/channel/UCyTLBzmZogV1y2D6bDkBX5g ) will also provide potential shareholders a ... DNA technology to an industry that is notorious for ...
(Date:3/29/2016)... 29, 2016 LegacyXChange, Inc. (OTC: ... and SelectaDNA/CSI Protect are pleased to announce our successful ... a variety of writing instruments, ensuring athletes signatures against ... collectibles from athletes on LegacyXChange will be assured of ... DNA. Bill Bollander , CEO states, ...
(Date:3/22/2016)... India , March 22, 2016 /PRNewswire/ ... market research report "Electronic Sensors Market for Consumer ... Proximity, & Others), Application (Communication & IT, ... Geography - Global Forecast to 2022", published ... industry is expected to reach USD 26.76 ...
Breaking Biology News(10 mins):
(Date:6/27/2016)... - BIOREM Inc. (TSX-V: BRM) ("Biorem" or "the Company") announces ... Clean Technology Fund I, LP and Clean Technology Fund ... venture capital funds which together hold approximately 59% of ... as converted basis), that they have entered into an ... in Biorem to TUS Holdings Co. Ltd. ("TUS") (en.tusholdings.com) ...
(Date:6/27/2016)... , ... June 27, 2016 , ... ... bring innovative medical technologies, services and solutions to the healthcare market. The company's ... of various distribution, manufacturing, sales and marketing strategies that are necessary to help ...
(Date:6/24/2016)... 24, 2016  Regular discussions on a range of subjects ... the two entities said Poloz. Speaking at a ... , he pointed to the country,s inflation target, which ... "In certain areas ... have common economic goals, why not sit down and address ...
(Date:6/23/2016)... ... June 23, 2016 , ... ... of its second eBook, “Clinical Trials Patient Recruitment and Retention Tips.” Partnering with ... in this eBook by providing practical tips, tools, and strategies for clinical researchers. ...
Breaking Biology Technology: