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:2/3/2016)... , Feb. 3, 2016 Vigilant Solutions announces ... Department in Missouri solved two ... reader (LPR) data from Vigilant Solutions. Brian ... in which the victim was walking out of a convenience store and witnessed ... next to his vehicle, striking his vehicle and leaving ...
(Date:2/2/2016)... Technology Enhancements Accelerate Growth of X-ray Imaging ... and computed radiography markets in Thailand ... Indonesia (TIM). It provides an in-depth analysis ... as regional market drivers and restraints. The study offers ... market attractiveness, both for digital and computed radiography. Market ...
(Date:2/1/2016)... 1, 2016  Wocket® smart wallet ( www.wocketwallet.com ) announces the launch ... Joey Fatone . Las Vegas , where Joey ... --> Las Vegas , where Joey appeared at the ... new video ad was filmed at the Consumer Electronics Show (CES2016) in ... booth to meet and greet fans. --> ...
Breaking Biology News(10 mins):
(Date:2/9/2016)... ... February 08, 2016 , ... ... Baruch S. Blumberg Institute at the Pennsylvania Biotechnology Center of Bucks County, 3805 ... The Commonwealth Medical College (TCMC) will hold an open house for participants to ...
(Date:2/9/2016)... (PRWEB) , ... February 09, 2016 , ... ... its latest innovations on its free and validated Electronic Data Capture (EDC) system ... the Outsourcing in Clinical Trials West Coast 2016 Conference in San Mateo, California ...
(Date:2/9/2016)... ... 2016 , ... The American Academy of Thermology (AAT) has announced ... AAT Member Certification Qualification Course for Technicians via a two part webinar on July ... a detailed review of hardware, software, and camera setup/operations, aligns with the in-person member ...
(Date:2/9/2016)... ... February 09, 2016 , ... PharmApprove ... Policy for the National Organization for Rare Disorders (NORD). Dorman will lead PharmApprove ... voices are heard throughout the drug regulatory review process. , “Adding Diane Dorman ...
Breaking Biology Technology: