#betzig

[frontal-cortex]

Live super-resolved microscopy: The next level

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Microscopy has been used for hundreds of years to decipher biological mechanisms. The advent of fluorescence microscopy and the ability to label specific proteins as well as to image live specimens allowed for even more discoveries. Boundaries have been pushed further by the development of super-resolution microscopy which awarded a Nobel Prize in Chemistry to Eric Betzig, Stefan Hell and William Moerner. It still bears notable problems that Eric Betzig cites in his new paper: fixed samples, phototoxicity and the need of an enormous photon budget. To bypass these limitations, they have been using SIM (Structured Illumination Microscopy) with the highest numerical aperture possible, and this way enhancing resolution while limiting the excitation to only of small portion of the cell volume and reducing phototoxicity. To achieve even higher resolution, patterned activation of photoswitchable fluorophores was used, allowing for further resolution enhancement and faster acquisition times. Still images convey the vast improvements over conventional optical microscopes but impressive videos can be seen on the online version.

Above : Two approaches for improved live-cell imaging at sub-100-nm resolution. (Left) Association of cortical actin (purple) with clathrin-coated pits (green), the latter seen as rings (inset) at 84-nm resolution via a combination of total internal reflection fluorescence and structured illumination microscopy at ultrahigh numerical aperture (high-NA TIRF-SIM). (Right) Progression of resolution improvement across the actin cytoskeleton of a COS-7 cell, from conventional, diffraction-limited TIRF (220-nm resolution), to TIRF-SIM (97-nm resolution), and nonlinear SIM based on the patterned activation of a reversibly photoswitchable fluorescent protein (PA NL-SIM, 62 nm resolution). Scale bars, 2 μm (left); 3 μm (right).

D. Li et al., Science 349, aab3500 (2015) DOI: 10.1126/science.aab3500

[frontal-cortex]

Movie 5, High NA SIM of actin / clathrin interactions

[stojdllab]

Wow. Just, wow. Using Nobel prize winning microscopy to watch live cells in action.

https://vimeo.com/136534107

Image: Interaction of filamentous actin (mApple-F-tractin, purple) with myosin IIA bipolar head groups (EGFP, myosin IIA, green) at 20-second intervals for 100 time points, as seen with high-NA TIRF-SIM. Credit: Li et al 2015

Read the original paper here.

[frontal-cortex]

Peering into the nanoworld

In the world of fundamental and applied research, fluorescence microscopy is an invaluable tool to witness biological phenomena and events. In order to do this, scientists use fluorescent proteins and tags to follow a particular protein. Thus, the ability to capture this emitted light is directly limited by its diffraction. It means that the resolution of conventional light microscopy is not supposed to exceed 200 nm. Last year, the Nobel Prize in Chemistry was awarded to Eric Betzig, William Moerner and Stefan Hell for their involvement in the development of super-resolved fluorescence microscopy, bringing fluorescence microscopy right down to the nanoscale. Not only does it sound cool, it also means that now we can precisely visualize individual molecules inside a cell, a thought that lied only in the realms of science fiction ten years ago. 

Although there are now many tricks to achieve super-resolution, two distinct techniques were awarded in 2014. Eric Betzig took part in the development of PALM (Photo-activated localization microscopy) [in the living room of a colleague]. This technique relies on photo-activable fluorescent proteins which will emit light at different time points. The image can thus be reconstructed later with much better accuracy. I suggest watching this video to better understand. An example is featured as the first picture above. A related technique called STORM (STochastic Optical Reconstruction Microscopy) is displayed in the second and third picture. A combination of both can be seen on the last picture.

A second type of super-resolved microscopy was mentioned : STED (STimulated Emission Depletion). As stated in its name, STED microscopy relies on a high intensity laser which will delete the halo around a fluorophore. See fourth and fifth picture above for an example (confocal on the left, STED on the right).

It sounds complicated, but these techniques relying on the basic principles of light and molecules allow to bypass a limit which seemed impossible to overcome a few years ago. The impact of these men and the improvements in terms of optics but also chemistry - with the apparition of new, photoactivable and more stable fluorophores - will propel scientific progress for years to come, taking microscopy to the next level. The “nano” one.

[paularead]

Flea Glasses and Hidden Spaces

Flea Glasses and Hidden Spaces

Imagine the excitement of using one of the world’s first magnifying glasses back in the 16th century. All those creatures and items too tiny for examination with the naked eye would have suddenly revealed some of their secrets.

Early magnifying glasses were so popular for looking at minuscule life forms such as fleas that they were sometimes called ‘flea glasses’. The workings of bodily and…

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