Thursday, April 20, 2006

Light Smaller Than Light: Rayleigh's Diffraction Barrier Broken

Regular posting resumes.

People who work with really really small things--in our own humble example, neurons--are generally acquainted with Rayleigh's diffraction barrier. It goes by a number of names: Rayleigh's law of resolution maxima, the , and so forth. But the gist of is that there is a hard limit to the maximum resolving power one can achieve via optical microscopy, partially due to the properties of visible light.

If you haven't encountered this idea before, click on the link above, as wiki can explain it better than I can. But the basic version: for our purposes, I think we can get away with describing the resolving power of a microscope as one's basic ability to distinguish between objects in a field being viewed at a certain magnification. This is dependent on a function whose variables include the wavelength of the light involved, the of the medium being viewed through, and the sine of the angular aperture. Examining our three variables: the sine function maxes out at 1 (although for practical reasons, the limit is generally 0.94). For reasons I cannot claim any knowledge of, the best refractive index achievable seems to be 1.56, with an oil immersion. And the lowest wavelength of visible light available is thus 187 nm. Therefore, the lowest wavelength that can be visualized into visible light is 187 nm. Thus, the minimum distance that can be between any two objects we would like to visualize is about 200 nm.

Now, I know what you're thinking: WOW, that's REALLY REALLY REALLY SMALL. And you're right.

But: let's compare it to a few numbers:
Size of an average neuron's soma (the cell body): 10-25 micrometers. That's around 50-125 times the size of our barrier. Not bad.
Size of an average (the processes that receive information from other cells) or an (the processes that send information to other cells): ~1 micrometer. Now, that's still five times the resolution barrier.

But at the same time, realize that this limit is the minimum space objects need to be separated by to be distinguishable from each other. This means that you're pretty much screwed when it comes to trying to visualize things smaller than the level of an individual dendrite or axon--which is where all the interesting stuff is happening. To be sure, there are a number of way to get around this. You can tag specific proteins with fluorescent dyes, so that only they will appear on the scope. But if it's a highly localized protein, then you're still in trouble. You can also visualize via electron microscopy, but the preparation required prevents you from working with living biological systems.

But this morning, thanks to Alex Palazzo, I discovered that a new technique--called stimulated emission depletion (STED) microscopy--has been developed to work around this limitation. Apparently Stefan Hell and his colleagues take his name literally*, as this technique can be used on fluorescently labelled preparations to achieve a resolution of approximately 50 nm. And, in theory there is no fixed limit to the resolution achievable by this technique. Last week, they published their first paper from practical use of this technique, and Nature has published a brief overview of the technique.

So how'd they do it?

I'm glad you asked.

From Nature's overview, by Garth Simpson:

The first beam excites molecules just as in traditional fluorescence microscopy — as the molecule absorbs the energy from the light, it is promoted up to a higher energetic state, and as it relaxes back to the ground state, it releases the energy in the form of light. The second beam, at a different wavelength, suppresses this fluorescence by 'stimulated emission', in which molecules are actively pumped down out of the excited state by light (in essence, absorption driven backwards). In sufficiently intense optical fields, stimulated emission becomes more efficient than fluorescence and is the dominant effect, drastically reducing the fluorescence.

In STED microscopy, the normal fluorescence is suppressed by a depletion beam that is shaped like a doughnut and contains a node of zero intensity — a hole — at the centre of the focus. So, any fluorescent light that is detected arises just from this hole of about 50 nm across where the depletion beam is absent (Fig. 1). Scanning this narrowed viewing point across the sample allows a higher-resolution image to be built up, much as a computer screen made up of smaller pixels will provide a sharper image...

But a picture is worth a thousand words, so here you go:

In the upper left corner, you can see a blue light produced by excitation of a fluorophore. In the upper right corner, you can see the orange STED beam. This is basically blocking the flourescence of the excited molecules in its range by knocking them down a peg (in terms of energy states) before they can release any light. Then in the lower left corner, you see a green light representing what's left after the two lights interact. And the graph in the bottom corner represents the flourescence profile after their interaction.

So what have they used this technique on to get me so enthused? Well, if you really really care you've probably already clicked on the link and found out. If not, you can either click on it now (if you're the impatient sort), or wait until later tonight for the exciting conclusion of our two-part post. Because, um, right now I've gotta get running to class.
*In German, "Hell" means "brightly."

Simpson GJ (2006) The diffraction barrier broken. Nature 440:879-880.
Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440:935-939.

EDITED at 7:30 PM because I accidentally linked to the U-of-M-access Nature pages.

EDITED on Wednesday, 26 April 2006 after a fundamental physics error was called to my attention.


At 26 April, 2006 14:44, Anonymous Plant Master Flash said...

Just wanted to comment that your original estimate on distance discrimination is quite likely exaggerated:

the lowest wavelength of visible light available is thus 187 nm

If you can see 187 nm light, I'm damn impressed, because not only is that short a wavelength in the UV spectrum, I suspect it is actually UV-C. Y'know, the sort of stuff that gives you cataracts and skin cancer.

At 26 April, 2006 15:44, Blogger The Neurophile said...

Check. It's just poor phrasing, due to not really thinking my way through the wiki article while trying to remind myself of the details.

Not that I wouldn't have really meant it, given the chance.

Consider it corrected (mostly because it now is).

At 30 October, 2015 00:39, Blogger ninest123 Ninest said...

ninest123 10.30
longchamp, gucci outlet, oakley sunglasses, michael kors outlet, ugg boots, cheap oakley sunglasses, michael kors outlet, kate spade outlet, oakley sunglasses, nike outlet, prada handbags, polo ralph lauren outlet, ray ban sunglasses, christian louboutin outlet, replica watches, longchamp outlet, kate spade handbags, tiffany jewelry, ugg boots, prada outlet, louis vuitton outlet, chanel handbags, michael kors, louboutin, coach outlet, nike free, polo ralph lauren outlet, louis vuitton, jordan shoes, tory burch outlet, longchamp outlet, michael kors outlet, replica watches, tiffany and co, michael kors outlet, oakley sunglasses, burberry, nike air max, louis vuitton, ugg boots, burberry outlet online, oakley sunglasses, michael kors outlet, ray ban sunglasses, louis vuitton, louboutin outlet, ray ban sunglasses, louis vuitton outlet, louboutin shoes

At 30 October, 2015 00:43, Blogger ninest123 Ninest said...

lacoste pas cher, abercrombie and fitch, vanessa bruno, sac guess, ray ban pas cher, nike free, nike huarache, nike roshe run, michael kors, hermes, new balance pas cher, converse pas cher, tn pas cher, timberland, longchamp, lululemon, ralph lauren pas cher, hollister, hollister, ray ban uk, air force, north face, nike roshe, air max, hollister pas cher, coach factory outlet, hogan, nike air max, vans pas cher, north face, longchamp pas cher, true religion outlet, nike free run uk, sac longchamp, michael kors, nike blazer, louboutin pas cher, air jordan pas cher, michael kors, oakley pas cher, nike air max, true religion jeans, ralph lauren uk, michael kors, nike trainers, nike air max, true religion jeans, mulberry, true religion jeans, coach purses, burberry

At 30 October, 2015 00:47, Blogger ninest123 Ninest said...

reebok shoes, soccer shoes, ferragamo shoes, oakley, ray ban, louboutin, beats by dre, valentino shoes, iphone 6s plus cases, mont blanc, herve leger, bottega veneta, asics running shoes, nike air max, north face outlet, ghd, hollister, ralph lauren, chi flat iron, s5 cases, nfl jerseys, new balance, lululemon, vans, nike roshe, iphone 5s cases, iphone cases, baseball bats, ipad cases, vans shoes, mcm handbags, iphone 6 plus cases, instyler, p90x workout, gucci, babyliss, soccer jerseys, timberland boots, birkin bag, celine handbags, insanity workout, iphone 6 cases, abercrombie and fitch, jimmy choo shoes, iphone 6s cases, wedding dresses, converse, mac cosmetics, hollister, north face outlet, nike air max, giuseppe zanotti, coach outlet

At 30 October, 2015 00:50, Blogger ninest123 Ninest said...

doke gabbana outlet, canada goose uk, thomas sabo, converse outlet, canada goose outlet, moncler, moncler outlet, swarovski, moncler, canada goose, doudoune canada goose, supra shoes, bottes ugg, pandora charms, moncler, links of london, hollister, juicy couture outlet, ugg pas cher, replica watches, karen millen, ugg,ugg australia,ugg italia, pandora charms, pandora jewelry, montre pas cher, louis vuitton, toms shoes, sac louis vuitton pas cher, canada goose, marc jacobs, coach outlet, michael kors outlet, michael kors handbags, lancel, canada goose outlet, juicy couture outlet, moncler, louis vuitton, wedding dresses, moncler, moncler, swarovski crystal, michael kors outlet online, louis vuitton, canada goose, barbour jackets, ugg,uggs,uggs canada, ugg boots uk, louis vuitton, pandora jewelry, moncler
ninest123 10.30

At 11 December, 2015 01:54, Blogger mmjiaxin said...

toms outlet
michael kors handbags
michael kors online outlet
tiffany and co
coach outlet
lacoste shirts
snapbacks wholesale
oakley sunglasses
ray-ban sunglasses
cleveland cavaliers
ugg boots
nike roshe
canada goose outelt
beats by dre
tiffany outlet
toms outlet store
abercrombie and fitch
air jordan 11
swarovski crystal
new balance shoes


Post a Comment

Send Haloscan trackback ping

<< Home

Academics Blog Top Sites