a photoactivatable fluorophore

June 24, 2008 at 7:45 am | | cool results, literature, news, single molecules

I have a chance to brag, so I’m going to take it. I just published a Communication in JACS.

The basic idea is to use an an azide to disrupt the push-pull character of a known fluorophore, rendering it dark. Because the azide is photoconvertible to an electron donor (the amine), you can photoactivate fluorescence.

The motivation for designing photoactivatable fluorophores that emit many photon include super-resolution schemes (described here and here).

Anyway that’s all. Just wanted to talk about myself a little. Which is what a blog is for.

22 Comments »

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  1. Congrats. Well, a few questions for you.

    First, a more general question:
    Why does the flurophore have to deactivated while you inject it? Is there something wrong with just injecting an active fluorophore into the cell? You may have explained that in your intro, but if so it went over me.

    Now about your system:
    In the fourth paragraph you talk about how you expect a reactive nitrene, resulting in azepine or bond insertion but end up with this amine.

    Where does the hydrogen for the amine come from?
    Just surrounding water?
    To test I suppose you could try and active the system in a glovebox system?
    If so, you would end up with hydroxyl ions floating around, and isn’t that not desirable for the system? I realize it wouldn’t be much, depending on how much you are putting in, but nonetheless.

    At the bottom of the second page, you say that the system is static because of bioconjugation to biomolecule due to nitrene insertion.

    I thought in the 4th paragraph you said that this isn’t what happened?

    Nice paper, interesting stuff; I’m sure most of these questions are just because I’m not too familiar with the field.

    Comment by Will — June 24, 2008 #

  2. congrats!

    Comment by sujit — June 24, 2008 #

  3. well, there’s a lot of info in the supporting material. that’s a problem with papers these days: all the real results are hidden in the supporting material. but you can’t publish letter like you used to, simply reporting an interesting (but totally unexplored) new result.

    anyway, the reason you want a dark fluorogen that can be photoactivated is because it gives you a handle for active control of the emitter: you can activate just a few fluorophores at a time and locate each of those to high precision. if you do this over and over, you can build up a very detailed image of the system you’re looking at. PALM and STORM need molecules with this active control to get high-resolution fluorescence images.

    as far as the reaction, the reason we don’t form azepines is probably because of the EWG on the aryl azide, which promotes forming the amine (according to the literature). the protons probably come from solution. we can favor nitro formation by aerating the solution; amine by deoxygenating.

    in solution, the nitrene formed mostly a primary amine by stealing those protons from solution. in the cell or embedded in the membrane, the reactive nitrene is adjacent to biomolecules and has the opportunity to insert into C-C bonds. at least that’s what we postulate, because we haven’t had a chance to study that bioconjugation reaction yet.

    Comment by sam — June 24, 2008 #

  4. oh, and i don’t think there’s much worry about producing one hydroxyl per molecule. any fluorophore in most experiments will probably produce several to hundreds of singlet O2 molecules before it eventually photobleaches (probably from reacting with one such activated O2).

    Comment by sam — June 24, 2008 #

  5. Wow, that is a lot of SI. S6 sure is a pretty column, and File5 is a nice demonstration.

    I see; all makes sense, thanks for taking the time to educate. Good work again.

    Comment by Will — June 24, 2008 #

  6. Congrats! I saw the structure in the ASAPs and was intrigued for about two seconds (until I read the title).
    Also, I just took the opportunity to Facebook stalk you. :D

    Comment by psi*psi — June 24, 2008 #

  7. you mean the title bored you? ;)

    Comment by sam — June 24, 2008 #

  8. Pretty much anything containing the phrase “live cells” makes me a little nauseous. ;)

    Comment by psi*psi — June 24, 2008 #

  9. *sigh*

    Comment by Jeremiah — June 25, 2008 #

  10. You had me at “fluorophore”….

    Comment by fiona — June 29, 2008 #

  11. Hi
    Congrats!this is very interesting…i m dumb struck by amazing stuff in your blog…thanks for publishing it….

    Comment by Neerali — July 2, 2008 #

  12. i have a major problem with your figure…the word bright is rather arbitrary. what’s bright to you may be dull to me. i wipe my ass with this communication (if it’s a big one, i’ll include the supporting info).

    Comment by John — July 3, 2008 #

  13. Alright! Way to go sam, congratulations!
    The author list is basically the Moerner lab all-stars!

    -Jaesuk

    Comment by jaesuk — July 3, 2008 #

  14. Hey! Very awesome. Do you know if your compound can fluoresce after Staudinger ligation or click chemistry?

    Comment by Nate — May 6, 2009 #

  15. Thanks, Nate. Yes, I do know. We are working on perfecting that!

    Comment by sam — May 6, 2009 #

  16. Cool. How about photoswitching? Do you know if your azido-fluorophore can photoswitch (as in dSTORM)?

    Comment by Nate — May 6, 2009 #

  17. why do you think dSTORM (or STORM or PALM or FPALM) needs photoSWITCHING? for static structures, photoACTIVATING is preferable: there’s not reason to relocalize a fluorophore that you’ve already localized.

    of course, for STED and structured illumination, robust switching is necessary.

    this fluorophore is irreversible.

    Comment by sam — May 7, 2009 #

  18. There actually may be advantages to relocalizing fluorophores, especially you’re not interested in precise counting.

    Imagine you’re interested in obtaining an idea of the geometry of a small structure (like the nuclear pore or mitochondrial cristae). You’d ideally like to have a very stringent threshold for localization precision of your fluorophores, because with an object of that diameter, it probably won’t do to reconstruct an image with points localized to less precision than ~30 nm. In general, there aren’t fluorophores that put out enough photons before photobleaching for you to get that precision 100% of the time. (If you know of one, let me know.)

    Reversibly photoswitchable fluorophores may only rarely emit very brightly, but occasionally they do blink very brightly and thus permit you to obtain high localization precision. By repeatedly photoswitching the same population of fluorophores and simply rejecting the data whenever you can’t get some arbitrary localization precision (say <10 nm or whatever), you can obtain the geometric information you’re actually looking for. Reconstructing the image, it may be prettier and more valuable if it’s composed of many points localized repeatedly with high precision than if you’ve only localized each component onece and the data includes a wide variation in localization precision.

    Again, I admit, reversible photoswitching is no good for counting, but it can serve other purposes. And if you’re attempting to count using irreversibly photoactivatable fluorophores, you still inevitably have to contend with the problem of undercounting. If it’s a fluorescent protein, you have no idea what proportion of FPs have folded properly, and if it’s a small-molecule dye, you may not know with certainty what your dye incorporation rate is per biological structure of interest. In either case, you also can’t know how many fluorophores have already photoactivated and bleached before imaging. In your final reconstructed image, you will have an underrepresentation of the components that comprise any given structure, based on the threshold you impose on fluorophore brightness for inclusion in your data set.

    Know what I’m saying? : )

    Comment by Nate — May 25, 2009 #

  19. Hi Nate. Thanks for your comment. I see what you mean: if you have several chances to localize, you can wait till a cycle with many photons.

    However, your example assumes that a photoswitching molecule emits many photons in one cycle. That is rarely the case. Often, a photoswitch emits as many photons in total before photobleaching, just split into many cycles. (For instance, the Cy3/Cy5/thiol photoswitch emits ~30,000 photons per cycle: enough to be localized most cycles, but it’s an order of magnitude less than what Cy5 emits without thiol. I think.)

    You’d rather have all 10^5 to 10^6 photons in one bunch, than split over many photoswitching cycles. There may be photoswitches that emit 10^5 photons per cycle over many cycles, but I don’t know of any off-hand.

    Of course, there are many cases where photoswitching is ideal: imaging a dynamic structure is a perfect example. Also, STED and structured-illumination require robust photoswitches.

    Comment by sam — May 26, 2009 #

  20. […] There are several photoactivatable fluorescent proteins, such as PA-GFP. Another example close to my heart is the azido DCDHF fluorophore: upon irradiation with blue light, an azide photoreacts into an […]

    Pingback by Everyday Scientist » photoactivation vs. photoswitching (part 1) — September 3, 2009 #

  21. Congratulations!

    Comment by Diane — October 24, 2011 #

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