Infrared fluorescent proteins

8 05 2009

Hunting for new fluorescent proteins in the coral reefs of the Caribbean and Australia is a task that a lucky few researchers have managed to get funding for. Scuba diving in some of the world’s most beautiful places; it’s not a bad gig, if you can get it.  Most fluorescent protein scientists are confined to a lab, mutating existing fluorescent proteins from jellyfish and coral. Shifting their excitation and emission spectra has allowed multiple fluorescent proteins to be used as molecular highlighters at the same time, since their colors are distinct from each other. Some members of this palette are shown in Brain Windows top image bar.  After over a decade of research, the spectrum is pretty well covered.  Except for one area…  The infrared.

The near-infrared band is an area of enormous importance to scientific researchers, because is contains the spectral window where the body becomes transparent. Hemoglobin in the blood strongly absorbs visible wavelengths shorter than 650nm, while water absorbs wavelengths above 900nm.  If a fluorescent protein could be found, or engineered to have excitation and emission within this window, we could use it to peer much deeper into the body. The near-IR light penetrates much more easily into and out of the tissue.  This is easily seen by pressing a flashlight against your hand.  Only the deep red light passes through. The quest for an infrared fluorescent protein has preoccupied several labs for a decade.

Efforts to push FPs out to the infrared resulted in mCherry, mPlummKate, among others.  The further red-shifting of these proteins is constrained by the space limitations of the beta-barrel structure of GFP-like proteins.  In general, the longer the resonance chain of the chromophore, the longer the wavelength of the chromophore’s excitation and emission spectra.  It has been difficult to extend the FP spectra beyond 650nm without adding an additional bond to the resonance structure, for which there is little space left in the protected center of the barrel.  


Wavelength of excitation and emission is longer in larger resonance structures.

Wavelength of excitation and emission is longer in larger resonance structures.

In Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome Shu et al. of Roger Tsien’s lab, looked beyond traditionally used fluorescent proteins to extend the palette into the near-IR.  They engineered a protein, IFP, which binds biliverdin, a natural product involved in aerobic respiration (and similar in structure to the phytocyanobilins discussed in our Photoactivated Transcription journal club post). Biliverdin is non-fluorescent in solution, but when bound to IFP, it is rigidized and becomes fluorescent, with excitation at 684nm and emission at 708nm.  IFP can then be fused to the protein of interest and visualized through thick absorbent tissue.  Even the liver, which is dense with heme, is easily seen through the skin when labelled with IFP.  


IFP1.1 expressed in mouse liver is clearly visible through the skin 


IFP1.1 expressed in mouse liver is clearly visible through the skin


IFP1.4 should immediately push forward the field of in-vivo imaging of cancer and other diagnostics, at least in animal models.  It’s not clear yet how useful it will be for in-vivo brain imaging, they show cultured neurons expressing IFP1.4 become fluorescent upon biliverdin application, but can biliverdin be effectively delivered to neurons in vivo? Like channelrhodopsin, there may be sufficent amounts of endogenous co-factor to make the protein useful without exogenous application. Perhaps of greater importance is the new engineering avenues IFP opens up.  This is an entirely new, two-component scaffold, with different characteristics from GFP that protein engineers will be able to optimize and exploit.   Over the next decade, IFP may spawn as diverse a set of tools as GFP has over the previous one.



6 responses

8 05 2009
Evolving infrared fluorescent proteins from Bacterial phytochrome - a new direction for rational engineering photochemical transducers | - CaiLog

[…] Brain Windows is presenting a recent paper from Roger Tsien’s lab about an infrared fluorescent protein IFP1.4. Check it out! Science 8 May 2009: Vol. 324. no. 5928, pp. 804 – 807 DOI: 10.1126/science.1168683 […]

8 05 2009

This is an exciting, really new direction for fluorescent proteins. Hopefully this will be as usefull as GFP. However the FlaSH and ReASH system was also very promising but is still not broadly adopted… Only time will tell !

8 05 2009

Ahhh yes, the last Science paper from the lab. From what I have seen, IFP is pretty clean and easy to use, unlike the fairly complex protocol optimization of FlAsH/ReAsH. No worries about arsenic and non-specific background. Should be more broadly adopted I would think.

26 05 2009
Gus Lott

Just read this short article in spectrum about a lead-selenide based photostimulus from 830nm laser light.

I bet there’s more detailed stuff in publications from the group at Case Western.

It’s stimulus instead of recording, but seemed relevant🙂


14 07 2009

Case Western has some really good people working on optical control of neurons. Many people forget that Stefan Herlitze, of Case Western, is also one of the pioneers of Channelrhodopsin. In his 2005 PNAS paper, he showed both neuronal excitation with ChR2 and neuronal inhibition with vertebrate rhodopsin.

3 08 2009

Really fascinating site and articles. Liked this article as it was less technically challenging for a lay person as myself to comprehend!. Amazing how ultimately nature provides the keys to so many technologically driven advances. From studying geckos grip patterns for gravity defying technology, the syringe for hypodermic needles from snake fangs, stem cell blastocysts formation in salamanders that lose an apendage to this research – pure genius and totally fun!

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