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708 nm, an extinction coefficient >90,000 M−1 cm−1 and a quantum yield of 0.07.
Although the excitation and emission characteristics of IFP1.4 compare favor-ably to those of existing fluorescent proteins for in vivo imaging, there are a wide range of additional considerations for proteins that are used as in vivo reporters. Given the properties of mammalian tissue, fluorescent markers must be highly efficient (with high quantum yields and photostability) and biocompatible. Brightness is a function of quantum yield and extinction coefficient, and as such IFP1.4 is about five-fold less bright than GFP. The photostability of the modified infrared fluorescent protein is greatly improved relative to the full-length parental bacteriophytochrome from D. radiodurans, and compares favorably to that of other fluorescent proteins. The toxicity of IFP and its fluorophore biliverdin has not been fully evaluated; however, biliverdin is a precursor to bilirubin, which is a toxin and the cause of pathological jaundice. Biliverdin is a known antioxidant and can reduce oxidative stress, but it can also alter mammalian physiology. Years of research on GFP and related proteins have produced a collection of reporter proteins with a
near-infrared reporters that can be expressed as markers of transcription, protein function and metabolism have not previously been described. The excitation wavelengths of pro-teins based on green fluorescent protein (GFP) and related proteins range from 380 to 590 nm, with emission maxima of 440–648 nm; the most red-shifted of these proteins is mKATE2, with excitation and emission peaks of 588 and 633 nm, respectively10—wavelengths that are far short of optimal for transmission through mammalian tissues (>700 nm). By contrast, the infrared fluorescent protein now reported by Shu et al. has spectral properties that are nearly 100 nm further into the near infrared.
Shu et al.8 began their development of a near-infrared fluorescent protein with clones of bacteriophytochrome from the extremo-philic bacterium Deinococcus radiodurans. The chromophore of this phytochrome is biliverdin, which is produced by heme oxyge-nases in mammalian cells and in the cells of other aerobic organisms; it can also be added exogenously to laboratory animals. Through rational design and directed mutagenesis, the authors engineered this bacteriophy-tochrome into a monomeric near-infrared fluorescent protein, called IFP1.4, with excitation and emission maxima of 684 and
Fluorescent and bioluminescent proteins have revolutionized biology by making it possible to carry out functional studies in living cells and animals1–5, but these pro-teins have limited utility in organisms that are relatively large and opaque, such as mam-mals6. Inspired by the work of Fischer and Lagarias7, who recognized the potential of phytochromes as genetically encoded report-ers, Shu et al.8 recently reported in Science the development of near-infrared reporter proteins from bacterial phytochromes. These reporters extend the utility of fluo-rescent proteins in vivo because they excite and emit in the near-infrared region of the electromagnetic spectrum, where mammals are less opaque (Fig. 1).
A detailed understanding of mammalian biology and disease will require the ability to noninvasively monitor molecular events in vivo. Current technology for such molecu-lar imaging with light relies on reporter pro-teins with optical properties in the visible region of the electromagnetic spectrum that can be detected from outside the body4,5. Fluorescent proteins have been widely used in cell culture and with great success in small transparent organisms such as flies and worms2, but their use in laboratory rodents4,5 has been limited by the absorbing and scattering properties, as well as the auto-fluorescence, of mammalian tissues (Fig. 1). Near-infrared wavelengths between 700 and 950 nm provide a window of opportunity for molecular optical imaging because hemoglo-bin, the primary absorber in mammalian tis-sue, predominantly absorbs light of shorter wavelengths and because autofluorescence is reduced in this part of the spectrum.
Research on in vivo optical imaging has been aimed at the development of dyes and imaging agents that excite and emit in the near-infrared region, and some of these are being translated to the clinic9. However, genetically encoded
The importance of being redBradley w Rice & Christopher H Contag
A near-infrared fluorescent protein opens a window into the mammalian body.
Bradley W. Rice is at Caliper Life Sciences, Alameda, California, USA and Christopher H. Contag is in the Departments of Pediatrics, Microbiology & Immunology and Radiology, and in the programs of Photomedicine and Molecular Imaging at Stanford University, Stanford, California, USA. email: [email protected]
Figure 1 The ‘transparent mouse’ experiment. The relative opacity of mammalian tissues is wavelength specific. (a) Light transmission is highest and autofluorescence is lowest in the near-infrared region of the spectrum. (b) Transillumination of a mouse with white light. (c) The spectra of the transmitted light in b at six different points. At 400–550 nm, the degree of signal transmission is very low, emphasizing the need for proteins that excite and emit at longer wavelengths. At ~525 nm (blue arrow), transmission increases owing to a drop in hemoglobin absorption in a narrow spectral range (490–550 nm). Many fluorescent and bioluminescent reporters emit with a peak near 560 nm. The green arrow at 600 nm marks the beginning of the ‘transparent window’ that is the target for optical imaging reagent development. This window is characterized by reduced absorbance and minimal autofluorescence. The yellow arrow highlights the extreme differences in transmission from point 12 relative to that of point 1, which are due to transmission through thicker and more complex tissues.
a b cTransmitted
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Transillumination through the entire body is extremely challenging with visible light, but is relatively straightforward in the near infra-red. The IFP1.4 protein is therefore a first step toward the development of imaging technolo-gies able to detect multifunctional infrared reporter proteins with a sensitivity that can reveal complex biological processes as they occur deep in mammalian tissues.
1. Giepmans, B.N., Adams, S.R., ellisman, M.H. & Tsien, R.Y. Science 312, 217–224 (2006).
2. Chalfie, M., Tu, Y., euskirchen, G., Ward, W.W. & Prasher, D.C. Science 263, 802–805 (1994).
3. Contag, C.H. et al. Photochem. Photobiol. 66, 523–531 (1997).
4. Takada, T. et al. Nat. Biotechnol. 15, 458–461 (1997).
5. Chishima, T. et al. Cancer Res. 57, 2042–2047 (1997).
6. Zhao, H. et al. J. Biomed. Opt. 10, 41210 (2005).7. Fischer, A.J. & Lagarias, J.C. Proc. Natl. Acad. Sci.
USA 101, 17334–17339 (2004).8. Shu, X. et al. Science 324, 804–807 (2009).9. Rasmussen, J.C., Tan, i.C., Marshall, M.v., Fife, C.e.
& Sevick-Muraca, e.M. Curr. Opin. Biotechnol. 20, 74–82 (2009).
10. Shcherbo, D. Biochem. J. 418, 567–574 (2009).11. Troy, T., Jekic-McMullen, D., Sambucetti, L. & Rice,
B. Mol. Imaging 3, 9–23 (2004).12. Zacharakis, G., Shih, H., Ripoll, J., Weissleder, R. &
Ntziachristos, v. Mol. Imaging 5, 153–159 (2006).
living laboratory rodents. Fluorescent pro-teins with shorter excitation and emission wavelengths are well suited for microscopic imaging, whereas bioluminescent proteins have signal-to-noise ratios in living mam-mals that make them exceptional in vivo reporters. For this reason many animal stud-ies have used fusion proteins comprised of short-emitting fluorescent proteins and bioluminescent reporters such as luciferase. Infrared fluorescent proteins could make it possible to simplify these constructs by using a single reporter for both microscopic and macroscopic imaging. Thus, infrared fluores-cent proteins will reduce or eliminate many of the differences between bioluminescence and fluorescence imaging in vivo11.
In tandem with advances in near-infrared fluorescent proteins, it will be important to develop compatible detection technologies and image processing software. Transillumination geometry (as opposed to reflection, or epi-illumination), for example, offers improved sensitivity and enables three-dimensional diffuse tomographic reconstruc-tions for localizing fluorescent reporters12.
tremendous breadth of capabilities. By con-trast, research on infrared fluorescent protein is in its infancy, and the report by Shu et al.8 is the first to describe the use of such proteins in living mammalian systems. As with GFP, continued work on IFP1.4 is likely to lead to a new set of reporters that are optimized for operation in the near infrared and for use in mammalian systems.
In vivo imaging studies by Shu et al.8 dem-onstrated detectable signals in the liver after adenovirus-mediated delivery of IFP1.4. Although qualitative in nature, these stud-ies revealed more intense signals with IFP1.4 than with mKATE as well as increased fluo-rescence upon exogenous addition of biliver-din, They also indirectly; revealed the effects of tissue absorption removal of overlying tis-sues, showed that the lower excitation and emission wavelengths of mKATE are more strongly affected by tissue absorption than those of IFP1.4.
A real strength of IFP1.4 is its ability to be imaged over a range of spatial scales, rang-ing from microscopic detection at subcel-lular resolution to macroscopic studies in
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