Photoacoustic imaging allows absorption-based high-resolution spectroscopic in vivo imaging at a depth beyond that of optical microscopy. Until recently, photoacoustic imaging has largely been restricted to visualizing the vasculature through endogenous haemoglobin contrast, with most non-vascularized tissues remaining invisible unless exogenous contrast agents are administered. Genetically encodable photoacoustic contrast is attractive as it allows selective labelling of cells, permitting studies of, for example, specific genetic expression, cell growth or more complex biological behaviours in vivo. In this study we report a novel photoacoustic imaging scanner and a tyrosinase-based reporter system that causes human cell lines to synthesize the absorbing pigment eumelanin, thus providing strong photoacoustic contrast. Detailed threedimensional images of xenografts formed of tyrosinase-expressing cells implanted in mice are obtained in vivo to depths approaching 10 mm with a spatial resolution below 100 μm. This scheme is a powerful tool for studying cellular and genetic processes in deep mammalian tissues.O ptical techniques such as fluorescence or bioluminescence imaging are widely used to visualize biological tissues in vivo 1-4 . However, strong optical scattering fundamentally limits the penetration depth or spatial resolution. Microscopy and other techniques that utilize ballistic photons 4 can provide cellular resolution, but only to sub-millimetre penetration depths, while diffuse optical methods such as fluorescence optical tomography 1 can provide greater penetration depths (on the scale of centimetres) but with only limited spatial resolution (on the scale of millimetres). Photoacoustic imaging (PAI) offers the prospect of overcoming these limitations 5-8 . Here, ultrasound waves generated by the absorption of laser light by tissue chromophores are used to produce images of biological tissues based on optical absorption. Because acoustic waves are scattered much less than photons in soft tissues, PAI avoids the depth and spatial resolution limitations of purely optical imaging techniques: depths of a few centimetres with scalable spatial resolution ranging from tens to hundreds of micrometres (depending on depth) are readily achievable.Although strong absorption by haemoglobin enables the acquisition of exquisite three-dimensional photoacoustic (PA) images of the vasculature 9-14 , most cells and tissues are relatively weakly absorbing at visible and near-infrared wavelengths and are thus indistinguishable in the absence of exogenous contrast. The latter can be provided by nanoparticle-or dye-based targeted contrast agents 15-17 , but these can present challenges in achieving effective specific targeting and clearance. The use of reporter genes to provide genetically encoded exogenous PA contrast would avoid these limitations and has the further advantage of providing opportunities to study more complex biological behaviours such as cell growth dynamics and intracellular processes such as gene expressi...

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SHOC2 is mutated in Noonan syndrome and plays a key role in the activation of the ERK-MAPK pathway, which is upregulated in the majority of human cancers. SHOC2 functions as a PP1-regulatory protein and as an effector of MRAS. Here we show that SHOC2 and MRAS form a complex with SCRIB, a polarity protein with tumor suppressor properties. SCRIB functions as a PP1-regulatory protein and antagonizes SHOC2-mediated RAF dephosphorylation through a mechanism involving competition for PP1 molecules within the same macromolecular complex. SHOC2 function is selectively required for the malignant properties of tumor cells with mutant RAS, and both MRAS and SHOC2 play a key role in polarized migration. We propose that MRAS, through its ability to recruit a complex with paradoxical components, coordinates ERK pathway spatiotemporal dynamics with polarity and that this complex plays a key role during tumorigenic growth.

Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λmax=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

Genetically expressed fluorescent proteins have been shown to provide photoacoustic contrast. However, they can be limited by low photoacoustic generation efficiency and low optical absorption at red and near infrared wavelengths, thus limiting their usefulness in mammalian small animal models. In addition, many fluorescent proteins exhibit low photostability due to photobleaching and transient absorption effects. In this study, we explore these issues by synthesizing and characterizing a range of commonly used fluorescent proteins (dsRed, mCherry, mNeptune, mRaspberry, AQ143, E2 Crimson) and novel non-fluorescent chromoproteins (aeCP597 and cjBlue and a non-fluorescent mutant of E2 Crimson). The photoacoustic spectra, photoacoustic generation efficiency and photostability of each fluorescent protein and chromoprotein were measured. Compared to the fluorescent proteins, the chromoproteins were found to exhibit higher photoacoustic generation efficiency due to the absence of radiative relaxation and ground state depopulation, and significantly higher photostability. The feasibility of converting an existing fluorescent protein into a non-fluorescent chromoprotein via mutagenesis was also demonstrated. The chromoprotein mutant exhibited greater photoacoustic signal generation efficiency and better agreement between the photoacoustic and the specific extinction coefficient spectra than the original fluorescent protein. Lastly, the genetic expression of a chromoprotein in mammalian cells was demonstrated. This study suggests that chromoproteins may have potential for providing genetically encoded photoacoustic contrast.

A highly efficient and rapid CuII‐mediated three‐component “click reaction” allows one‐pot assembly of dual optical and nuclear labeling reagents. Proof‐of‐concept imaging studies demonstrate that the distribution of the dual‐labeled antibody A5B7 can be interrogated from the cellular to the macroscopic level using a combination of optical and nuclear imaging techniques.

A high yielding, scalable and convergent synthesis of infra-luciferins and investigation of their potential for near-infrared bioluminescence imaging.

Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to nearinfrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to l max = 706 nm with different Fluc mutants. This emission is the most redshifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.Bioluminescence imaging (BLI) has revolutionized molecular genetic imaging in biomedical research as a cheap and easy means to longitudinally image the genetic behavior of life and disease processes in whole mammals. [1][2][3][4] As they produce the brightest form of bioluminescence, [5] genes from coleopterans are commonly used to localize, track, and quantify cells and molecular or functional events in vivo. [6][7][8] In a well-studied reaction, [9] beetle luciferin (1, Figure 1 a) is adenylated by firefly luciferase (Fluc) and this reacts with molecular oxygen to produce an excited state species, oxyluciferin* (2), which decays to release a photon with a high quantum yield (l max = 558 nm).[5] However, absorption of visible light by hemoglobin (Hb) and melanin restricts image resolution and signal penetration at this wavelength. Between l = 600-800 nm, the absorption of light by Hb decreases by a factor of approximately 50, resulting in less attenuation of red light. This wavelength range is within what is termed the "bio-optical window" and there has been much focus on engineering red-shifted Fluc enzymes that have maximum emission wavelengths in this range, [10][11][12][13][14][15] but these have peaked at wavelengths less than l = 645 nm.The most red-shifted luciferin analogues to date [16] are based upon amino derivatives (Figure 1 b), for example cyclic aminoluciferin (3 a: l max = 599 nm; 3 b: l max = 607 nm), [17] seleno-d-aminoluciferin (4: l max = 600 nm), [18] and a rationally designed 4-(dimethylamino)phenyl derivative conjugated to a thiazoline group (5: l max = 675 nm). [19] In particular cyclic aminoluciferin derivative 3 a has been shown to give improved bioluminescence imaging compared to luciferin (LH 2 ; 1) at dilute concentrations where the intracellular concentration of the luciferin or analogue is limiting.[20] Nearinfrared emission has been detected with an aminoluciferin Cy5 conjugate, but this is due to bioluminescence resonance energy transfer (BRET), [21] meaning that the conjugate cannot be used for multip...