Near-infrared Fluorescent Markers Research Articles

Supervised Autonomous Electrosurgery for Soft Tissue Resection.

Surgical resection is the current clinical standard of care for treating squamous cell carcinoma. Maintaining an adequate tumor resection margin is the key to a good surgical outcome, but tumor edge delineation errors are inevitable with manual surgery due to difficulty in visualization and hand-eye coordination. Surgical automation is a growing field of robotics to relieve surgeon burdens and to achieve a consistent and potentially better surgical outcome. This paper reports a novel robotic supervised autonomous electrosurgery technique for soft tissue resection achieving millimeter accuracy. The tumor resection procedure is decomposed to the subtask level for a more direct understanding and automation. A 4-DOF suction system is developed, and integrated with a 6-DOF electrocautery robot to perform resection experiments. A novel near-infrared fluorescent marker is manually dispensed on cadaver samples to define a pseudotumor, and intraoperatively tracked using a dual-camera system. The autonomous dual-robot resection cooperation workflow is proposed and evaluated in this study. The integrated system achieves autonomous localization of the pseudotumor by tracking the near-infrared marker, and performs supervised autonomous resection in cadaver porcine tongues (N=3). The three pseudotumors were successfully removed from porcine samples. The evaluated average surface and depth resection errors are 1.19 and 1.83mm, respectively. This work is an essential step towards autonomous tumor resections.

Near-infrared dye marking for thoracoscopic resection of small-sized pulmonary nodules: comparison of percutaneous and bronchoscopic injection techniques

BackgroundMinimally invasive video-assisted thoracoscopic surgery for small-sized pulmonary nodules is challenging, and image-guided preoperative localisation is required. Near-infrared indocyanine green fluorescence is capable of deep tissue penetration and can be distinguished regardless of the background colour of the lung; thus, indocyanine green has great potential for use as a near-infrared fluorescent marker in video-assisted thoracoscopic surgery.MethodsThirty-seven patients with small-sized pulmonary nodules, who were scheduled to undergo video-assisted thoracoscopic wedge resection, were enrolled in this study. A mixture of diluted indocyanine green and iopamidol was injected into the lung parenchyma as a marker, using either computed tomography-guided percutaneous or bronchoscopic injection techniques. Indications and limitations of the percutaneous and bronchoscopic injection techniques for marking nodules with indocyanine green fluorescence were examined and compared.ResultsIn the computed tomography-guided percutaneous injection group (n = 15), indocyanine green fluorescence was detected in 15/15 (100%) patients by near-infrared thoracoscopy. A small pneumothorax occurred in 3/15 (20.0%) patients, and subsequent marking was unsuccessful after a pneumothorax occurred. In the bronchoscopic injection group (n = 22), indocyanine green fluorescence was detected in 21/22 (95.5%) patients. In 6 patients who underwent injection marking at 2 different lesion sites, 5/6 (83.3%) markers were successfully detected.ConclusionEither computed tomography-guided percutaneous or bronchoscopic injection techniques can be used to mark pulmonary nodules with indocyanine green fluorescence. Indocyanine green is a safe and easily detectable fluorescent marker for video-assisted thoracoscopic surgery. Furthermore, the bronchoscopic injection approach enables surgeons to mark multiple lesion areas with less risk of causing a pneumothorax.Trial RegistrationUMIN-CTR R000027833 accepted by ICMJE. Registered 5 January 2013.

Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (~710nm). They are small monomers (~15kDa) and not only show excellent photostability, but are also thermostable up to 80°C, tolerate acid down to pH2 and high concentrations of denaturants. The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers. In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy, alone or fused to target proteins, is exemplified in several mammalian cells, including, human cell lines, in the nematode, Caenorhabditis elegans and, at low pH, in Lactobacillus lactis.

Macroscopic-imaging technique for subsurface quantification of near-infrared markers during surgery.

Obtaining accurate quantitative information on the concentration and distribution of fluorescent markers lying at a depth below the surface of optically turbid media, such as tissue, is a significant challenge. Here, we introduce a fluorescence reconstruction technique based on a diffusion light transport model that can be used during surgery, including guiding resection of brain tumors, for depth-resolved quantitative imaging of near-infrared fluorescent markers. Hyperspectral fluorescence images are used to compute a topographic map of the fluorophore distribution, which yields structural and optical constraints for a three-dimensional subsequent hyperspectral diffuse fluorescence reconstruction algorithm. Using the model fluorophore Alexa Fluor 647 and brain-like tissue phantoms, the technique yielded estimates of fluorophore concentration within ±25% of the true value to depths of 5 to 9 mm, depending on the concentration. The approach is practical for integration into a neurosurgical fluorescence microscope and has potential to further extend fluorescence-guided resection using objective and quantified metrics of the presence of residual tumor tissue.

From Binary Cu2S to ternary Cu-In-S and quaternary Cu-In-Zn-S nanocrystals with tunable composition via partial cation exchange.

We present an approach for the synthesis of ternary copper indium sulfide (CIS) and quaternary copper indium zinc sulfide (CIZS) nanocrystals (NCs) by means of partial cation exchange with In(3+) and Zn(2+). The approach consists of a sequential three-step synthesis: first, binary Cu2S NCs were synthesized, followed by the homogeneous incorporation of In(3+) by an in situ partial cation-exchange reaction, leading to CIS NCs. In the last step, a second partial exchange was performed where Zn(2+) partially replaced the Cu(+) and In(3+) cations at the surface, creating a ZnS-rich shell with the preservation of the size and shape. By careful tuning reaction parameters (growth and exchange times as well as the initial Cu(+):In(3+):Zn(2+) ratios), control over both the size and composition was achieved. This led to a broad tuning of photoluminescence of the final CIZS NCs, ranging from 880 to 1030 nm without altering the NCs size. Cytotoxicity tests confirmed the biocompatibility of the synthesized CIZS NCs, which opens up opportunities for their application as near-infrared fluorescent markers in the biomedical field.

The Photochemistry of Bacteriophytochrome: Key to its Use as a Deep-Tissue Fluorescence Probe

Bacteriophytochromes (Bphs) are red-light photoreceptors that regulate a variety of bacterial responses. Their photosensory core consists of PAS, GAF and PHY domains and covalently binds biliverdin (BV). The Bph light activation mechanism involves isomerization around the BV C15=C16 double bond, resulting in a flip of its D-ring. In an important recent development, PAS-GAF variants were engineered for use as near-infrared fluorescent markers in mammalian tissues (Shu et al. Science 2009). Here, we report the photochemistry of two Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) that have distinct photoconversion and fluorescence properties. We applied ultrafast spectroscopy on P3 and P2 PAS-GAF proteins and the P3 D216A and P2 D202A PAS-GAF-PHY proteins. In these mutants a conserved aspartate which connects BV with the PHY domain through extensive hydrogen-bond networks, was replaced by alanine. The excited-state lifetime of P3 and P2 PAS-GAF was significantly larger than their PAS-GAF-PHY counterparts. Mutation of the conserved Asp to Ala in PAS-GAF-PHY had a similar but larger effect. In particular, the fluorescence quantum yield of the P3 D216A mutant was 0.066, higher than that of wild type P3 (0.043) and similar to the engineered Bph of Shu et al. We conclude that elimination of a key hydrogen-bond interaction between Asp and a conserved Arg in the PHY domain is responsible for the excited-state lifetime increase. H/D exchange resulted in a 1.5 - 1.7 fold increase of excited-state lifetime. The results are rationalized with a reaction model where excited-state deactivation of BV proceeds via excited-state proton transfer from the BV pyrrole nitrogens to the backbone of the conserved Asp or a bound water. This work may aid in rational structure- and mechanism-based conversion of P3 and other BPhs into efficient near-IR fluorescent markers.

Fluorescence quantum yield and photochemistry of bacteriophytochrome constructs

Bacteriophytochromes (Bphs) are red-light photoreceptor proteins with a photosensory core that consists of three distinct domains, PAS, GAF and PHY, and covalently binds biliverdin (BV) to a conserved cysteine in the PAS domain. In a recent development, PAS-GAF variants were engineered for use as a near-infrared fluorescent marker in mammalian tissues (Tsien and co-workers, Science, 2009, 324, 804-807). Here, we report the fluorescence quantum yield and photochemistry of two highly-related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We applied ultrafast spectroscopy to wild type P3 and P2 PAS-GAF proteins and their P3 D216A, Y272F and P2 D202A PAS-GAF-PHY mutant proteins. In these mutants hydrogen-bond interactions between a conserved aspartate (Asp) which connects the BV chromophore with the PHY domains are disrupted. The excited-state lifetime of the truncated P3 and P2 PAS-GAF proteins was significantly longer than in their PAS-GAF-PHY counterparts that constitute the full photosensory core. Mutation of the conserved Asp to Ala in the PAS-GAF-PHY protein had a similar but larger effect. The fluorescence quantum yields of the P3 D216A and Y272F mutants were 0.066, higher than that of wild type P3 (0.043) and similar to the engineered Bph of Tsien and co-workers. We conclude that elimination of a key hydrogen-bond interaction between Asp and a conserved Arg in the PHY domain is responsible for the excited-state lifetime increase in all Bph variants studied here. H/D exchange resulted in a 1.4-1.7 fold increase of excited-state lifetime. The results support a reaction model in which deactivation of the BV chromophore proceeds via excited-state proton transfer from the BV pyrrole nitrogens to the backbone of the conserved Asp or to a bound water. This work may aid in rational structure- and mechanism-based conversion of constructs based on P3 and other BPhs into efficient near-IR, deep tissue, fluorescent markers.

Angular domain fluorescence imaging for small animal research

We describe a novel macroscopic fluorescent imaging technique called angular domain fluorescence imaging (ADFI) applicable to the detection of fluorophores embedded in biological tissues. The method exploits the collimation detection capabilities of an angular filter array (AFA). The AFA uses the principle of acceptance angle filtration to extract minimally scattered photons emitted from fluorophores deep within tissue. Our goal was to develop an ADFI system for imaging near-infrared fluorescent markers for small animal imaging. According to the experimental results, the ADFI system offered higher resolution and contrast compared to a conventional lens and lens-pinhole fluorescent detection system. Furthermore, ADFI of a hairless mouse injected with a fluorescent bone marker revealed vertebral structural and morphometric data that correlated well with data derived from volumetric x-ray computed tomography images. The results suggested that ADFI is a useful technique for submillimeter mapping of the distribution of fluorescent biomarkers in small animals.

Optimized and Far-Red-Emitting Variants of Fluorescent Protein eqFP611

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.

Preparation of luminescent Cy5 doped core-shell SFNPs and its application as a near-infrared fluorescent marker

Cy5 dye is widely used as a biomarker in the research fields of life science because of its excitation at wavelengths above 600 nm where autofluorescence of bio-matter is much reduced. However, Cy5 dye could not be encapsulate into silica directly to form stable nanoparticles by using of the traditional methods. In this paper, an improved method had been developed to prepare Cy5 dye doped core-shell silica fluorescent nanoparticles (SFNPs), employing biomolecules conjugated Cy5 as the core material and silica coating produced from the hydrolysis TEOS (tetraethyl orthosilicate) in the water-in-oil microemulsion. To obtain stable Cy5 dye doped SFNPs with core-shell structure, five kinds of biomolecules with different iso-electric point (p I) have been selected to conjugate Cy5 for preparation of core-shell SFNPs. Results demonstrated that very bright and photostable Cy5 doped core-shell SFNPs could be both prepared by use of positive polysine conjugated Cy5 or IgG conjugated Cy5 as the core material, respectively. IgG conjugated Cy5 doped core-shell SFNPs was selected as a demonstration to be characterized and applied as a near-infrared fluorescent marker in cell recognition. The results showed that Cy5 doped core-shell SFNPs prepared by conjugating with a positive biomolecules IgG as the core material were luminescent and stable. About 110 Cy5 dye molecules could be doped in one nanoparticle with size of 42 ± 5 nm. The breast cancer cells had been selectively recognized by use of the near-infrared fluorescent marker based on the Cy5–IgG doped core-shell SFNPs. And the results demonstrated that this Cy5 doped core-shell SFNPs fluorescence marker was superior to the pure Cy5 dye marker for cell recognition in photostability and detection sensitivity.