NIR fluorescent ytterbium compound for in vivo fluorescence molecular imaging

We have developed a new NIR fluorescent probe based on an ytterbium(III) (E)‐1‐(pyridin‐2‐yl‐diazenyl)naphthalen‐2‐ol (PAN) complex. This probe emits near‐infrared luminescence derived from the Yb ion through excitation of the PAN moiety with visible light (λ_ex = 530 nm, λ_em = 975 nm). The results support the possible utility of the probe for in vivo fluorescence molecular imaging. Copyright © 2009 John Wiley & Sons, Ltd.     

Computed tomography for in vivo deep over‐1000 nm near‐infrared fluorescence imaging

This study aims to develop a novel cross‐sectional imaging of fluorescence in over‐1000 nm near‐infrared (OTN‐NIR), which allows in vivo deep imaging, using computed tomography (CT) system. Cylindrical specimens of composite of OTN‐NIR fluorophore, NaGdF₄ co‐doped with Yb³⁺ and Ho³⁺ (ex: 980 nm, em: 1150 nm), were embedded in cubic agar (10.5–12 mm) or in the peritoneal cavity of mice and placed on a rotatable stage. When the fluorescence from inside of the samples was serially captured from multiple angles, the images were disrupted by the reflection and refraction of emitted light on the sample‐air interface. Immersing the sample into water filled in a rectangular bath suppressed the disruption at the interface and successfully reconstructed the position and concentration of OTN‐NIR fluorophores on the cross‐sectional images using a CT technique. This is promising as a novel three‐dimensional imaging technique for OTN‐NIR fluorescent image projections of small animals captured from multiple angles.     

Rhodobacter sphaeroides,a novel tumor‐targeting bacteria that emits natural near‐infrared fluorescence

Several optical imaging techniques have been used to monitor bacterial tropisms for cancer. Most such techniques require genetic engineering of the bacteria to express optical reporter genes. This study investigated a novel tumor‐targeting strain of bacteria, Rhodobacter sphaeroides 2.4.1 (R. sphaeroides), which naturally emits near‐infrared fluorescence, thereby facilitating the visualization of bacterial tropisms for cancer. To determine the penetration depth of bacterial fluorescence, various numbers of cells (from 10⁸ to 10¹⁰ CFU) of R. sphaeroides and two types of Escherichia coli, which stably express green fluorescent protein (GFP) or red fluorescent protein (RFP), were injected s.c. or i.m. into mice. Bacterial tropism for cancer was determined after i.v. injection of R. sphaeroides (10⁸ CFU) into mice implanted s.c. with eight types of tumors. The intensity of the fluorescence signal in deep tissue (muscle) from R. sphaeroides was much stronger than from E. coli‐expressing GFP or RFP. The near‐infrared fluorescence signal from R. sphaeroides was visualized clearly in all types of human or murine tumors via accumulation of bacteria. Analyses of C‐reactive protein and procalcitonin concentrations and body weights indicated that i.v. injection of R. sphaeroides does not induce serious systemic immune reactions. This study suggests that R. sphaeroides could be used as a tumor‐targeting microorganism for the selective delivery of drugs to tumor tissues without eliciting a systemic immune reaction and for visualizing tumors.     

Optical fluorescence imaging with shortwave infrared light emitter nanomaterials for in vivo cell tracking in regenerative medicine

In vivo tracking and monitoring of adoptive cell transfer has a distinct importance in cell‐based therapy. There are many imaging modalities for in vivo monitoring of biodistribution, viability and effectiveness of transferred cells. Some of these procedures are not applicable in the human body because of low sensitivity and high possibility of tissue damages. Shortwave infrared region (SWIR) imaging is a relatively new technique by which deep biological tissues can be potentially visualized with high resolution at cellular level. Indeed, scanning of the electromagnetic spectrum (beyond 1000 nm) of SWIR has a great potential to increase sensitivity and resolution of in vivo imaging for various human tissues. In this review, molecular imaging modalities used for monitoring of biodistribution and fate of administered cells with focusing on the application of non‐invasive optical imaging at shortwave infrared region are discussed in detail.     

A novel near-infrared fluorescence imaging probe for in vivo neutrophil tracking

The development and validation of a multiscopic near-infrared fluorescence (NIRF) probe, cinnamoyl-F-(D)L-F-(D)L-F-PEG-cyanine7 (cFlFlF-PEG-Cy7), that targets formyl peptide receptor on neutrophils using a mice ear inflammation model is described. Acute inflammation was induced in mice by topical application of phorbol-12-myristate-13-acetate to left ears 24 hours before probe administration. Noninvasive NIRF imaging was longitudinally performed up to 24 hours following probe injection. The in vivo neutrophil-targeting specificity of the probe was characterized by a blocking study with preadministration of excess nonfluorescent peptide cFlFlF-PEG and by an imaging study with a scrambled peptide probe cLFFFL-PEG-Cy7. NIRF imaging of mice injected with cinnamoyl-L-F-F-F-L-PEG-cyanine7 (cFlFlF-PEG-Cy7) revealed that the fluorescence intensity for inflamed left ears was approximately fourfold higher than that of control right ears at 24 hours postinjection. In comparison, the ratios acquired with the scrambled probe and from the blocking study were 1.5- and 2-fold at 24 hours postinjection, respectively. Moreover, a microscopic immunohistologic study confirmed that the NIRF signal of cFlFlF-PEG-Cy7 was associated with activated neutrophils in the inflammatory tissue. With this probe, in vivo neutrophil chemotaxis could be correlatively imaged macroscopically in live animals and microscopically at tissue and cellular levels.     

A novel near-infrared fluorescence imaging probe for in vivo neutrophil tracking

AbstractWe delivered adenovirus vector (Ad) via intravitreous injection and monitored transgene (luciferase) expression in living mice (BALB/c) at multiple time points. In vivo live imaging technology was able to assess dynamically intraocular luciferase expression in a single animal population throughout the entire experiment period. Using this information, we were able to determine the optimal time point for readministration of Ad into the eyes and to dynamically study the time course of expression of a second Ad administration. Optical imaging demonstrated the limited period of transgene expression in eyes. Significant transgene signal was also detected in livers. The repeat intraocular delivery of the adenovirus resulted in significant blunting of transgene expression in both eyes and livers compared to the initial delivery. Periocular corticosteroid (triamcinolone acetonide) injection combined with initial Ad delivery was effective to rescue luciferase expression on repeat Ad vector delivery. However, this effect was not observed when corticosteroid was combined with repeat Ad delivery. Although corticosteroid enhanced ocular transgene expression, it also increased transgene expression in liver, which has potential safety implications. This dynamic transgene expression in eyes was successfully traced and monitored via a live imaging technique.     

Interaction of metastasis-inducing S100A4 protein in vivo by fluorescence lifetime imaging microscopy

Elevated levels of the calcium-binding regulatory protein, S100A4, have been shown to be causative of a metastatic phenotype in models of cancer metastasis and to be associated with reduced patient survival in breast cancer patients. Recombinant S100A4 protein interacts in vitro in a calcium-dependent manner with the heavy chain of non-muscle myosin isoform A at a protein kinase C phosphorylation site. At present, the mechanism of metastasis induction by S100A4 in vivo is almost completely unknown. The binding of S100A4 to a C-terminal recombinant fragment of non-muscle myosin heavy chain in living HeLa cells has now been shown using confocal microscopy, fluorescence lifetime imaging microscopy and time-correlated single-photon counting. The association between S100A4 and non-muscle myosin heavy chain was studied by determining fluorescence resonance energy transfer-derived changes in the fluorescence lifetime of enhanced cyan fluorescent protein fused to S100A4 in the presence of a recombinant fragment of the C-terminal region of non-muscle myosin heavy chain (rNMMHCIIA) fused to enhanced yellow fluorescent protein. There was no interaction between the non-muscle myosin heavy chain fragment and a calcium-binding-deficient mutant of S100A4 protein which has been shown to be defective in the induction of metastasis in model systems in vivo. The results demonstrate, for the first time, not only direct interaction between S100A4 and a target rNMMHCIIA in live mammalian cells, but also that the interaction between S100A4 and the non-muscle myosin heavy chain in vivo could contribute to the mechanism of metastasis induction by a high level of S100A4 protein.     

Perfusion‐based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near‐infrared molecular probes

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near‐infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor‐selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor‐to‐background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real‐time intraoperative procedures.      

Development of a molecular bioswitch using fluorescence lifetime imaging: Incremental activation of fluorescein diacetate

Molecular bioswitches offer an invaluable asset in the shift from systemic to targeted treatments. Within the growing arsenal of switches are imaging probes that functionalize only in given locations or situations. Acetate esters are a common fluorescent example, known to activate upon interaction with esterases. Fluorescein diacetate (FDA) is one such fluorophore used in cell viability assays. These assays rely on the fact that the compound begins colorless and with no fluorescent signature whatsoever, and only after internalization into cells it is possible to detect a fluorescence signal. In this study, using fluorescence intensity (FI) and fluorescence lifetime (FLT) imaging, FDA is shown to be fluorescent even when unactivated. Furthermore, the FLT is shown to change with pH. Finally, the ability to image FDA in different environments simulated by tissue‐imitating phantoms is explored. Altogether, the ability of FDA to serve as a bioswitch when measured using FLT imaging microscopy (FLIM) is assessed. The combination of a spectrum of FDA activation and FLIM serves as a bioswitch, where biologically relevant stimulation can generate detectable and incremental variations.      

Enhancement of near‐infrared up‐conversion and blue down‐conversion luminescence in LuNbO4:Yb3+,Tm3+ with Ga3+ and Ta5+ substitutions

Under a 980‐nm excitation, the up‐conversion (UC) spectra of LuNbO₄:Yb³⁺,Tm³⁺ powders exhibited predominantly near‐infrared bands (~805 nm) of Tm³⁺ through an energy transfer process from Yb³⁺ to Tm³⁺. Regarding the down‐conversion (DC) luminescence of the powders, the photoluminescence excitation spectra consisted of a broad charge transfer band (270 nm) due to [NbO₄]^3? and sharp band (360 nm) of Tm³⁺, while the corresponding emission spectra exhibited a blue emission at 458 nm. Upon substitution of Ga³⁺ and Ta⁵⁺ for Lu³⁺ and Nb⁵⁺, respectively, both UC and DC luminescence properties were significantly enhanced. For the Ga³⁺ substitution, the increased emission intensity could be explained by the crystal field asymmetry surrounding the Tm³⁺ ions induced by the large difference in ionic radius between Ga³⁺ and Lu³⁺. For the Ta⁵⁺ substitution, we believe that an M′‐LuTaO₄ substructure was formed in the host, which led to the formation of a TaO₆ octahedral coordination instead of a NbO₄ tetrahedral coordination. Consequently, the crystal symmetry of the local structure was modified, and thus the UC and DC luminescence properties were enhanced. The dual‐mode (UC and DC) luminescence demonstrates that LuNbO₄:Yb³⁺,Tm³⁺ has a great potential in the fields of temperature sensing probes, anti‐counterfeiting, and bioapplications.     

Indocyanine green labeling for optical and photoacoustic imaging of mesenchymal stem cells after in vivo transplantation

The transplantation of mesenchymal stem cells (MSCs) holds great promise for the treatment of a plethora of human diseases, but new noninvasive procedures are needed to monitor the cell fate in vivo. Already largely used in medical diagnostics, the fluorescent dye indocyanine green (ICG) is an established dye to track limited numbers of cells by optical imaging (OI), but it can also be visualized by photoacoustic imaging (PAI), which provides a higher spatial resolution than pure near infrared fluorescence imaging (NIRF). Because of its successful use in clinical and preclinical examinations, we chose ICG as PAI cell labeling agent. Optimal incubation conditions were defined for an efficient and clinically translatable MSC labeling protocol, such that no cytotoxicity or alterations of the phenotypic profile were observed, and a consistent intracellular uptake of the molecule was achieved. Suspensions of ICG‐labeled cells were both optically and optoacoustically detected in vitro, revealing a certain variability in the photoacoustic spectra acquired by varying the excitation wavelength from 680 to 970 nm. Intramuscular engraftments of ICG‐labeled MSCs were clearly visualized by both PAI and NIRF over few days after transplantation in the hindlimb of healthy mice, suggesting that the proposed technique retains a considerable potential in the field of transplantation‐focused research and therapy. Stem cells were labeled with the Food and Drug Administration (FDA)‐approved fluorescent dye ICG, and detected by both PAI and OI, enabling to monitor the cell fate safely, in dual modality, and with good sensitivity and improved spatial resolution.      

A novel diagnostic method for human immunodeficiency virus type-1 in plasma by near-infrared spectroscopy

Presently, the diagnosis of virus infections is based mainly on serological assays. Although polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) have been increasingly used for the diagnosis of such viral infections, the risk of transfusion-transmitted blood-borne viruses remains. Furthermore, PCR and ELISA are expensive and time-consuming, and sometimes cause falsepositive or false-negative results. Therefore, a rapid, accurate and cost-effective diagnostic procedure is needed. We subjected plasma from individuals infected with human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immune deficiency syndrome (AIDS), as well as plasma from uninfected individuals as a control to near-infrared (NIR) spectroscopy, which may provide a rapid diagnostic method for HIV-1 infection without using any reagent. NIR spectra in the 600-1,000 nm region for plasma from pre-serologically HIV-1-infected individuals and healthy donors were subjected to partial least squares (PLS) regression analysis and leave-out cross-validation to develop a multivariate model to estimate the concentration of HIV-1. Simultaneously, the same plasma samples were examined for HIV-1 p24 by ELISA. The results obtained by the NIR spectroscopy model for HIV-1 yielded a good correlation with those obtained by the reference method (HIV-1 p24 ELISA). These results suggest that NIR spectroscopy using plasma could provide a rapid, accurate, cost-effective tool for large-scale diagnosis of HIV-1 infection.     

In vivo imaging of CT26 mouse tumours by using cmHsp70.1 monoclonal antibody

The major stress‐inducible heat shock protein 70 (Hsp70) is frequently present on the cell surface of human tumours, but not on normal cells. Herein, the binding characteristics of the cmHsp70.1 mouse monoclonal antibody (mAb) were evaluated in vitro and in a syngeneic tumour mouse model. More than 50% of the CT26 mouse colon carcinoma cells express Hsp70 on their cell surface at 4°C. After a temperature shift to 37°C, the cmHsp70.1‐fluorescein isothiocyanate mAb translocates into early endosomes and lysosomes. Intraoperative and near‐infrared fluorescence imaging revealed an enrichment of Cy5.5‐conjugated mAb cmHsp70.1, but not an identically labelled IgG1 isotype‐matched control, in i.p. and s.c. located CT26 tumours, as soon as 30 min. after i.v. injection into the tail vein. Due to the rapid turnover rate of membrane‐bound Hsp70, the fluorescence‐labelled cmHsp70.1 mAb became endocytosed and accumulated in the tumour, reaching a maximum after 24 hrs and remained detectable at least up to 96 hrs after a single i.v. injection. The tumour‐selective internalization of mAb cmHsp70.1 at the physiological temperature of 37°C might enable a targeted uptake of toxins or radionuclides into Hsp70 membrane‐positive tumours. The anti‐tumoral activity of the cmHsp70.1 mAb is further supported by its capacity to mediate antibody‐dependent cytotoxicity.     

Red‐shifted emission from 1,2‐dioxetane‐based chemiluminescent reactions

Commercial chemiluminescent reagents emit across a broad portion of the electromagnetic spectrum (400–500 nm). A challenge to the use of chemiluminescence to monitor biological processes is the presence of interfering substances in the biological optical window. In the present study, longer wavelength emitting fluorophores (the organic dyes Alexa 568 and Alexa 647), and a semiconductor nanoparticle (QDOT800) were used to red‐shift the emission from commercially available 1,2‐dioxetane‐based chemiluminescent substrate reactions. By adding non‐conjugated fluorescent emitters into chemiluminescent reaction mixtures, an emission peak occurred at the predicted wavelength of the fluorescent emitter. The excitation and emission from QDOT800 was preserved in the presence of a 100 µm‐thick glass barrier separating it from the chemiluminescent reaction components. The maximum tissue phantom penetration by QDOT800 emission was 8.5 mm; in comparison, the native chemiluminescent emission at 500 nm was unable to penetrate the thinnest tissue phantom of 2.5 mm. The described method for red‐shifted emissions from chemiluminescent reactions does not require direct interaction between the chemiluminescent reaction and the fluorescent emitters. This suggests that the mechanism of chemiluminescent excitation of fluorophores and QDOT800 is not exclusive to chemiluminescence resonance energy transfer or sensitized chemiluminescence, but rather by broad energization from the native chemiluminescent emission. Copyright © 2014 John Wiley & Sons, Ltd.     

Two‐channel near‐infrared fluorescence Ag+ ion sensing of a new star‐shaped dendrimer

A new near‐infrared fluorescence sensor PDI‐PD for Ag⁺ ions was successfully prepared and its structure characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high‐resolution mass spectrometry; matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (HRMS MALDI‐TOF). The probe exhibited rapid, sensitive, and selective two‐channel fluorescence responses towards Ag⁺ ions and protons. The probe has a marked high binding affinity and high sensitivity for Ag⁺, with a detection limit of 1.4 × 10^?6 M. An approximately five‐fold enhanced core emission at 784 nm was attributed to fluorescence resonance energy transfer (FRET). The enhanced core emission of the probe with Ag⁺ ions based on photo‐induced electron transfer and FRET is discussed. In addition, the probe presented a visible colour change. All experimental results demonstrated that PDI‐PD is an efficient tool for the selective, sensitive and rapid detection of Ag⁺ ions and protons using two‐channel fluorescence responses.     

Dual‐function fluorescent probe for cancer imaging and therapy

To date, several fluorescent probes modified by a single targeting agent have been explored. However, studies on the preparation of dual‐function quantum dot (QD) fluorescent probes with dual‐targeting action and a therapeutic effect are rare. Here, a dual‐targeting CdTe/CdS QD fluorescent probe with a bovine serum albumin–glycyrrhetinic acid conjugate and arginine‐glycine‐aspartic acid was successfully prepared that could induce the apoptosis of liver cancer cells and showed enhanced targeting in in vitro cell imaging. Therefore, the as‐prepared fluorescent probe in this work is an efficient diagnostic tool for the simultaneous detection of liver cancer and breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Gold nanoclusters as novel optical probes for in vitro and in vivo fluorescence imaging

Fluorescent probes play an important role in the development of fluorescence-based imaging techniques for life sciences research. Gold nanoclusters (AuNCs) are a novel type of fluorescent nanomaterials which have attracted great interest in recent years. Composed of only a few atoms, these ultrasmall AuNCs exhibit quantum confinement effects and molecule-like properties. Fluorescent AuNCs have an attractive set of features including ultrasmall size, good biocompatibility and photostability, and tunable emission in the red to near-infrared spectral region, which make them promising as fluorescent labels for biological imaging. Examples of their application include live cell labeling, cancer cell targeting, cellular apoptosis monitoring, and in vivo tumor imaging. Here, we present a brief overview of recent advances in utilizing these emissive ultrasmall AuNCs as optical probes for in vitro and in vivo fluorescence imaging.     

有机荧光探针辅助的近红外荧光成像技术在宫颈癌中的应用

荧光成像已被广泛应用于生物医学和临床诊断领域.近红外(Near-infrared,NIR,700-1700 nm)荧光成像在NIR波段对生物组织显影,与可见光波段(400-760 nm)的传统荧光成像相比,更有助于提高成像的信噪比和灵敏度.高质量的荧光成像需要借助良好的荧光探针,纳米技术的快速发展使具备良好荧光特性的有...     

Transmission in near‐infrared optical windows for deep brain imaging

Near‐infrared (NIR) radiation has been employed using one‐ and two‐photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR‐low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.

     

A novel analytical method for in vivo phosphate tracking

Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (P(i)) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows P(i)-dependent increases in FRET efficiency. FLIPPi affinity mutants cover P(i) changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na(+)/P(i) co-transporter exhibited FRET changes when perfused with 100 microM P(i), demonstrating concentrative P(i) uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of P(i) metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P(i) during cell migration.