Optical Contrast Agents Research Articles

Probing polyvinylpyrrolidone-passivated graphene oxide nanoflakes as contrast agents inside tissue-like phantoms via multimodal confocal microscopy

Beside attractive electrical, thermal and mechanical properties, graphene oxide (GO) exhibits visible and near-infrared (NIR) photoluminescence (PL) and well-defined fingerprint Raman bands which are remarkable optical signatures to implement GO as new contrast agent for the visualization of cells or tissue, including cancer tumors. However, the biomedical use of GO as optical contrast agent is to some extent hindered by the intrinsic low emission efficiency especially at neutral pH. Herein, we successfully modulate the PL of GO nanoflakes in acidic and neutral medium by passivating them with polyvinylpyrrolidone (PVP), an amphiphilic and biocompatible polymer, thus improving the PL at pH relevant for biomedical applications. We demonstrate the potential of as-fabricated PVP-GO nanocomposites to operate as dual Raman-PL contrast agents inside tissue-like agarose-phantoms via scanning confocal Raman microscopy (CRM) under excitation at 532 nm. Super-resolution re-scan confocal microscopy (RCM) was further employed to investigate the distribution of PVP-GO inside biological phantoms at 3D level under three excitation lines (405, 488, and 561 nm). Finally, two-photon excited fluorescence lifetime imaging microscopy (TPE-FLIM) at 810 nm excitation reveals the ability of PVP-GO to serve as NIR-activatable contrast agent inside tissue-like phantom. Notably, PVP coating empowers GO nanoflakes not only with enhanced optical signature, but also with excellent dispersibility inside biological phantoms, thus offering improved labeling performance of as-designed imaging contrast agent.

Bacteria eat nanoprobes for aggregation-enhanced imaging and killing diverse microorganisms

Currently optical-based techniques for in vivo microbial population imaging are limited by low imaging depth and highly light-scattering tissue; and moreover, are generally effective against only one specific group of bacteria. Here, we introduce an imaging and therapy strategy, in which different bacteria actively eat the glucose polymer (GP)-modified gold nanoparticles through ATP-binding cassette (ABC) transporter pathway, followed by laser irradiation-mediated aggregation in the bacterial cells. As a result, the aggregates display ~15.2-fold enhancement in photoacoustic signals and ~3.0-fold enhancement in antibacterial rate compared with non-aggregated counterparts. Significantly, the developed strategy allows ultrasensitive imaging of bacteria in vivo as low ~105 colony-forming unit (CFU), which is around two orders of magnitude lower than most optical contrast agents. We further demonstrate the developed strategy enables the detection of ~107 CFU bacteria residing within tumour or gut. This technique enables visualization and treatment of diverse bacteria, setting the crucial step forward the study of microbial ecosystem.

Single-step acid-catalyzed synthesis of luminescent colloidal organosilica nanobeads

We present a single-step, room-temperature synthesis of fluorescent organosilica nanobeads (FOS NBs). The FOS NBs were synthesized under aqueous conditions using (3-aminopropyl)triethoxysilane (APTES) as the silicon source in the presence of l-ascorbic acid (L-AA). In the APTES/L-AA/water ternary phase, the hydrolysis and condensation reaction of APTES occurred under acidic conditions to form spherical FOS NBs with an average diameter of 426.8 nm. FOS NBs exhibit excellent colloidal stability in aqueous media. The formation of FOS NBs was complete within a 10 min reaction time, which indicates potential for large-scale mass-production synthesis of luminescent colloidal NBs. The FOS NBs exhibited blue photoluminescence (PL) under UV excitation in the absence of an additional high temperature calcination process or with the incorporation of any fluorophores. This phenomenon is attributed to the presence of carbon-containing defects, which act as luminescent centers formed by the reaction between amino groups in the APTES and l-ascorbic acid reductant. Finally, the results of a cytotoxicity test and cellular uptake experiments revealed that the FOS NBs showed potential as optical contrast agents for bioimaging.Graphical

A Prostate-Specific Membrane Antigen-Targeted Near-Infrared Conjugate for Identifying Pulmonary Squamous Cell Carcinoma during Resection.

Pulmonary squamous cell carcinoma is the second most common lung cancer subtype and has a low 5-year survival rate at 17.6%. Complete resection with negative margins can be curative, but a high number of patients suffer early postoperative recurrence due to inadequate disease clearance at the index operation. Intraoperative molecular imaging (IMI) with tumor-targeted optical contrast agents is effective in improving resection completeness for other tumor types, but there are no IMI tracers targeted to pulmonary squamous cell carcinoma. In this report, we describe the use of a novel prostate-specific membrane antigen (PSMA)-targeted near-infrared conjugate (OTL78) to identify pulmonary squamous cell carcinoma. We identified PSMA as a viable target by examining its expression in human lung tumor specimens from a surgical cohort. Ninety-four percent of tumors expressed PSMA in either the pulmonary squamous cells or the tumor neovasculature. Using in vitro and in vivo models, we found that OTL78 reliably localized pulmonary squamous cell carcinoma in a PSMA-dependent manner. Finally, we found that IMI with OTL78 markedly improved surgeons' ability to identify residual disease after surgery in a preclinical model. Ultimately, this novel optical tracer may aid surgical resection of pulmonary squamous cell carcinoma and potentially improve long-term outcomes.

Progress and Perspective of Solid-State Organic Fluorophores for Biomedical Applications.

Fluorescent organic dyes have been extensively used as raw materials for the development of versatile imaging tools in the field of biomedicine. Particularly, the development of solid-state organic fluorophores (SSOFs) in the past 20 years has exhibited an upward trend. In recent years, studies on SSOFs have focused on the development of advanced tools, such as optical contrast agents and phototherapy agents, for biomedical applications. However, the practical application of these tools has been hindered owing to several limitations. Thus, in this Perspective, we have provided insights that could aid researchers to further develop these tools and overcome the limitations such as limited aqueous dispersibility, low biocompatibility, and uncontrolled emission. First, we described the inherent photophysical properties and fluorescence mechanisms of conventional, aggregation-induced emissive, and precipitating SSOFs with respect to their biomedical applications. Subsequently, we highlighted the recent development of functionalized SSOFs for bioimaging, biosensing, and theranostics. Finally, we elucidated the potential prospects and limitations of current SSOF-based tools associated with biomedical applications.

In vivo multimodal imaging of hyaluronan-mediated inflammatory response in articular cartilage

One driving factor in the progression to posttraumatic osteoarthritis (PTOA) is the perpetuation of the inflammatory response to injury into chronic inflammation. Molecular imaging offers many opportunities to complement the sensitivity of current imaging modalities with molecular specificity. The goal of this study was to develop and characterize agents to image hyaluronan (HA)-mediated inflammatory signaling. We developed optical (Cy5.5-P15-1) and magnetic resonance contrast agents (Gd-DOTA-P15-1) based in a hyaluronan-binding peptide (P15-1) that has shown anti-inflammatory effects on human chondrocytes, and validated them in vitro and in vivo in two animal models of PTOA. In vitro studies with a near infrared (NIR) Cy5.5-P15-1 imaging agent showed a fast and stable localization of Cy5.5-P15-1 on chondrocytes, but not in synovial cells. In vivo NIR showed significantly higher retention of imaging agent in PTOA knees between 12 and 72h (n=8, Cohen's d>2 after 24h). NIR fluorescence accumulation correlated with histologic severity in cartilage and meniscus (ρ between 0.37 and 0.57, P<0.001). By using in vivo magnetic resonance imaging with a Gd-DOTA-P15-1 contrast agent in 12 rats, we detected a significant decrease of T1 on injured knees in all cartilage plates at 48h (-15%, 95%-confidence interval (CI)=[-18%,-11%]) while no change was observed in the controls (-2%, 95%-CI=[-5%,+1%]). This study provides the first in vivo evidence that hyaluronan-related inflammatory response in cartilage after injury is a common finding. Beyond P15-1, we have demonstrated that molecular imaging can provide a versatile technology to investigate and phenotype PTOA pathogenesis, as well as study therapeutic interventions.

Nanotechnology in Tumor Biomarker Detection: The Potential of Liganded Nanoclusters as Nonlinear Optical Contrast Agents for Molecular Diagnostics of Cancer.

Simple SummaryThe key factor in preventing premature death from cancer is an early and accurate diagnosis. While common diagnostic procedures are successful in the detection and rough description of a tumor mass, a deeper insight into cancer’s molecular features is needed to optimize the treatment and increase the chances of survival. Nanotechnology can aid the molecular diagnostics of cancers through a design of nanomaterials that can simultaneously recognize specific cancer-associated molecules, so-called tumor biomarkers, and allow for their visualization by different imaging techniques. With a recent explosion in the production of various nanomaterials, the selection of the most suitable nanomaterial for tumor biomarker detection becomes a challenge. In this article, we review recent advances in the molecular diagnostics of cancer using nanotechnology and focus on liganded noble metal quantum nanoclusters, a class of ultrasmall nanomaterials with distinctive structural and optical properties, as tools in tumor biomarker detection.Cancer is one of the leading causes of premature death, and, as such, it can be prevented by developing strategies for early and accurate diagnosis. Cancer diagnostics has evolved from the macroscopic detection of malignant tissues to the fine analysis of tumor biomarkers using personalized medicine approaches. Recently, various nanomaterials have been introduced into the molecular diagnostics of cancer. This has resulted in a number of tumor biomarkers that have been detected in vitro and in vivo using nanodevices and corresponding imaging techniques. Atomically precise ligand-protected noble metal quantum nanoclusters represent an interesting class of nanomaterials with a great potential for the detection of tumor biomarkers. They are characterized by high biocompatibility, low toxicity, and suitability for controlled functionalization with moieties specifically recognizing tumor biomarkers. Their non-linear optical properties are of particular importance as they enable the visualization of nanocluster-labeled tumor biomarkers using non-linear optical techniques such as two-photon-excited fluorescence and second harmonic generation. This article reviews liganded nanoclusters among the different nanomaterials used for molecular cancer diagnosis and the relevance of this new class of nanomaterials as non-linear optical probe and contrast agents.

Evaluation of OTL38-Generated Tumor-to-Background Ratio in Intraoperative Molecular Imaging-Guided Lung Cancer Resections.

Cancer surgery has multiple challenges including localizing small lesions, ensuring negative margins, and identifying synchronous cancers. One of the tools proposed to address these issues is intraoperative molecular imaging (IMI). An important consideration in IMI is the quantification of the tumor fluorescence during the procedure and using that data to add clinical value. Currently, the most commonly cited measure of quantification is the tumor-to-background ratio (TBR). Our goal was to evaluate the clinical value of TBR measured with OTL38 NIR tracer during a lung cancer resection. Intraoperative data was retrospectively reviewed from a prospectively collected 5-year database. Between 2015 and 2020, 279 patients were included in the study. For standardization, all patients underwent infusion of the same targeted molecular optical contrast agent (OTL38) for lung cancer resections; then, the mean fluorescence intensity of the tumors and background tissues were calculated. To evaluate the clinical efficacy of the TBR calculation, the results were correlated with patient, biologic, tumor, and technological factors. For pulmonary surgery, patient factors such as gender, age, smoking history, and time from infusion of OTL38 to surgery did not have any statistical significance in predicting the TBR during surgery. In addition, TBR measurements did not correlate with location of the tumor in the lung (p= 0.123). There was no statistical correlation of preoperative positron emission tomography measurements (standardized uptake value) with intraoperative TBR. However, there was statistically significant negative correlation of in situ TBR measurement and the distance of the lesion from the surface of the organ (p< 0.001). Adenocarcinoma spectrum lesions overall had statistically significant correlation with in situ fluorescence compared to other NSCLC malignancies (p< 0.01) but TBR measurements could not identify histopathologic subtype on univariate analysis (p= 0.089). There was a tendency for in situ fluorescence for moderately and well-differentiated adenocarcinoma spectrum lesions, but this was not statistically significant. When comparing the in situ TBR of benign to malignant nodules in the lung, there was no statistically significant association (p= 0.145). In subset analysis, adenocarcinoma spectrum lesions tend to fluoresce at brighter with OTL38 compared to other histologic subtypes. In our various iterations, the results of our retrospective analysis did not show that TBR measurements during OTL38-guided surgery provide clinically useful information about the nature of the nodule or cancer. The true value of IMI is in the ability for the surgeon to use the fluorescence to guide the surgeon to the tumor and margins, but that sophisticated quantification of the amount of fluorescence may not have clinical utility.

Flash Scanning Volumetric Optoacoustic Tomography for High Resolution Whole‐Body Tracking of Nanoagent Kinetics and Biodistribution

AbstractTracking of biodynamics across entire living organisms is essential for understanding complex biology and disease progression. The presently available small‐animal functional and molecular imaging modalities remain constrained by factors including long image acquisition times, low spatial resolution, limited penetration or poor contrast. Here flash scanning volumetric optoacoustic tomography (fSVOT), a new approach for high‐speed imaging of fast kinetics and biodistribution of optical contrast agents in whole mice that simultaneously provides reference images of vascular and organ anatomy with unrivaled fidelity and contrast, is presented. The imaging protocol employs continuous overfly scanning of a spherical matrix array transducer, accomplishing a 200 µm resolution 3D scan of the whole mouse body within 45 s without relying on signal averaging. This corresponds to an imaging speed gain of more than an order of magnitude compared with existing state‐of‐the‐art implementations of comparable resolution performance. Volumetric tracking and quantification of gold nanoagent and near infrared (NIR)‐II dye kinetics and their differential uptake in various organs are demonstrated. fSVOT thus offers unprecedented capabilities for multiscale imaging of pharmacokinetics and biodistribution with high contrast, resolution, and speed.

A colorful approach towards developing new nano-based imaging contrast agents for improved cancer detection.

Providing physicians with new imaging agents to help detect cancer with better sensitivity and specificity has the potential to significantly improve patient outcomes. Development of new imaging agents could offer improved early cancer detection during routine screening or help surgeons identify tumor margins for surgical resection. In this study, we evaluate the optical properties of a colorful class of dyes and pigments that humans routinely encounter. The pigments are often used in tattoo inks and the dyes are FDA approved for the coloring of foods, drugs, and cosmetics. We characterized their absorption, fluorescence and Raman scattering properties in the hopes of identifying a new panel of dyes that offer exceptional imaging contrast. We found that some of these coloring agents, coined as "optical inks", exhibit a multitude of useful optical properties, outperforming some of the clinically approved imaging dyes on the market. The best performing optical inks (Green 8 and Orange 16) were further incorporated into liposomal nanoparticles to assess their tumor targeting and optical imaging potential. Mouse xenograft models of colorectal, cervical and lymphoma tumors were used to evaluate the newly developed nano-based imaging contrast agents. After intravenous injection, fluorescence imaging revealed significant localization of the new "optical ink" liposomal nanoparticles in all three tumor models as opposed to their neighboring healthy tissues (p < 0.05). If further developed, these coloring agents could play important roles in the clinical setting. A more sensitive imaging contrast agent could enable earlier cancer detection or help guide surgical resection of tumors, both of which have been shown to significantly improve patient survival.

Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time.

High resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to significant scattering and autofluorescence in tissue at visible (VIS, 350–700 nm) and near-infrared (NIR, 700–1000 nm) wavelengths. Here, we enable real-time, non-invasive multicolor imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1000–2000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established structure-photophysical property relationships for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable NIR/SWIR excitation and single-channel detection, facilitating video-rate multicolor SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-color imaging with high temporal and spatial resolutions.

AND-gate contrast agents for enhanced fluorescence-guided surgery

The surgical resection of tumours requires the precise location and definition of the margins between lesions and normal tissue. However, this is made difficult by irregular margin borders. Although molecularly targeted optical contrast agents can be used to define tumour margins during surgery in real time, the selectivity of the contrast agents is often limited by the target being expressed in both healthy and tumour tissues. Here, we show that AND-gate optical imaging probes requiring the processing of two substrates by multiple tumour-specific enzymes produce a fluorescent signal with significantly improved specificity and sensitivity to tumour tissue. We evaluated the performance of the probes in mouse models of mammary tumours and of metastatic lung cancer, and during fluorescence-guided robotic surgery. Imaging probes relying on multivariate activation to selectively target complex patterns of enzymatic activity should be useful in disease detection, treatment and monitoring.

Parallelized Monte Carlo Photon Transport Simulations for Arbitrary Multi-Angle Wide-Field Illumination in Optoacoustic Imaging

Optoacoustic imaging (OAI) or photoacoustic imaging can resolve the distribution of tissue chromophores and optical contrast agents deep inside tissue from the optoacoustic detection with multi-spectral illumination. The development of a fast and accurate modeling method for the photon transport in OAI is necessary to quantitatively evaluate the tissue optical parameters. This paper presents a parallelized Monte Carlo modeling method especially for OAI (MCOAI) to simulate photon transport in bio-tissues with arbitrary multi-angle wide-field illumination. The performance of the MCOAI method is verified by comparison with the graphics processing unit (GPU)-accelerated MCX method in the typical cases with the pencil beam and ring source illumination. The simulation results demonstrate the GPU-based MCOAI method has equivalent accuracy and significantly improved computation efficiency, compared to the MCX method. The simulations with cylindrical and hemispherical source illumination further illustrate that the MCOAI method can effectively implement three-dimensional photon transport simulation for typical illumination geometries of OAI systems. A cross-section of Digimouse is selected as a realistic heterogeneous phantom illuminated with six different light sources usually employed in OAI, in order to prove the necessity of establishing photon transport modeling in OAI for the quantitative visualization in deep tissues. The MCOAI method can provide a powerful tool to efficiently establish photon transport modeling with arbitrary illumination modes for OAI applications.

Intraoperative near-infrared imaging can identify canine mammary tumors, a spontaneously occurring, large animal model of human breast cancer.

IntroductionCurrent methods of intraoperative margin assessment in breast conserving surgery are impractical, unreliable, or time consuming. We hypothesized that intraoperative near-infrared (NIR) imaging with an FDA-approved NIR optical contrast agent could identify canine mammary tumors, a spontaneous large animal model of human breast cancer, during surgery.MethodsDogs with mammary tumors underwent a standard of care lumpectomy or mastectomy with wide surgical margins 20 hours after indocyanine green administration (3 mg/kg IV). During surgery, NIR imaging was performed on tumors and wound margins in situ and tumors and lymph nodes ex vivo. Following resection, the wound bed was examined for residual fluorescence. Fluorescence intensity was determined by signal-to-background ratio (SBR). All tumors, areas of residual fluorescence, and lymph nodes underwent histopathologic analysis.ResultsThere were 41 mammary tumors in 16 female dogs. Twenty tumors were malignant and 21 were benign. Twenty-eight tumors were fluorescent (mean SBR 1.5±0.2). Sensitivity of fluorescence for all malignant tumors was 80% (16/20) and 93.3% (14/15) for malignant tumors > 2 cm. Specificity for malignancy was low (< 2cm = 55%; > 2cm = 30%). Tumors > 2 cm were more likely to be fluorescent (OR 6.05, 95% CI 1.50–24.44, P = 0.011) but not more likely to be malignant (OR 3.09, 95% CI 0.86–11.14, P = 0.085) than tumors ≤ 2 cm. Four out of seven inguinal lymph nodes excised in the mastectomy specimen fluoresced. All four drained malignant tumors; however only 2/4 contained metastatic disease.ConclusionSystemic ICG accumulates reliably in malignant canine mammary tumors > 2 cm. Although no tumor margins fluoresced, a wider margin of normal tissue is removed in canine mastectomy, making direct comparisons with breast conserving surgery difficult. Targeted NIR imaging agents are likely required to improve detection of smaller tumors and improve the specificity of NIR imaging for residual disease and metastatic lymph node detection.

Image-guided tumor surgery: The emerging role of nanotechnology.

Surgical resection is a mainstay treatment for solid tumors. Yet, methods to distinguish malignant from healthy tissue are primarily limited to tactile and visual cues as well as the surgeon's experience. As a result, there is a possibility that a positive surgical margin (PSM) or the presence of residual tumor left behind after resection may occur. It is well-documented that PSMs can negatively impact treatment outcomes and survival, as well as pose an economic burden. Therefore, surgical tumor imaging techniques have emerged as a promising method to decrease PSM rates. Nanoparticles (NPs) have unique characteristics to serve as optical contrast agents during image-guided surgery (IGS). Recently, there has been tremendous growth in the volume and types of NPs used for IGS, including clinical trials. Herein, we describe the most recent contributions of nanotechnology for surgical tumor identification. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Implantable Materials and Surgical Technologies > Nanoscale Tools and Techniques in Surgery Diagnostic Tools > in vivo Nanodiagnostics and Imaging.

A Spectral Demixing Model for Triplex In Vivo Imaging of Optical Coherence Tomography Contrast Agents

A Spectral Demixing Model for Triplex <i>In Vivo</i> Imaging of Optical Coherence Tomography Contrast Agents

Colonic Carcinoma Diagnosis using Chitosan Nanoparticles Based on the Optical Properties.

Medical diagnosis using optical techniques and contrast agents is a promising method where it is safe and unexpansive technique. Every tissue can be distinguished by its optical absorption and scattering properties that are related to many physiological changes and it is a sign for cancerous cells. Characterizing the light propagation in the human tissues is a vital issue in early cancer diagnosis for more effective therapeutic. In this work, the glowing effect of chitosan nanoparticles has been observed. Also, the light propagation in each of colon cancer (Caco-2 cell line) and normal cells (WI-38 cell line) at 650 nm and 808 nm in the absence and in the presence of chitosan nanoparticles was studied to study its effect in differentiate the cancer cells from the normal cells. Chitosan nanoparticles were characterized by the dynamic light scattering and transmission electronic Microscope (TEM). A Monte-Carlo simulation model was applied to obtain spatially resolved steady state diffuse reflectance measurements for each of the examined cells. Furthermore, the optical fluence rate distribution at the tissue surface were used to reconstruct the image using the diffuse equation using the finite element method. Chitosan nanoparticles appeared its glowing effect. The proposed diffuse reflectance curves and fluence rate images show different features regarding for each of Caco-2 cell line and WI-38 cell line that promises to be effective in medical diagnosis.

Methods for analysis of near-infrared (NIR) quenched-fluorescent contrast agents in mouse models of cancer.

Optical contrast agents containing near-infrared (NIR) fluorophores are useful for visualizing biological landmarks, enzyme activities and biological processes in live animals and humans. Activatable (smart) quenched-fluorescent probes are sensors that become fluorescent after processing by an enzyme or in response to a physiological change (i.e., pH, ROS, etc.). Recently, there has been increased interest in developing activatable probes for research and clinical applications. This requires evaluation using in vivo animal models to gain insights into the pharmacodynamic and pharmacokinetic properties of a given probe. Important parameters to measure when evaluating quenched-fluorescent probes are signal brightness and signal-to-background ratios, which define the sensitivity and specificity of a probe. In this chapter, we discuss methods to evaluate activatable quenched-fluorescent probes in mouse models of cancer. Quantification of fluorescent signal intensity, calculation of tumor-to-background ratios, comparison of fluorescent activation in specific organ compartments, and fluorescence scanning of sectioned tissue will be discussed.

Microfluidic chip enabled one-step synthesis of biofunctionalized CuInS2/ZnS quantum dots.

Biofunctionalized quantum dots (QDs) are effective target fluorescent labels for bioimaging. However, conventional synthesis of biofunctionalized I-III-VI core-shell CuInS2/ZnS QDs requires complex bench-top operations, resulting in limited product performance and variety, and is not amenable to a 'one-step' approach. In this work, we have successfully demonstrated a fully automated method for preparing denatured bovine serum albumin (dBSA)-CuInS2/ZnS QDs by introducing microfluidic (MF) chips to synthesize biofunctionalized QDs, hence establishing a 'one-step' procedure. We have also studied and optimized the reaction synthesis parameters. The emission wavelength of the dBSA-CuInS2/ZnS QDs is located in the near-infrared range and can be tuned from 650 to 750 nm by simply varying the reaction parameters. In addition, the 'one-step'-synthesized dBSA-CuInS2/ZnS QDs have a long average fluorescence lifetime of 153.76 ns and a small particle size of 5 ± 2 nm. To demonstrate the applicability of the 'one-step'-synthesized dBSA-CuInS2/ZnS QDs in bioimaging studies, we modified the QDs with folic acid and hyaluronic acid, and then performed target bioimaging and cytotoxicity tests on macrophages, liver cancer cells and pancreatic cancer cells. The cell images show that the red emission signals originate from the QDs, which indicates that the dBSA-CuInS2/ZnS QDs prepared by the MF approach are suitable optical contrast agents for target bioimaging. This 'one-step' MF-based QD synthesis approach could serve as a rapid, cost-effective, and small-scale nanocrystal production platform for complex QD formulations for a wide range of bioapplications.

Multimodal optical contrast agents as new tools for monitoring and tuning nanoemulsion internalisation into cancer cells. From live cell imaging to in vivo imaging of tumours.

Tailor-made NIR emitting dyes were designed as multimodal optical probes. These asymmetric amphiphilic compounds show combined intense absorption in the visible region, NIR fluorescence emission, high two-photon absorption in the NIR (with the maximum located around 1000 nm) as well as large Stokes' shift values and second-harmonic generation ability. Thanks to their structure, high loading into nanoemulsions (NEs) could be achieved leading to very high one- and two-photon brightness. These dyes were demonstrated to act as multimodal contrast agents able to generate different optical modalities of interest for bioimaging. Indeed, the uptake and carrier behaviour of the dye-loaded NEs into cancer cells could be monitored by simultaneous two-photon fluorescence and second-harmonic generation optical imaging. Multimodal imaging provided deep insight into the mechanism and kinetics of dye internalisation. Quite interestingly, the nature of the dyes was also found to influence both the kinetics of endocytosis and the internalisation pathways in glioblastoma cancer cells. By modulating the charge distribution within the dyes, the NEs can be tuned to escape lysosomes and enter the mitochondria. Moreover, surface functionalization with PEG macromolecules was realized to yield stealth NIRF-NEs which could be used for in vivo NIRF imaging of subcutaneous tumours in mice.