Brightness of fluorescent organic nanomaterials.

Brightness is a fundamental property of fluorescent nanomaterials reflecting their capacity to absorb and emit light. In sensing materials, brightness is crucial for high-sensitivity (bio)molecular detection, while in optical bioimaging it ensures high spatial and temporal resolution. Fluorescent organic nanoparticles (NPs) are particularly attractive because of their superior brightness compared to organic dyes. With the ever-growing diversity of organic nanomaterials, it is important to establish universal principles for measuring and estimating their brightness. This tutorial review provides definitions of brightness and describes the major approaches to its analysis based on ensemble and single-particle techniques. We present the current chemical approaches to fight Aggregation-Caused Quenching (ACQ) of fluorophores, which is a major challenge in the design of bright organic nanomaterials. The main classes of fluorescent organic NPs are described, including conjugated polymer NPs, aggregation-induced emission NPs, and NPs based on neutral and ionic dyes. Their brightness and other properties are systematically compared. Some brightest examples of bulk solid-state emissive organic materials are also mentioned. Finally, we analyse the importance of brightness and other particle properties in biological applications, such as bioimaging and biosensing. This tutorial will provide guidelines for chemists on the design of fluorescent organic NPs with improved performance and help them to estimate and compare the brightness of new nanomaterials with literature reports. Moreover, it will help biologists to select appropriate materials for sensing and imaging applications.

Simultaneous multipurpose fluorescence imaging with IRDye® 800BK during laparoscopic surgery.

BACKGROUND: IRDye® 800BK is a fluorophore, currently undergoing clinical translation, which has both biliary and renal clearance. To date, there is no description of a fluorophore, which can be simultaneously used for non-invasive, near-infrared fluorescence-based (NIRF) visualization of different structures and perfusion evaluation. The purpose of this study was to evaluate IRDye® 800BK for the simultaneous assessment of bowel perfusion, lymphography, ureter and bile duct delineation. METHODS: Six pigs received a 0.15 mg/kg dye as a single bolus intravenous injection (IV). With the FLER (fluorescence-based enhanced reality) software, fluorescence intensity (FI) of 5 regions of interest (ROI) in an ischemic bowel loop was measured along with the time to reach the FI peak, and capillary lactate was measured from the same ROI, followed by the assessment of the ureters and bile ducts for a maximal duration of 180 min after dye administration. In 3 animals, the procedure was initiated via gastroscopic injection of a 0.6 mg (1 mg/mL) dye in the gastric submucosa followed by lymphography in a NIRF setting. RESULTS: Excellent visualization of the ureters and bowel perfusion was obtained under NIRF imaging. Additionally, the bile duct and gastric lymph ducts and nodes were visualized. A positive correlation was found between the time to peak FI in the ischemic bowel loop and the corresponding capillary lactate levels (rho 0.59, p < 0.001). CONCLUSION: In this study, we successfully demonstrated the simultaneous multipurpose IRDye® 800BK applicability during laparoscopic surgery. This fluorophore has the potential to become a powerful and versatile image-guided surgery tool.

Preoperative endoscopic marking of the gastrointestinal tract using fluorescence imaging: submucosal indocyanine green tattooing versus a novel fluorescent over-the-scope clip in a survival experimental study.

BACKGROUND: Intraoperative localization of endoluminal lesions is can be difficult during laparoscopy. Preoperative endoscopic marking is therefore necessary. Current methods include submucosal tattooing using visible dyes, which in case of transmural injection can impair surgical dissection. Tattooing using indocyanine green (ICG) coupled to intraoperative near-infrared (NIR) laparoscopy has been described. ICG is only visible under NIR-light, therefore, it doesn't impair the surgical workflow under white light even if there is spillage. However, ICG tattoos have rapid diffusion and short longevity. We propose fluorescent over-the-scope clips (FOSC), using a novel biocompatible fluorescent paint, as durable lesion marking. METHODS: In six pigs, gastric and colonic endoscopic tattoos using 0.05 mg/mL of ICG and markings using the fluorescent OSC were performed (T0). Simultaneously, NIR laparoscopy was executed. Follow-up laparoscopies were conducted at postoperative day (POD) 4-6 (T1) and POD 11-12 (T2). During laparoscopy, fluorescence intensity was assessed. In one human cadaver, FOSC was used to mark a site on the stomach and on the sigmoid colon, respectively. Intraoperative detection during NIR laparoscopy was assessed. RESULTS: Gastric and colonic ICG tattooing and OSC markings were easily visible using NIR laparoscopy on T0. All FOSC were visible at T1 and T2 in both stomach and colon, whereas the ICG tattooing at T1 was only visible in the stomach of 2 animals and in the colon of 3 animals. At T2, tattoos were not visible in any animal. FOSC were still visible in both stomach and colon of the human cadaver at 10 days. CONCLUSION: Endoscopic marking using FOSC can be an efficient and durable alternative to standard methods.

Hybrid fluorescent magnetic gastrojejunostomy: an experimental feasibility study in the porcine model and human cadaver.

BACKGROUND: Laparoscopic gastrojejunostomies are time consuming and require a specific training. Alternatively, sutureless anastomosis can be achieved using endoscopically delivered magnetic rings. Our aim was to assess the feasibility and reproducibility of an endo-laparoscopic gastrojejunostomy technique, using a pair of magnets coated with a near-infrared fluorescent biocompatible polymeric material. METHODS: Five pigs (3 acute and 2 survival models) and one human anatomical specimen were included. In the survival models, the distal ring was inserted into the duodenum endoscopically, and it was fixed to a thread clipped to the gastric mucosa. Twenty-four hours later, a two-port laparoscopy was performed using a near-infrared (NIR) laparoscope. The magnet position in the jejunum was detected with the transluminal fluorescence of the dye. Magnetic interaction with the metallic tip of the laparoscopic grasper allowed to capture the ring and bring the bowel loop to the future anastomotic site on the gastric wall. The proximal magnet was inserted into the stomach endoscopically and released when magnetic interaction started, allowing for a precise connection with the distal ring. The animals were followed up for 12 days and underwent control endoscopies and radiograms. In the acute animals, the anastomotic procedure was repeated 24 times. Finally, the procedure was performed in the human anatomical specimen. RESULTS: There were no technical problems, and magnetic connection could be precisely directed at both the anterior and posterior gastric walls. No complications occurred during the survival period and the anastomoses were patent on day 5. Transluminal fluorescence enabled a rapid detection of the magnet. CONCLUSIONS: Hybrid-reduced port magnetic gastrojejunostomy using a pair of fluorescently coated magnetic rings was feasible, reproducible, and easy to perform in both porcine and cadaver models.

A Super-Resolution Probe To Monitor HNO Levels in the Endoplasmic Reticulum of Cells.

Selective detection of nitroxyl (HNO), which has recently been identified as a reactive nitrogen species, is a challenging task. We report a BODIPY-based luminescence ON reagent for detection of HNO in aqueous solution and in live RAW 264.7 cells, based on the soft nucleophilicity of the phosphine oxide functionality toward HNO. The probe shows high selectivity to HNO over other reactive oxygen/nitrogen and sulfur species. Luminescence properties of the BODIPY-based chemodosimetric reagent make it an ideal candidate for use as a reagent for super-resolution structured illumination microscopy. The viability of the reagent for biological in vivo imaging application was also confirmed using Artemia as a model.

Ultrabright Fluorescent Polymeric Nanofibers and Coatings Based on Ionic Dye Insulation with Bulky Counterions.

Preparation of bright fluorescent materials based on polymers is hampered by a fundamental problem of aggregation-caused quenching (ACQ) of encapsulated dyes. Here, ultrabright fluorescent polymeric nanofibers and coatings are prepared based on a concept of ionic dye insulation with bulky hydrophobic counterions that overcomes the ACQ problem. It is found that bulky hydrophobic counterion perfluorinated tetraphenylborate can boost >100-fold the fluorescence quantum yields of cationic dye octadecyl rhodamine B at high loading (30 wt %) in biocompatible poly(methyl methacrylate) (PMMA). The concept is applicable to both rhodamine and cyanine dyes, which results in bright fluorescent polymeric materials of four different colors spanning from blue to near-infrared. It allows for preparation of electrospun polymeric nanofibers with >50-fold higher dye loading by mass (30 wt %, >20-fold higher molarity for rhodamine dyes) while preserving good fluorescence quantum yields (31%), which implies drastic improvement in their fluorescence brightness. The counterion-based polymeric materials are also validated as coatings of model medical devices, such as stainless steel fiducials and 3D-printed stents of complex geometry. Spin-coated fluorescent polymeric films loaded with a dye paired with bulky counterions exhibit excellent biocompatibility and low toxicity. Moreover, counterion-modified materials show much better stability against dye leakage in the presence of living cells and a serum-containing medium, compared to materials based on the dye with a small inorganic anion. Overall, by pushing the barriers of ACQ, our counterion approach emerges as a powerful tool to develop ultrabright fluorescent polymeric materials ranging from nano- and macroscale.

Intraoperative ureter identification with a novel fluorescent catheter.

Iatrogenic ureteral injuries (IUI) occur in 0.5-1.3% of cases during abdominal surgery. If not recognized intraoperatively, IUI increase morbidity/mortality. A universally accepted method to prevent IUI is lacking. Near-infrared fluorescent imaging (NIRF), penetrating deeper than normal light within the tissue, might be useful, therefore ureter visualization combining NIRF with special dyes (i.e. IRDye 800BK) is promising. Aim of this work is to evaluate the detection of ureters using stents coated with a novel biocompatible fluorescent material (NICE: near-infrared coating of equipment), during laparoscopy. female pigs underwent placement of NICE-coated stents (NS). NIRF was performed, and fluorescence intensity (FI) was computed. Successively, 0.15 mg/kg of IRDye 800BK was administered intravenously, and FI was computed at different timepoints. Ureter visualization using NS only was further assessed in a human cadaver. Both methods allowed in vivo ureter visualization, with equal FI. However, NS were constantly visible whereas IRDye 800BK allowed visualization exclusively during the ureteral peristaltic phases. In the human cadaver, NS provided excellent ureter visualization in its natural anatomical position. NS provided continuous ureteral visualization with similar FI as the IRDye 800BK, which exclusively allowed intermittent visualization, dependent on ureteral peristalsis. NS might prove useful to visualize ureters intraoperatively, potentially preventing IUI.

Recent Advances in Fluorescent Probes for Detection of HOCl and HNO.

It is known that reactive oxygen (ROS) and nitrogen (RNS) species play a diverse role in various biological processes, such as inflammation, signal transduction, and neurodegenerative injury, apart from causing various diseases caused by oxidative and nitrosative stresses, respectively, by ROS and RNS. Thus, it is very important to quantify the concentration level of ROS and RNS in live cells, tissues, and organisms. Various small-molecule-based fluorescent/chemodosimetric probes are reported to quantify and map the effective distribution of ROS/RNS under in vitro/in vivo conditions with a great spatial and temporal resolution. Such reagents are now appreciated as an excellent tool for aiding breakthroughs in modern redox biology. This mini-review is a brief, but all-inclusive, account of such molecular probes that have been developed recently.

Near-infrared fluorescent coatings of medical devices for image-guided surgery.

Rapidly expanding field of image-guided surgery needs new materials for near-infrared imaging with deep tissue penetration. Here, we introduce near-infrared coating of equipment (NICE) for image-guided surgery based on a series of lipophilic cyanine-7.5 dyes with bulky hydrophobic counterions and a biocompatible polymer, poly(methyl methacrylate). The NICE material exhibits superior brightness (15-20-fold higher) and photostability compared to fluorescent coatings based on commonly used indocyanine green (ICG). It can be deposited on different surfaces and devices, such as steel and gold fiducials, silicone and PVC catheters, polymeric surgical sutures and gauzes. Such coated medical devices show excellent stability in air and buffer for ≥150 days. Accelerated ageing revealed their shelf-life of ≥3 years. They are also stable in serum-containing media, whereas ICG-based coating shows rapid dye leakage. NICE is compatible with standard sterilization protocols based on ethylene oxide and vapor. Moreover, our coating material is biocompatible, where cultured cells spread effectively without signs of cytotoxicity. Ex vivo studies suggest that NICE on fiducials can be visualized as deep as 0.5 cm, and NICE on catheters enables their visualization inside ureters and esophagus. Finally, NICE on different medical devices has been validated for image-guided surgery in porcine and human cadaver models. Thus, the developed NIR coating material emerges as a powerful tool for a variety of medical applications.

Solvatochromic Near-Infrared Probe for Polarity Mapping of Biomembranes and Lipid Droplets in Cells under Stress.

Can polarity-sensitive fluorescent dyes monitor the response of live cells to fundamental stress conditions, such as deprivation from nutrition and oxidative stress? To address this question, we developed a push-pull dioxaborine probe (DXB-NIR) for biomembranes and lipid droplets featuring strong solvatochromism in the far-red to near-infrared region, high fluorescence brightness, photostability, and two-photon absorption cross section, reaching 13800 GM at 930 nm. In model membranes, DXB-NIR exhibits unprecedented 80 nm shift between liquid ordered and disordered membrane phases, allowing robust imaging of separated membrane microdomains. Two-color imaging of live cells with DXB-NIR enables polarity mapping in plasma membranes, endoplasmic reticulum, and lipid droplets, which reveals that starvation and oxidative stress produce an increase in the local polarity, and this change is different for each of the studied cell compartments. Thus, by pushing the limits of existing solvatochromic dyes, we introduce a concept of polarity mapping for monitoring the response of cells to stress.

A fluorogenic BODIPY molecular rotor as an apoptosis marker.

Based on a BODIPY molecular rotor and a zinc-dipicolylamine receptor, we designed a fluorogenic probe for the detection of apoptosis. Being poorly emissive in solution and with healthy cells, it selectively binds phosphatidylserine of early apoptotic cells and internalizes into late apoptotic cells, lighting up its green fluorescence.