A Highly Bright Near-Infrared Afterglow Luminophore for Activatable Ultrasensitive In Vivo Imaging.

Afterglow luminescence imaging probes, with long-lived emission after cessation of light excitation, have drawn increasing attention in biomedical imaging field owing to their elimination of autofluorescence. However, current afterglow agents always suffer from an unsatisfactory signal intensity and complex systems consisting of multiple ingredients. To address these issues, this study reports a near-infrared (NIR) afterglow luminophore (TPP-DO) by chemical conjugation of an afterglow substrate and a photosensitizer acting as both an afterglow initiator and an energy relay unit into a single molecule, resulting in an intramolecular energy transfer process to improve the afterglow brightness. The constructed TPP-DO NPs emit a strong NIR afterglow luminescence with a signal intensity of up to 108  p/s/cm2 /sr at a low concentration of 10 µM and a low irradiation power density of 0.05 W/cm2 , which is almost two orders of magnitude higher than most existing organic afterglow probes. The highly bright NIR afterglow luminescence with minimized background from TPP-DO NPs allows a deep tissue penetration depth ability. Moreover, we develop a GSH-activatable afterglow probe (Q-TPP-DO NPs) for ultrasensitive detection of subcutaneous tumor with the smallest tumor volume of 0.048 mm3 , demonstrating the high potential for early diagnosis and imaging-guided surgical resection of tumors.

[A Standard Method for Performance Detection of Fluorescence Imaging System].

Fluorescence imaging now becomes an intraoperative navigation technique that gaining popularity in surgery and clinical research. However, at present, there is no mature and reliable method or other related guidance documents for the detection of fluorescence imaging performance. The performance analysis and quality supervision of products on the market could not be performed, which affects their clinical use and image quality. In this paper, a standard method of fluorescence imaging performance testing for fluorescence imaging system is proposed. Several kinds of fluorescence imaging performance parameters affecting fluorescence images are defined strictly. We also recommend scientific and feasible methods for their detections and analyses, which are verified by practical examples. This paper aims to provide a feasible reference standard for fluorescence performance evaluation.

A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells.

BACKGROUND: GPCRs are considered essential for various physiological processes and have been the most productive drug targets. Therefore, development of the methods of GPCR ligands screening is a high priority for pharmaceutical industries and research institutions. METHODS: We developed a potential method (piggyBac-TANGO) based on the TANGO and PRESTO-TANGO assays. The system was optimized with a piggyBac transposon as a transgene vehicle, and eGFP was used as a reporter instead of luciferase. The assay was validated in the HEK 293T and U87-MG cell lines and antagonist activities of the compounds were assessed. The transgene copy number and long-term stability were evaluated by qPCR. Then, we performed a DRD2-targeted screening for natural products using the piggyBac-TANGO assay. RESULTS: The validation assay showed that using the piggyBac transposon as a transgene vehicle produced high signal-to-background ratio and stable readout confirmed by investigation of the transgene copy number and long-term stability. Use of eGFP instead of luciferase as a reporter enabled to create a high throughput system suitable for live cells. Moreover, the piggyBac-TANGO assay permitted versatile detection of antagonist activity of compounds and was not limited to a particular cell type. With the use of the piggyBac-TANGO assay, we have successfully identified a novel agonist of DRD2. CONCLUSION: Thus, the results indicate that the piggyBac-TANGO method is a user-friendly, robust and imaging-based assay that provides a novel approach to high throughput GPCR-targeted ligand screening and drug development.

Low-Dose Evans Blue Dye for Near-Infrared Fluorescence Imaging in Photothrombotic Stroke Model.

Background: Evans blue dye (EBD) is the most common indicator to analyze the extent of blood-brain barrier (BBB) breakdown in several neurological disease models. However, the high-dose of EBD (51.9 mg/kg) is usually required for visualization of blue color by the human eye that brings potential safety issues. Methods: To solve this problem, low-dose of EBD was applied for the near-infrared (NIR) fluorescence-assisted quantitation of BBB breakdown in photothrombotic stoke model. Animals were allocated to seven dose groups ranging from 1.35 nmol (5.19 µg/kg) to 13.5 µmol (51.9 mg/kg) EBD. Results: EBD was undetectable in the non-ischemic brain tissue, and the fluorescence signals in the infarcted hemisphere seemed proportional to the injected dose in the dose range. Although the maximum fluorescence signals in brain tissue were obtained with the injections of 1.35 nmol ~ 13.5 µmol EBD, the background signals in the neighboring brain tissues were significantly increased as well. Since the high concentration of EBD is necessary for color-based identification of the infarcted lesion in brain tissues, even 10-fold diluted could not be distinguished visually by naked eye. Conclusions: NIR fluorescence-assisted method could potentially provide new opportunities to study BBB leakage just using small amount of EBD in different pathological conditions and to test the efficacy of various therapeutic strategies to protect the BBB.

Multivalent Mannose-Decorated NIR Nanoprobes for Targeting Pan Lymph Nodes.

Lymphadenectomy is a prerequisite for most malignancies to define the precise staging of cancer, as well as resect the possible metastases completely. While it improves prognosis, lymphadenectomy often causes postoperative edema or bleeding because of unclear surgical margins. In this study, we synthesized near-infrared (NIR) fluorescent nanoprobes with conjugating various mannose moieties on the surface to target macrophages in the lymph node. Armed with these NIR nanoprobes, we demonstrated the feasibility of intraoperative pan lymph nodes (PLN) mapping and real-time optical imaging under the NIR fluorescence imaging system. We found that even single mannose-conjugated ZW800-1 showed specific uptake in lymph nodes within 4 h, and multiple mannose-employed polyrotaxanes highlighted PLN efficiently with low background signals in major organs. This technology can help surgeons perform lymphadenectomy with ease and safety by identifying all regional lymph nodes proficiently after a single intravenous injection of NIR nanoprobes.

Fluorometric determination of lead(II) and mercury(II) based on their interaction with a complex formed between graphene oxide and a DNAzyme.

The authors have designed a DNAzyme where graphene oxide (GO) interacts with the ssDNA stem loop region. The DNAzyme strand and substrate strand are hybridized and bind to the surface of GO which act as a signal reporter, while GO act as a strong quencher. The presence of Pb(II) ion disturbs the GO-DNAzyme complex and causes internal cleavage of the DNAzyme complex. On addition of Thioflavin T (ThT) as a quadruplex inducer, fluorescence intensity (best measured at excitation/emission peaks of 425/490 nm) is strongly enhanced. Subsequent addition of Hg(II) to ThT/G-quadruplex complex decreases fluorescence because the G-quadruplex is unwinding to form a T-Hg(II)-T dsDNA system. Therefore, the change in fluorescence intensity of ThT is directly correlated to the concentration of Pb(II) and Hg(II). As a result, the assay is highly selective and sensitive. The limits of detection are 96 pM for Pb(II) and 356 pM for Hg(II). Moreover, the method was applied to the detection of the two ions in spiked real samples and gave satisfactory results. Graphical abstract A label free sensitive and selective "on-off" fluorescent assay for detection of Pb(II) and Hg(II) based on graphene oxide -DNAzyme complex with fluorogenic dye thioflavin T. The limits of detection are 96 pM (Pb2+) and 356 pM (Hg2+).

A dual-mode lateral flow immunoassay by ultrahigh signal-to background ratio SERS probes for nitrofurazone metabolites ultrasensitive detection.

In this work, an ultra-sensitive lateral flow immunoassay (LFIA) with SERS/colorimetric dual signal mode was constructed for the detection of nitrofurazone metabolites, an antibiotic prohibited in animal-origin foods. Au@4-MBN@AgNRs nano-sandwich structural signal tag integrates the unique advantages of high signal-to-background ratio and anti-matrix interference through geometric control of SERS tag and nanoengineering adjustment of chemical composition. Under the optimal conditions, the detection limits of nitrofurazone metabolites by SERS/colorimetric dual-mode LFIA were 20 pg/mL (colorimetric mode) and 0.08 pg/mL (SERS mode). Excitingly, the vLOD of the colorimetric signal improved by a factor of 100 compared to Au NPs-based LFIA. In this study, the proposed dual-mode LFIA was successfully applied to the on-site real-time detection of honey, milk powder, and chicken. It is anticipated that with low background interference and anti-matrix interference output signal, our proposed dual-mode strategy can pave an innovative pathway for the fabrication of a powerful biosensor.

Machine-Learning-Assisted Rational Design of Si─Rhodamine as Cathepsin-pH-Activated Probe for Accurate Fluorescence Navigation.

High-performance fluorescent probes stand as indispensable tools in fluorescence-guided imaging, and are crucial for precise delineation of focal tissue while minimizing unnecessary removal of healthy tissue. Herein, machine-learning-assisted strategy to investigate the current available xanthene dyes is first proposed, and a quantitative prediction model to guide the rational synthesis of novel fluorescent molecules with the desired pH responsivity is constructed. Two novel Si─rhodamine derivatives are successfully achieved and the cathepsin/pH sequentially activated probe Si─rhodamine─cathepsin-pH (SiR─CTS-pH) is constructed. The results reveal that SiR─CTS-pH exhibits higher signal-to-noise ratio of fluorescence imaging, compared to single pH or cathepsin-activated probe. Moreover, SiR─CTS-pH shows strong differentiation abilities for tumor cells and tissues and accurately discriminates the complex hepatocellular carcinoma tissues from normal ones, indicating its significant application potential in clinical practice. Therefore, the continuous development of xanthene dyes and the rational design of superior fluorescent molecules through machine-learning-assisted model broaden the path and provide more advanced methods to researchers.

Intraoperative near infrared fluorescence guided identification of the ureters using low dose methylene blue: a first in human experience.

PURPOSE: Near infrared fluorescence imaging is a promising technique that offers real-time visual information during surgery. In this study we report the first clinical results to our knowledge of ureteral imaging using near infrared fluorescence after a simple peripheral infusion of methylene blue. Furthermore, we assessed the optimal timing and dose of methylene blue. MATERIALS AND METHODS: A total of 12 patients who underwent lower abdominal surgery were included in this prospective feasibility study. Near infrared fluorescence imaging was performed using the Mini-FLARE™ imaging system. To determine optimal timing and dose, methylene blue was injected intravenously at doses of 0.25, 0.5 or 1 mg/kg after exposure of the ureters. Imaging was performed for up to 60 minutes after injection. RESULTS: In all patients both ureters could be clearly visualized within 10 minutes after infusion of methylene blue. The signal lasted at least up to 60 minutes after injection. The mean signal-to-background ratio of the ureter was 2.27 ± 1.22 (4), 2.61 ± 1.88 (4) and 3.58 ± 3.36 (4) for the 0.25, 0.5 and 1 mg/kg groups, respectively. A mixed model analysis was used to compare signal-to-background ratios among dose groups and times, and to assess the relationship between dose and time. A significant difference among time points (p <0.001) was found. However, no difference was observed among dose groups (p = 0.811). CONCLUSIONS: This study demonstrates the first successful use of near infrared fluorescence using low dose methylene blue for the identification of the ureters during lower abdominal surgery.

Surface-Enhanced Raman Scattering Bioimaging with an Ultrahigh Signal-to-Background Ratio under Ambient Light.

Surface-enhanced Raman scattering (SERS) nanoprobes have attracted particular interests in the field of bioimaging owing to their high sensitivity and specificity of the fingerprint spectrum. However, the limited signal-to-background ratio (SBR) in SERS imaging and the requirement to perform imaging in a dark environment have largely hindered its biomedical application. To circumvent this, we have developed a type of bio-orthogonal nanoprobes for SERS imaging with an ultrahigh SBR and ambient light anti-interference ability. The core-shell nanoprobes exhibit strongly enhanced Raman signals and depress the background from photoluminescence of metallic nanoparticles by off-resonance excitation and from the Raman scattering and auto-fluorescence of tissues by near-infrared laser excitation. Such nanoprobes have achieved an SBR of over 100 in SERS bioimaging, 5 times higher than the traditional on-resonant nanoprobes, and their bio-orthogonal signal in the Raman-silent region renders the anti-interference capability under ambient light. The development of these SERS probes opens up a new era for the future applications of Raman imaging in clinical medicine.

Visualization of Acute Inflammation through a Macrophage-Camouflaged Afterglow Nanocomplex.

Acute inflammation is a basic innate, immediate, and stereotyped immune response to injury, which is characterized by rapid recruitment of immune cells to the vasculature and extravasation into the damaged parenchyma. Visualization of acute inflammation plays an important role in monitoring the disease course and understanding pathogenesis, which lacks specific targeted and observing tools in vivo. Here, we report a Trojan horse strategy of a macrophage-camouflaged afterglow nanocomplex (UCANPs@RAW) to specifically visualize acute inflammation. Due to the advantages of optical antibackground interference elimination, as well as particular immune homing and long-term tracking capacity, UCANPs@RAW demonstrates an excellent acute inflammatory recognition ability. In an arthritis model, previously intravenously injected UCANPs@RAW could directionally migrate from the liver to the inflammation site as soon as 3 h after the model was induced, which could be continuously lighted for at least 36 h with the highest imaging signal-to-background ratio (SBR) as 382 at the time point of 9 h. Additionally, UCANPs@RAW is observed to penetrate the blood-brain barrier and image the deep brain inflamed region covered by the thick skull in an acute brain inflammation model with an SBRmax of 258, which is based on the strong recruiting ability of macrophages to immune response. In view of this smart nanocomplex, our strategy holds great potential for inflammatory detection and treatments.

Tumor microenvironment enhanced NIR II fluorescence imaging for tumor precise surgery navigation via tetrasulfide mesoporous silica-coated Nd-based rare-earth nanocrystals.

In vivo fluorescent imaging by using the new contrast agents emitted at short-wavelength infrared region (NIR II, 1000-1700 â€‹nm) presents an unprecedent advantages in imaging sensitivity and spatial resolution over traditional near-infrared (NIR) light. Recently, Nd-based rare-earth nanocrystals have attracted considerable attention due to the high quantum yield (∼40%) of their emission at NIR II. However, undesirable capture by reticuloendothelial system to bring strong background signal is unsatisfying for tumor discrimination. Here, GSH-sensitive tetrasulfide bond incorporated mesoporous silica shell has entrusted onto Nd-based down-conversion nanocrystals (DCNPs) surface to totally quench the fluorescence of DCNPs. After RGD conjugation on the silica surface, the NIR II contrast agents could actively target to liver tumors. Then tetrasulfide bonds can be broken during the silica framework decomposing in cytoplasm under high GSH concentration to result in NIR II fluorescence explosive recover. Benefiting from this specific response under tumor microenvironment, the NIR II signal in other organs was markedly reduced, while the signal-to-background ratio is prominently enhanced in tumors. Then, solid liver tumors were successfully resected under the guidance of our GSH responsive NIR II fluorescent imaging with no recurrence after 20-day of surgery. Meanwhile, by combining with the ignorable side effects, the Nd-based nanoprobes vastly improved the imaging resolution of tumor margin, opening a paradigm of NIR II fluorescent imaging-guided surgery.

Collimator and energy window optimization for YTTRIUM-90 bremsstrahlung SPECT imaging.

The optimal collimator and energy window for Yttrium-90 bremsstrahlung SPECT imaging was investigated in the study. Yttrium-90 images were acquired with a dual-head gamma camera, equipped with parallel hole collimators and 90Y vial for different energy windows ranging from 56 to 232 keV. Image quality parameters (sensitivity, %FOV, and S/B) were examined for the energy window and collimator combinations. It is concluded that the optimal SPECT imaging was achieved using FBP Method with a HEGP collimator and the energy window of 90-110 keV.

Study on the Functionalization and Signaling Efficiency of the Hybridization Chain Reaction Using Traditional and Single Molecular Characterizations.

As an important enzyme-free amplifier, the hybridization chain reaction (HCR) uses an ssDNA to trigger cycled displacement interactions between substrate hairpins and finally form elongated dsDNA concatamer mixtures. In many cases, to provide a signal probe or advanced function, additional oligonucleotides (named hairpin tails) have to be extended upon classic HCR hairpin substrates, but by doing so the HCR assembly efficiency and signal-to-noise ratio (SNR) may get seriously reduced. In this Article, a rational and general model that may guide the study on HCR functionalization and signaling efficiency is provided. We rationally design a four-hairpin model HCR system (4H-HCR) in which one or more hairpin substrates are appended with additional tails as a signaling probe. After HCR assembly, two adjacent tails are supposedly integrating into a full G-quadruplex structure to provide the evidence or signal for the assembly. A systematic study has been applied to reveal the relationship between the "tail-design" with assembly efficiency and SNR. A clear design rule-set guiding the optimized assembly and signal has been provided for traditional electrophoresis and G-quadruplex-enhanced fluorescence signal. Importantly, solid-state nanopore single molecular detection has been innovatively introduced and recommended as an "antirisk" and "mutual benefit" readout to traditional G-quadruplex signaling. Nanopore detection can provide a clear signal distinguished before and after the HCR reaction, especially when the traditional G-quadruplex-enhanced signal only provides low SNR. The G-quadruplex, in turn, may enhance the nanopore signal amplitude via increasing the diameter of the HCR products.

A Novel Method That Allows SNP Discrimination with 160:1 Ratio for Biosensors Based on DNA-DNA Hybridization.

Highly sensitive (high SBR) and highly specific (high SNP discrimination ratio) DNA hybridization is essential for a biosensor with clinical application. Herein, we propose a method that allows detecting multiple pathogens on a single platform with the SNP discrimination ratios over 160:1 in the dynamic range of 101 to 104 copies per test. The newly developed SWAT method allows achieving highly sensitive and highly specific DNA hybridizations. The detection and discrimination of the MTB and NTM strain in the clinical samples with the SBR and SNP discrimination ratios higher than 160:1 indicate the high clinical applicability of the SWAT.

A hybrid region growing tumour segmentation method for low contrast and high noise Nuclear Medicine (NM) images by combining a novel non-linear diffusion filter and global gradient measure (HNDF-GGM-RG).

Poor spatial resolution and low signal-to-noise ratio (SNR) along with the finite image sampling constraint make lesion segmentation on Nuclear Medicine (NM) images (e.g., PET-Positron Emission Tomography) a challenging task. Since the size, signal-to-background ratio (SBR) and SNR of lesion vary within and between patients, performance of the conventional segmentation methods are not consistent against statistical fluctuations. To overcome these limitations, a hybrid region growing segmentation method is proposed combining non-linear diffusion filter and global gradient measure (HNDF-GGM-RG). The performance of the algorithm is validated on PET images and compared with the 40%-fixed threshold and a state-of-the-art active contour (AC) methods. Segmented volume, dice similarity coefficient (DSC) and percentage classification error (% CE) were used as the quantitative figures of merit (FOM) using the torso NEMA phantom that contains six different sizes of spheres. A 2:1 SBR was created between the spheres and background and the phantom was scanned with a Siemens TrueV PET-CT scanner. 40T method is SNR dependent and overestimates the volumes ( ≈ 4.5  times ) . AC volumes match with the true volumes only for the largest three spheres. On the other hand, the proposed HNDF-GGM-RG volumes match closely with the true volumes irrespective of the size and SNR. Average DSC of 0.32 and 0.66 and % CE of 700% and 160% were achieved by the 40T and AC methods respectively. Conversely, average DSC and %CE are 0.70 and 60% for HNDF-GGM-RG and less dependent on SNR. Since two-sample t-test indicates that the performance of AC and HNDF-GGM-RG are statistically significant for the smallest three spheres and similar for the rest, HNDF-GGM-RG can be applied where the size, SBR and SNR are subject to change either due to alterations in the radiotracer uptake because of treatment or uptake variability of different radiotracers because of differences in their molecular pathways.

A Highly Sensitive Strategy for Fluorescence Imaging of MicroRNA in Living Cells and in Vivo Based on Graphene Oxide-Enhanced Signal Molecules Quenching of Molecular Beacon.

In situ imaging of microRNA (miRNA) in living cells and in vivo is beneficial for promoting the studies on miRNA-related physiological and pathological processes. However, the current strategies usually have a low signal-to-background ratio, which greatly affects the sensitivity and imaging performance. To solve this problem, we developed a highly sensitive strategy for fluorescence imaging of miRNA in living cells and in vivo based on graphene oxide (GO)-enhanced signal molecule quenching of a molecular beacon (MB). 2Cy5-MB was designed by coupling two Cy5 molecules onto the opposite ends of MB. The fluorescence intensities of two Cy5 molecules were reduced because of the self-quenching effect. After adsorbing on the GO surface, the fluorescence quenching of the molecules was enhanced by fluorescence resonance energy transfer. This double-quenching effect significantly reduced the fluorescence background. In the presence of one miRNA molecule, the fluorescence signals of two Cy5 molecules were simultaneously recovered. Therefore, a significantly enhanced signal-to-background ratio was obtained, which greatly improved the detection sensitivity. In the presence of miRNA, the fluorescence intensity of 2Cy5-MB-GO recovered about 156 times and the detection limit was 30 pM. Compared with 1Cy5-MB-GO, the elevated fluorescence intensity was enhanced 8 times and the detection limit was reduced by an order of magnitude. Furthermore, fluorescence imaging experiments demonstrated that 2Cy5-MB-GO could visually detect microRNA-21 in various cancer cells and tumor tissues. This simple and effective strategy provides a new sensing platform for highly sensitive detection and simultaneous imaging analysis of multiple low-level biomarkers in living cells and in vivo.

Folate receptor overexpression can be visualized in real time during pituitary adenoma endoscopic transsphenoidal surgery with near-infrared imaging.

OBJECTIVE Pituitary adenomas account for approximately 10% of intracranial tumors and have an estimated prevalence of 15%-20% in the general US population. Resection is the primary treatment for pituitary adenomas, and the transsphenoidal approach remains the most common. The greatest challenge with pituitary adenomas is that 20% of patients develop tumor recurrence. Current approaches to reduce recurrence, such as intraoperative MRI, are costly, associated with high false-positive rates, and not recommended. Pituitary adenomas are known to overexpress folate receptor alpha (FRα), and it was hypothesized that OTL38, a folate analog conjugated to a near-infrared (NIR) fluorescent dye, could provide real-time intraoperative visual contrast of the tumor versus the surrounding nonneoplastic tissues. The preliminary results of this novel clinical trial are presented. METHODS Nineteen adult patients who presented with pituitary adenoma were enrolled. Patients were infused with OTL38 2-4 hours prior to surgery. A 4-mm endoscope with both visible and NIR light capabilities was used to visualize the pituitary adenoma and its margins in real time during surgery. The signal-to-background ratio (SBR) was recorded for each tumor and surrounding tissues at various endoscope-to-sella distances. Immunohistochemical analysis was performed to assess the FRα expression levels in all specimens and classify patients as having either high or low FRα expression. RESULTS Data from 15 patients (4 with null cell adenomas, 1 clinically silent gonadotroph, 1 totally silent somatotroph, 5 with a corticotroph, 3 with somatotrophs, and 1 somatocorticotroph) were analyzed in this preliminary analysis. Four patients were excluded for technical considerations. Intraoperative NIR imaging delineated the main tumors in all 15 patients with an average SBR of 1.9 ± 0.70. The FRα expression level of the adenomas and endoscope-to-sella distance had statistically significant impacts on the fluorescent SBRs. Additional considerations included adenoma functional status and time from OTL38 injection. SBRs were 3.0 ± 0.29 for tumors with high FRα expression (n = 3) and 1.6 ± 0.43 for tumors with low FRα expression (n = 12; p < 0.05). In 3 patients with immunohistochemistry-confirmed FRα overexpression (2 patients with null cell adenoma and 1 patient with clinically silent gonadotroph), intraoperative NIR imaging demonstrated perfect classification of the tumor margins with 100% sensitivity and 100% specificity. In addition, for these 3 patients, intraoperative residual fluorescence predicted postoperative MRI results with perfect concordance. CONCLUSIONS Pituitary adenomas and their margins can be intraoperatively visualized with the preoperative injection of OTL38, a folate analog conjugated to NIR dye. Tumor-to-background contrast is most pronounced in adenomas that overexpress FRα. Intraoperative SBR at the appropriate endoscope-to-sella distance can predict adenoma FRα expression status in real time. This work suggests that for adenomas with high FRα expression, it may be possible to identify margins and to predict postoperative MRI findings.

Intraoperative near-infrared imaging with receptor-specific versus passive delivery of fluorescent agents in pituitary adenomas.

OBJECTIVE: Intraoperative molecular imaging with tumor-targeted fluorescent dyes can enhance resection rates. In contrast to visible-light fluorophores (e.g., 5-aminolevulinic-acid), near-infrared (NIR) fluorophores have increased photon tissue penetration and less contamination from tissue autofluorescence. The second-window ICG (SWIG) technique relies on passive accumulation of indocyanine green (ICG) in neoplastic tissues. OTL38, conversely, targets folate receptor overexpression in nonfunctioning pituitary adenomas. In this study, we compare the properties of these 2 modalities for NIR imaging of pituitary adenomas to better understand the potential for NIR imaging in neurosurgery. METHODS: A total of 39 patients with pituitary adenomas were enrolled between June 2015 and January 2018 in 2, sequential, IRB-approved studies. Sixteen patients received systemic ICG infusions 24 hours prior to surgery, and another 23 patients received OTL38 infusions 2-3 hours prior to surgery. NIR fluorescence signal-to-background ratio (SBR) was recorded during and after resection. Immunohistochemistry was performed on the 23 adenomas resected from patients who received OTL38 to assess expression of folate receptor-alpha (FRα). RESULTS: All 16 adenomas operated on after ICG administration demonstrated strong NIR fluorescence (mean SBR 4.1 ± 0.69 [SD]). There was no statistically significant difference between the 9 functioning and 7 nonfunctioning adenomas (p = 0.9). After administration of OTL38, the mean SBR was 1.7 ± 0.47 for functioning adenomas, 2.6 ± 0.91 for all nonfunctioning adenomas, and 3.2 ± 0.53 for the subset of FRα-overexpressing adenomas. Tissue identification with white light alone for all adenomas demonstrated 88% sensitivity and 90% specificity. SWIG demonstrated 100% sensitivity but only 29% specificity for both functioning and nonfunctioning adenomas. OTL38 was 75% sensitive and 100% specific for all nonfunctioning adenomas, but when assessment was limited to the 9 FRα-overexpressing adenomas, the sensitivity and specificity of OTL38 were both 100%. CONCLUSIONS: Intraoperative imaging with NIR fluorophores demonstrates highly sensitive detection of pituitary adenomas. OTL38, a folate-receptor-targeted fluorophore, is highly specific for nonfunctioning adenomas but has no utility in functioning adenomas. SWIG, which relies on passive diffusion into neoplastic tissue, is applicable to both functioning and nonfunctioning pituitary adenomas, but it is less specific than targeted fluorophores. Thus, targeted and nontargeted NIR fluorophores play important, yet distinct, roles in intraoperative imaging. Selectively and intelligently using either agent has the potential to greatly improve resection rates and outcomes for patients with intracranial tumors.

Near-infrared fluorescent image-guided surgery for intracranial meningioma.

OBJECTIVE Meningiomas are the most common primary tumor of the central nervous system. Complete resection can be curative, but intraoperative identification of dural tails and tumor remnants poses a clinical challenge. Given data from preclinical studies and previous clinical trials, the authors propose a novel method of localizing tumor tissue and identifying residual disease at the margins via preoperative systemic injection of a near-infrared (NIR) fluorescent contrast dye. This technique, what the authors call "second-window indocyanine green" (ICG), relies on the visualization of ICG approximately 24 hours after intravenous injection. METHODS Eighteen patients were prospectively identified and received 5 mg/kg of second-window ICG the day prior to surgery. An NIR camera was used to localize the tumor prior to resection and to inspect the margins following standard resection. The signal to background ratio (SBR) of the tumor to the normal brain parenchyma was measured in triplicate. Gross tumor and margin specimens were qualitatively reported with respect to fluorescence. Neuropathological diagnosis served as the reference gold standard to calculate the sensitivity and specificity of the imaging technique. RESULTS Eighteen patients harbored 15 WHO Grade I and 3 WHO Grade II meningiomas. Near-infrared visualization during surgery ranged from 18 to 28 hours (mean 23 hours) following second-window ICG infusion. Fourteen of the 18 tumors demonstrated a markedly elevated SBR of 5.6 ± 1.7 as compared with adjacent brain parenchyma. Four of the 18 patients showed an inverse pattern of NIR signal, that is, stronger in the adjacent normal brain than in the tumor (SBR 0.31 ± 0.1). The best predictor of inversion was time from injection, as the patients who were imaged earlier were more likely to demonstrate an appropriate SBR. The second-window ICG technique demonstrated a sensitivity of 96.4%, specificity of 38.9%, positive predictive value of 71.1%, and a negative predictive value of 87.5% for tumor. CONCLUSIONS Systemic injection of NIR second-window ICG the day before surgery can be used to visualize meningiomas intraoperatively. Intraoperative NIR imaging provides higher sensitivity in identifying meningiomas than the unassisted eye. In this study, 14 of the 18 patients with meningioma demonstrated a strong SBR compared with adjacent brain. In the future, reducing the time interval from dye injection to intraoperative imaging may improve fluorescence at the margins, though this approach requires further investigation. Clinical trial registration no.: NCT02280954 ( clincialtrials.gov ).