Direct Fluorescence Research Articles

A Direct Non-destructive Method for Determination of Sulfur in Ore Samples Using EDXRF Spectrometry.

A method has been developed for direct non-destructive energy dispersive X-ray fluorescence (EDXRF) determination of sulfur in solid uranium ores and intermediates, obtained from the alkaline recovery process for uranium from its ores. The method involves thorough grinding of a few mg of solid powder samples to fine particle size and mixing the fine powder thus obtained with a few drops of 10% collodion solution in amyl acetate to make a paste. A very small amount of this paste was transferred with the help of the pestle tip, spread uniformly in the form of thin slurry on Mylar films, and dried to make very thin sample specimens on thin Mylar film supports. These specimens were presented for EDXRF measurements. A calibration plot was made by plotting the intensity ratios of S Kα and Rayliegh scattered peak of the excitation source (Ge Kα) against sulphur percent in the certified reference materials (CRMs). It was found that the precision obtained using this methodology was within 5% (±1σ) and the deviation of the EDXRF analytical results from the expected values of CRM was within 7%. The developed method was successfully applied for the determination of sulfur in the samples obtained from the different stages of the uranium ore processing using alkaline based leaching method.

Identification of inhibitors of UDP-galactopyranose mutase via combinatorial in situ screening.

An in situ screening assay for UDP-galactopyranose mutase (UGM, an essential enzyme of M. tuberculosis cell wall biosynthesis) has been developed to discover novel UGM inhibitors. The approach is based on the amide-forming reaction of an amino acid core with various cinnamic acids, followed by a direct fluorescence polarization assay to identify the best UGM binders without isolation and purification of the screened ligands. This assay allows us to perform one-pot high-throughput synthesis and screening of enzyme inhibitors in a 384-well plate format. UGM ligands were successfully identified by this technology and their inhibition levels were established from pure synthetic compounds in vitro and in a whole cell antibacterial assay. This study provides a blueprint for designing enamide structures as new UGM inhibitors and anti-mycobacterial agents.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

Versatile Multiplex Endogenous RNA Detection with Simultaneous Signal Normalization Using Mesoporous Silica Nanoquenchers.

Detection of endogenous tumor-related RNA is vital for cancer diagnostics. Despite advancements made, live-cell RNA detection still faces numerous problems, such as low signal output and cell-to-cell variations arising from differences in probe uptake. To address these issues, we designed a versatile and highly sensitive mRNA/miRNA nanosensor featuring, for the first time, signal amplification and in-built signal normalization. Using dye-loaded mesoporous silica nanoquenchers (qMSNs) capped with target-corresponding antisense oligos (ASOs), direct fluorescence "Turn-ON" with signal amplification was achieved upon target binding. By readily varying the capping ASOs as well as cargo dyes, a suite of RNA nanosensors for multiplex target detection could be easily prepared. Further modification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA-responsive molecular beacons (MBs) onto our nanosensor enabled dual detection of target RNA and GAPDH mRNA, allowing for target signal normalization using GAPDH as a reference. We demonstrated that this newly developed nanosensor could successfully differentiate between noncancer and cancer cells, as well as accurately monitor the relative expression levels of multiple tumor-related RNAs simultaneously in different cancer cell lines, with a high degree of specificity and sensitivity, functioning as a noninvasive "qPCR mimic" imaging tool in live cells.

Examination of Giardia intestinalis with Direct Microscopy and Direct Fluorescent Antibody in Patients with Diarrhea.

In this study, our objective was to compare direct microscopic examination and direct fluorescence antibody (DFA) method for Giardia diagnosis in stool samples and to evaluate the possible risk factors related to Giardia infections. Stool samples of 185 patients with diarrhoea collected between June 2019 and July 2019 in Erzurum Yakutiye Research Hospital were included in the study. Microscopic examination of the samples was performed with native-lugol, and they were subsequently scanned by the indirect fluorescent assay microscope using the DFA method at 100-200X magnification. In addition, all patients filled a questionnaire prepared to determine the possible risk factors related to Giardia infection. The age of the 185 participating patients who belonged to different groups was between 0 and 94 years. Giardia spp. cysts were detected in five stool samples (2.7%) using direct microscopic examination. Nine samples (4.9%) were DFA-positive. The incidence of giardiasis was noted to be 7.5% in children, 3.8% in adults, 7.3% in people living in rural areas, 2.9% in people living in urban areas, 10% in people having pets and 4.2% in people who do not have pets. By taking the DFA method as a reference, the sensitivity and specificity of the microscopic examination were found to be 44.4% and 99.4%, respectively. The Giardia positivity rate was higher in children, those living in rural areas, those having pets and those using well water as drinking water.

MMP activation–associated aminopeptidase N reveals a bivalent 14-3-3 binding motif

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

Laboratory measurements of stomatal NO<sub>2</sub> deposition to native California trees and the role of forests in the NO<sub>x</sub> cycle

Abstract. Both canopy-level field measurements and laboratory studies suggest that uptake of NO2 through the leaf stomata of vegetation is a significant sink of atmospheric NOx. However, the mechanisms of this foliar NO2 uptake and their impact on NOx lifetimes remain incompletely understood. To understand the leaf-level processes affecting ecosystem-scale atmosphere–biosphere NOx exchange, we have conducted laboratory experiments of branch-level NO2 deposition fluxes to six coniferous and four broadleaf native California trees using a branch enclosure system with direct laser-induced fluorescence (LIF) detection of NO2. We report NO2 foliar deposition that demonstrates a large degree of inter-species variability, with maximum observed deposition velocities ranging from 0.15 to 0.51 cm s−1 during the daytime, as well as significant stomatal opening during the night. We also find that the contribution of mesophyllic processing to the overall deposition rate of NO2 varies by tree species but has an ultimately inconsequential impact on NOx budgets and lifetimes. Additionally, we find no evidence of any emission of NO2 from leaves, suggesting an effective unidirectional exchange of NOx between the atmosphere and vegetation.

Metallo-graphene enhanced upconversion luminescence for broadband photodetection under polychromatic illumination

We report electrostatically conjugated core- silica (SiO2) shell upconversion nanoparticle ([email protected]2), and gold nanorod (AuNR) nanocomposite (NC) combined with graphene to demonstrate > 200-fold UC fluorescence enhancement. Plasmonic AuNR and graphene, resulted in enhanced fluorescence in UCNP with a maximum for the SiO2 shell thickness of 7 nm supported by finite difference time domain simulation of electric field distribution. In addition to the conventionally reported spectroscopic evidence, the plasmon aided UC fluorescence enhancement was demonstrated by direct confocal fluorescence imaging also, which was corroborated by a ~40% decrease in fluorescence lifetime. Finally, we have fabricated a NC/graphene hybrid photodetector (PD) that showed broadband (455–980 nm) photoresponse, with photoresponsivity of ~5000 AW−1, and response times of 80–200 ms under 980 nm illumination. The multiphoton infrared (IR, ~980 nm) absorbing UCNPs also show interesting high energy (blue (B), green (G), and red (R)) photoresponse which is now conclusively attributed to one-photon absorption in the UCNPs. We report, probably for the first time, the performance of the hybrid PD under monochromatic and polychromatic illumination of B, G, R, B + G, B + R, G + R, and B + G + R, among others. The photocurrent under polychromatic conditions is dominated by the strongest monochromatic response, and does not scale with net power of the illumination. The loss in photocurrent is attributed to saturation in absorption, and photothermal heating. The fast response of the PD device has been demonstrated while detecting high frequency modulated AC remote controller signals, and attributed to the fast charge sweeping by the AuNRs.

Real-Time Parallel Artificial Membrane Permeability Assay Based on Supramolecular Fluorescent Artificial Receptors.

Parallel artificial membrane permeability assay (PAMPA) is a screening tool for the evaluation of drug permeability across various biological membrane systems in a microplate format. In PAMPA, a drug candidate is allowed to pass through the lipid layer of a particular well during an incubation period of, typically, 10–16 h. In a second step, the samples of each well are transferred to a UV-Vis–compatible microplate and optically measured (applicable only to analytes with sufficient absorbance) or sampled by mass-spectrometric analysis. The required incubation period, sample transfer, and detection methods jointly limit the scalability of PAMPA to high-throughput screening format. We introduce a modification of the PAMPA method that allows direct fluorescence detection, without sample transfer, in real time (RT-PAMPA). The method employs the use of a fluorescent artificial receptor (FAR), composed of a macrocycle in combination with an encapsulated fluorescent dye, administered in the acceptor chamber of conventional PAMPA microplates. Because the detection principle relies on the molecular recognition of an analyte by the receptor and the associated fluorescence response, concentration changes of any analyte that binds to the receptor can be monitored (molecules with aromatic residues in the present example), regardless of the spectroscopic properties of the analyte itself. Moreover, because the fluorescence of the (upper) acceptor well can be read out directly by fluorescence in a microplate reader, the permeation of the drug through the planar lipid layer can be monitored in real time. Compared with the traditional assay, RT-PAMPA allows not only quantification of the permeability characteristics but also rapid differentiation between fast and slow diffusion events.

Characterization of the Vibriocidal Activity of Chitosan Microparticles: A Potential Therapeutic Agent for Emerging Multidrug-Resistant Cholera Infections.

Due to increasing reports of multidrug-resistant (MDR) Vibrio cholerae O1, the goal of this study was to characterize the in vitro antimicrobial activity of chitosan microparticles (CMs) to evaluate their potential as a novel therapeutic agent for cholera. We examined the antimicrobial activity of CMs against toxigenic V. cholerae O1 using direct enumeration, microscopy, and fluorescence microplate assays. Bacterial viability kinetics were measured with different concentrations of CMs, solution pH, and salt content using a live/dead staining technique. Growth inhibition of CM-exposed V. cholerae strains was conducted using a redox-sensitive stain and compared between wild-type and isogenic outer membrane (OM) mutants. CM concentrations above 0.1 wt % were sufficient to kill V. cholerae O1 suspensions with approximately 108 CFU/mL within 3 h. The nonviable cells demonstrated increased OM permeability that corresponded to gross morphological changes observed through scanning electron microscopy. CMs exhibited dose-dependent bactericidal activity that increased predictably at lower pH and decreased with salt addition. V. cholerae O1 strains lacking O-antigen were twice as susceptible to growth inhibition by CMs, whereas those with glycine modification to lipid A were ten times more resistant. We propose that CMs exert vibriocidal activity via electrostatic surface interactions between their positively charged amine groups and the negatively charged Gram-negative bacterial OM, resulting in disruption, increased permeability, decreased redox metabolism, and subsequent loss of cellular viability. Further research should be conducted in vivo to evaluate the efficacy of CMs as luminal agents to treat infections caused by MDR, toxigenic V. cholerae and other diarrheal pathogens.

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia-lyase and a TAT-penetrating peptide.

Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a trans-activator of transcription (TAT) cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 ± 0.04 and 0.34 ± 0.03 nmol•mg-1 •s-1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7 ± 6.2%, 28.2 ± 5.7%, and 33.9 ± 2.9% compared to the nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 μgml-1 even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.

Development of biotinylated polyclonal anti-ribonucleoprotein IgG for detection of rabies virus antigen by direct rapid immunohistochemical test

The direct rapid immunohistochemical test (dRIT) has been recommended for laboratorial diagnosis of rabies, especially in developing countries. The absence of commercial primary antibodies, however, still represents a major limitation to its wider use in testing. We describe here the development of a biotinylated polyclonal antibody against Rabies lyssavirus (RABV) ribonucleoprotein (RNP) and its use as a primary reagent in dRIT. Anti-RNP polyclonal horse IgG was purified by ionic exchange chromatography followed by immunoaffinity column chromatography, and its affinity, diagnostic sensitivity, and specificity were evaluated. CNS samples (120) of suspected rabies cases in different animal species were tested by dRIT, with the positive (n = 14) and negative (n = 106) results confirmed by direct fluorescence antibody testing (dFAT). Comparing the results of dRIT and dFAT, we found that the biotinylated anti-RNP IgG delivered 100% diagnostic specificity and sensibility for rabies diagnosis. Our findings show that the biotinylated anti-RNP polyclonal IgG can be produced with the quality required for application in dRIT. This work represents an important step in efforts to diagnose rabies in developing countries.

Two 3d-4f metal-organic frameworks as fluorescent sensor array for the discrimination of phosphates based on different response patterns

Both organic and inorganic phosphates play important roles in biological activities. Considering the conventional methods are not very useful for identifying them from their analogues, the sensor array strategy has emerged in recent years, which is able to distinguish chemical species with similar structure or chemical properties. We propose a fluorescence sensor array based on two 3d-4f metal-organic frameworks (MOFs) by recording their direct one-step fluorescence changes with the addition of various phosphates. With the introduction of d-metal ions in the sensor unit synthesis process, two 3d-4f MOFs display higher sensitivity and noticeable fluorescence differences to five phosphates than the homometallic MOFs. Phosphates show diverse affinities to sensor units, forming the basis of differential sensing. The signals are much easier to collect, enabling the sensor array to simply accomplish the identification. Through statistical analysis, the sensor array is proved to be excellent in discriminating individual phosphate and mixture. Discrimination of five phosphates is successfully performed in serum samples to illustrate the practicability of the sensor array. This work presents an efficient and reliable approach for simultaneous detection of different phosphates, which can be extended to devise other array-based sensing methods.

Molecularly imprinted polymers hydrogel for the rapid risk-category-specific screening of food using SPE followed by fluorescence spectrometric detection

In fluorescence measurement, one pressing concern today is to construct sensors that can withstand various disturbances for highly selective and sensitive detection of trace analytes in complicated samples. Molecularly imprinted polymers (MIPs) with tailored binding sites are preferred to be recognition elements in sensors for effective target detection. Here, MIPs combined with solid-phase extraction (SPE), followed by direct fluorescence detection on the same extraction medium, was proposed for rapid onsite selective screening for risk categories in food safety without sample pretreatment. A novel surface solid-phase fluorescence substrate with special adsorption property for rhodamine B (RhB) or sulfamethoxazole (SMX) was synthesized by wrapping the MIPs in the deformable hydrogel prepared by polyvinyl alcohol (PVA). Parameters that affected the extraction capacity of the analytes were explored. MIPs-PVA hydrogel showed a good selectivity (imprinting factor α = 2.95 with RhB-MIPs-PVA hydrogel), reproducibility (RSD ≤ 16.5% with 5 ppm RhB) and sensitivity (0.1 ppm to RhB) with a short analysis time of less than 5 min including sampling. The proposed method is very promising as a rapid, onsite screening protocol for food safety.

Determination of nereistoxin-related insecticide via quantum-dots-doped covalent organic frameworks in a molecularly imprinted network.

Quantum-dots-doped covalent organic frameworks in a molecularly imprinted network (QDs-doped COFs@MIP) were developed for detection of nereistoxin (NRT)-related insecticide in tap water. The preparation of QDs-doped COFs@MIP was easy to accomplish via one-pot synthesis at room temperature. QDs-doped COFs@MIP quenched by targeting thiosultap due to the photoinduced charge transfer. A Brunauer-Emmett-Teller surface area of 186.20m2g-1 and a maximum adsorption capacity of 771mgg-1 of the QDs-doped COFs@MIP exhibited good selectivity and adsorption capacity. Direct fluorescence determination was established over the range 5-100μgL-1 (R2 = 0.9959) with a detection limit of 1.60μgL-1. Furthermore, 86.5-106.5% recoveries of spiked tap water were achieved. The determination system was feasible for tracing the NRT-related insecticide with high accuracy and good repeatability and reproducibility. Graphical abstract .

Direct fluorescence imaging of lignocellulosic and suberized cell walls in roots and stems.

Investigating plant structure is fundamental in botanical science and provides crucial knowledge for the theories of plant evolution, ecophysiology and for the biotechnological practices. Modern plant anatomy often targets the formation, localization and characterization of cellulosic, lignified or suberized cell walls. While classical methods developed in the 1960s are still popular, recent innovations in tissue preparation, fluorescence staining and microscopy equipment offer advantages to the traditional practices for investigation of the complex lignocellulosic walls. Our goal is to enhance the productivity and quality of microscopy work by focusing on quick and cost-effective preparation of thick sections or plant specimen surfaces and efficient use of direct fluorescent stains. We discuss popular histochemical microscopy techniques for visualization of cell walls, such as autofluorescence or staining with calcofluor, Congo red (CR), fluorol yellow (FY) and safranin, and provide detailed descriptions of our own approaches and protocols. Autofluorescence of lignin in combination with CR and FY staining can clearly differentiate between lignified, suberized and unlignified cell walls in root and stem tissues. Glycerol can serve as an effective clearing medium as well as the carrier of FY for staining of suberin and lipids allowing for observation of thick histological preparations. Three-dimensional (3D) imaging of all cell types together with chemical information by wide-field fluorescence or confocal laser scanning microscopy (CLSM) was achieved.

Development of gold nanoclusters based direct fluorescence restoration approach for sensitive and selective detection of pesticide

Pesticide contamination of drinking water is of high concern due to their hazardous effects on ecosystems. Several detection methods are available which are associated with many drawbacks, e.g. slow response time and laborious procedural techniques. This limits their practicability. Therefore, there is a dire need to develop quick, simpler but equally sensitive and practical methods for pesticide detection. Herein, we report a highly sensitive assay using fluorescent AuNCs for the detection of a pesticide i.e., 2-methyl-4-chlorophenoxyacetic acid (MCPA). For this purpose, three different fluorescent Au nanoclusters stabilized by histidine (His-AuNCs), lysozyme (Lys-AuNCs) and RNase (RNase-AuNCs) were systematically studied as optical probes for the detection of MCPA using a turn-on assay. The fluorescence of these AuNCs was initially quenched with Cu2+ ions followed by the quantitative recovery of their fluorescent signals in the presence of MCPA. The sensitivity and selectivity of these clusters for MCPA were in a linear dynamic range, i.e., 110–180 nM, 100–170 nM and 90–160 nM in case of His-AuNCs, Lys-AuNCs and RNase-AuNCs, respectively. Also, the aforesaid clusters were having a different limit of detection (LOD) by each type, i.e., highest by His-AuNCs (21.87), intermediate by Lys-AuNCs (12.97) and lowest by RNase-AuNCs (9.26 nM). Hence, the RNase-AuNCs are found to be the most sensitive, whereas His-AuNCs are least sensitive for the detection of MCPA. These findings offer a rapid, sensitive and selective assay for MCPA detection and have potential implications for the on-site, real-time monitoring of noxious pollutants.

Molecular and Direct Detection Tests for Treponema pallidum Subspecies pallidum: A Review of the Literature, 1964-2017.

Direct detection methods for Treponema pallidum include dark-field microscopy (DFM), direct fluorescence antibody (DFA) testing, immunohistochemistry (IHC), and nucleic acid amplification tests (NAATs). Here, we reviewed the relevant syphilis diagnostic literature to address 2 main questions with respect to T. pallidum direct detection techniques: “What are the performance characteristics for each direct detection test for T. pallidum and what are the optimal specimen types for each test?” and “What options are available for T. pallidum molecular epidemiology?” To answer these questions, we searched 5 electronic databases (OVID Medline, OVID Embase, CINAHL, Cochrane Library, and Scopus) from 1964 to 2017 using relevant search terms and identified 1928 articles, of which 37 met our inclusion criteria. DFM and DFA sensitivities ranged from 73% to 100% in cases of primary syphilis; and while sensitivity using silver stain histopathology for T. pallidum was generally low (0%–41%), higher performance characteristics were observed for T. pallidum–specific IHC (49–92%). Different genes have been targeted by T. pallidum–specific NAATs, with the majority of studies indicating that sensitivity is primarily dependent on the type of collected biological sample, with highest sensitivity observed in primary lesion exudate (75–95%). Given the rising incidence of syphilis, the development of direct, Food and Drug Administration–cleared T. pallidum NAATs should be considered an immediate priority.

Label-Free Sensitive Detection of Steroid Hormone Cortisol Based on Target-Induced Fluorescence Quenching of Quantum Dots.

We discovered that several types of steroid hormones quench the fluorescence of quantum dots (QDs) at close proximity. Inspired by the finding, we developed a new type of biosensor for the sensitive detection of cortisol via direct fluorescence quenching of functionalized QD probes directly induced by the capture of target cortisol without additional reporter reagents. The detection selectivity was provided by cortisol-selective aptamers or anticortisol antibodies conjugated on the QD surfaces. With the magnetic nanoparticle labeling, the new sensing method enabled rapid cortisol sensing at physiologically relevant concentrations and yielded the detection limit of ∼1 nM for aptamer-based sensors and ∼100 pM for antibody-based sensors. We also evaluated the new detection method using saliva samples with an optimized sample preparation process under the assistance of magnetic manipulation. The result showed a satisfying recovery rate for spiked saliva tests. The facile sensing technology offers an appealing approach for the detection of steroid hormones in point-of-care settings.

Near-infrared photoimmunotherapy of cancer: a new approach that kills cancer cells and enhances anti-cancer host immunity.

Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed hybrid cancer therapy that directly kills cancer cells as well as producing a therapeutic host immune response. Conventional immunotherapies, such as immune-activating cytokine therapy, checkpoint inhibition, engineered T cells and suppressor cell depletion, do not directly destroy cancer cells, but rely exclusively on activating the immune system. NIR-PIT selectively destroys cancer cells, leading to immunogenic cell death that initiates local immune reactions to released cancer antigens from dying cancer cells. These are characterized by rapid maturation of dendritic cells and priming of multi-clonal cancer-specific cytotoxic T cells that kill cells that escaped the initial direct effects of NIR-PIT. The NIR-PIT can be applied to a wide variety of cancers either as monotherapy or in combination with conventional immune therapies to further activate anti-cancer immunity. A global Phase 3 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03769506) of NIR-PIT targeting the epidermal growth factor receptor (EGFR) in patients with recurrent head and neck cancer is underway, employing RM1929/ASP1929, a conjugate of anti-EGFR antibody (cetuximab) plus the photo-absorber IRDye700DX (IR700). NIR-PIT has been given fast-track recognition by regulators in the USA and Japan. A variety of imaging methods, including direct IR700 fluorescence imaging, can be used to monitor NIR-PIT. As experience with NIR-PIT grows, additional antibodies will be employed to target additional antigens on other cancers or to target immune-suppressor cells to enhance host immunity. NIR-PIT will be particularly important in patients with localized and locally advanced cancers and may help such patients avoid side-effects associated with surgery, radiation and chemotherapy.