Fluorescence Imaging Research Articles

Development of Polar BODIPY-Tetrazines for Rapid Pretargeted Fluorescence Imaging.

Polar BODIPY-tetrazine dyes were developed and clicked in vivo to a preaccumulated trans-cyclooctene-modified anti-TAG72 monoclonal antibody CC49 (CC49-TCO). The in vivo click performance was evaluated using an in-house developed ex vivo blocking assay. All tested polar BODIPY structures exhibited excellent in vivo binding, confirming that the turn-on tetrazine dyes successfully clicked in vivo to pretargeted CC49-TCO. Fluorescence imaging showed high tumor-to-muscle ratios of 4:1. This proof-of-concept study indicates that the pretargeting concept based on turn-on probes could be used for cancer treatments, such as photodynamic or -thermal therapy.

Machine learning-assisted laccase-like activity nanozyme for intelligently onsite real-time and dynamic analysis of pyrethroid pesticides

The intelligently efficient, reliable, economical and portable onsite assay toward pyrethroid pesticides (PPs) residues is critical for food safety analysis and environmental pollution traceability. Here, a fluorescent nanozyme Cu-ATP@ [Ru(bpy)3]2+ with laccase-like activity was designed to develop a versatile machine learning-assisted colorimetric and fluorescence dual-modal assay for efficient onsite intelligent decision recognition and quantification of PPs residues. In the presence of alkaline phosphatase (ALP), the laccase-like activity of Cu-ATP@ [Ru(bpy)3]2+ was enhanced to oxidize colorless o-phenylenediamine (OPD) into dark-yellow 2,3-diaminophenazine (DAP) via electron transfer, appearing a new yellow fluorescence at 550 nm. Meanwhile, the red fluorescence of Cu-ATP@ [Ru(bpy)3]2+ at 600 nm was quenched due to the internal filter effect (IFE) of DAP towards Cu-ATP@ [Ru(bpy)3]2+. However, the selective inhibition of PPs toward ALP activity enabled to observe a dual-modal response of PPs concentration-dependent decrease in colorimetric signal and enhancement in the fluorescence intensity ratio of F600 nm/F550 nm. On this basis, both the colorimetric and fluorescence images were captured and processed with a home-made WeChat applet-installed smartphone to extract the corresponding image color information, thus achieving machine learning-assisted onsite real-time and dynamic intelligent decision recognition and quantification of PPs residues in real samples, which shows a promising potential in safeguarding food safety and environmental health.

Microplastic retention in green walls for nature-based and decentralized greywater treatment

In wastewater treatment, two issues have recently received increased attention: nature-based solutions for addressing urban water stress through decentralized treatment and re-use; and emerging pollutants such as microplastics (MPs). At the interface of these, this study investigated living green walls for greywater treatment and their potential for MP removal.A large, pilot-scale green wall was irrigated with greywater (a mix of water collected from laundry, dishwasher, bathroom sinks, and synthetic greywater), and effluent from planted and unplanted sections was compared. MPs >50 μm were analyzed using μRaman spectroscopy and supplementary fluorescence microscopy imaging. The green wall proved efficient for the reduction of chemical oxygen demand (COD) (around 80%), removal of total suspended solids (TSS) (around 90%) and MPs, especially for MPs of the non-polar, hydrophobic polymer type polystyrene and MPs sized 100–500 μm. MP removal was improved in the planted (50–60%) compared to the unplanted section (20%), especially for the size fraction 100–500 μm. Physical filtration by the green wall growing media (a mix of perlite with a grain size of 1–5 mm, and coconut fiber), which was further enhanced by plant roots decreasing the effective pore size, can be considered the most important removal mechanism. Charge-mediated adsorption cannot be expected as MPs and growing media mix were both negatively charged at the prevailing water pH (7–8). Fluorescence imaging for MP analysis, using a merged UV/blue light fluorograph, overestimated MP concentrations in greywater (hundreds of MPs per sample were identified by fluorescence imaging versus tens of MPs by μRaman spectroscopy) and would benefit from further improvement before it can be reliably applied as a cheaper and faster alternative methodology for MP analysis.

A cross-reactive imaging matrix of membrane protein profiling for single-cell analysis

Membrane proteins play a critical role in cellular processes such as chemical transport, signal transduction, and enzymatic catalysis. A comprehensive understanding of membrane protein types and accurately measuring their expression levels are essential for advancing cell biology. However, current methods often fail to characterize the types and amounts of multiple membrane proteins in specific cell types due to inherent measurement limitations. Here, we have developed a single-cell image analysis technique based on a cross-reactive imaging matrix using membrane protein tags. This technique allows quantitative and qualitative analysis of multiple membrane protein types and their expression levels within single cells. As a proof of concept, we used four specific DNA aptamers to tag four different membrane proteins in seven different cell types. The four proteins are MUC1, EGFR, HeR2, and TLR4. The seven cell lines are 3t3, 4T1, A549, CT26, HeLa, Hepa1–6, Raw, respectively. We captured a comprehensive image matrix using wide-field fluorescence imaging, allowing precise quantification of protein expression at the single-cell level. Our approach introduces a multi-labeling analysis method for membrane proteins and reveals the influence of membrane protein diversity on cell heterogeneity analysis. We found that the accuracy of cell heterogeneity recognition can be affected by adjusting the number of membrane proteins. As the diversity of membrane protein species increases, the accuracy of cell type recognition increases. Our approach facilitates the evaluation of heterogeneity in diverse cells, providing new insights into cellular diversity and a powerful tool for studying single-cell biology.

A chitosan-based near-infrared ratiometric fluorescent nanoprobe created by molecular assembly with applications in hypochlorous acid detection in live mouse

Hypochlorous acid (HClO/ClO−) is a key reactive oxidative species (ROS) in the body. The HClO/ClO− concentrations are imbalanced during cancer formation due to the ROS stress response. This paper introduces a novel chitosan-based self-calibration fluorescent nanoprobe (ChCyNil) constructed by molecular assembly for the ratiometric detection of HClO/ClO−. Two chromophores with different fluorescence characteristics and HClO/ClO− sensitivity were labeled on chitosan, and nanoparticles were prepared by a self-assembly strategy for HClO/ClO− detection. ChCyNil exhibits several advantages, such as dual near-infrared emissions at 670 nm and 845 nm, tunable fluorescence intensity, self-calibration fluorescence, and good biocompatibility, improving its accuracy in HClO/ClO− detection. Our study confirmed that ChCyNil exhibits a well-assembled spheroidal nanostructure and good photophysical properties in solution. The fluorescence imaging properties were further proved by detecting endogenous HClO/ClO− produced by LPS/PMA stimuli in cells and zebrafish. In addition, ChCyNil was used to detect the fluorescence behavior of HClO/ClO− in tumors of live mice. The successful design and fabrication of ChCyNil have presented a new strategy for constructing detection tools with improved fluorescence properties for HClO/ClO− in live animals.

Clinical features, surgical treatment strategy, and feasibility of minimally invasive surgery for synchronous and metachronous multiple colorectal cancers: A 14-year single-center experience.

Patients with a history of colorectal cancer (CRC) are at increased risk of developing secondary synchronous/metachronous CRCs. The role of minimally invasive surgery (MIS) for multiple CRCs remains unclear. This study aimed to evaluate the short-term outcomes of MIS in patients with multiple CRCs and elucidate their clinical characteristics. This retrospective study reviewed CRC patients who underwent MIS between 2010 and 2023. Multiple CRC cases were categorized into synchronous and metachronous cohorts. Demographics, pathological findings, and perioperative outcomes were analyzed. Propensity score matching (PSM) analysis was performed as appropriate. A total of 1,272 patients met the inclusion criteria, with 99 (7.8%) having multiple CRCs (75 synchronous and 24 metachronous). Multiple CRC patients had a higher prevalence of strong family history (8.1% vs. 1.0%, P < 0.001) and right-sided colon cancer (55.6% vs. 34.4%, P < 0.001) compared to solitary CRC patients. MSI-high/MMR-deficient status, including Lynch syndrome, was frequently observed among patients with multiple CRCs. Synchronous CRCs requiring double-anastomosis were associated with longer operation times (P = 0.03) and increased blood loss (P = 0.03) compared to those with a single-anastomosis. In the metachronous cohort, repeat operation patterns were categorized based on tumor location and sacrificed arteries. Preservation of the left-colic artery avoided extended colectomy in some patients. Patients with multiple CRC involving rectal cancer had a higher anastomotic leakage (AL) rate (17.6% vs. 5.7%, P < 0.01); however, this difference in AL rate disappeared after PSM (8.8% vs. 8.8%, P = 1.0). In patients with multiple CRCs, AL has not been observed ever since the indocyanine green fluorescence imaging was implemented. MIS is feasible for multiple CRCs, with perioperative outcomes comparable to those for solitary CRCs. Preservation of critical arteries may benefit patients at high risk of secondary CRCs, particularly those with a strong family history of CRC, right-sided tumors, or MSI-high/MMR-deficient profiles, including Lynch syndrome.

A New Vision in Endometriosis Surgery: The Impact of Near-Infrared Indocyanine Green Fluorescence Imaging

A New Vision in Endometriosis Surgery: The Impact of Near-Infrared Indocyanine Green Fluorescence Imaging

A terphenyl derivative-based colorimetric-fluorimetric probe for the detection of lysine and arginine and their bioimaging

Lysine (Lys) and arginine (Arg) play a crucial role in the human diets, medical diagnostics, and functional biomaterial synthesis. The imbalance of intake for these two amino acids causes various diseases. Although many analytical techniques have been reported for the detection of amino acids, there are still some issues such as the need for bulky instruments, professional operators, sensitivity to be improved, and real-time detection. Here, a novel colorimetric-fluorimetric probe based on a terphenyl derivative (TPT) has been synthesized for the precise detection of Lys and Arg. In the EtOH-H2O solution of TPT, the NH2 group at the chain end of Lys/Arg undergoes a nucleophilic addition reaction with CN groups of benzothiazole groups of the probe TPT, which breaks the initial long-conjugated system of the probe molecule. As a result, blue shifts can be observed for both UV–vis absorption spectra and fluorescence spectra, accompanying with color changes of the TPT solution. The UV–vis absorption peak of TPT solution shifts from ∼410 nm to ∼325 nm, and the solution color changes from light-yellow to colorless. The fluorescence emission shifts from ∼580 nm to ∼470 nm and the bright-yellow TPT solution changes to blue under the irradiation of 365 nm UV light. For colorimetric method, the limits of detection (LoD) are 0.82 μM and 0.90 μM for Lys and Arg, respectively. For fluorimetric method, they are 2.02 nM and 1.62 nM for Lys and Arg, respectively. In addition, TPT has good selectivity and anti-interference for Lys and Arg. The synthesized probe TPT has been successfully used for the precise detection of Lys and Arg in drugs and for fluorescence imaging of living cells. This work demonstrates that terphenyl-based derivatives are promising organic probes for the detection of Lys and Arg, providing a new way for designing other amino acids probes.

BrightMice: a low-cost do-it-yourself instrument, designed for in vivo fluorescence mouse imaging.

In vivo fluorescent imaging represents a potent means for real-time probe quantification, facilitating insights into disease pathophysiology and therapeutic responses. Nonetheless, accurate signal quantification remains challenging due to inherent factors like light scattering and tissue absorption. Existing imaging systems, though sophisticated, often entail high costs and are typically restricted to well-funded laboratory settings. This study introduces BrightMice, an innovative in vivo fluorescent imaging system that harnesses 3D printing and consumer-grade digital cameras. Tailored for various fluorophores such as EYFP and E2-crimson, the system showcases both adaptability and effectiveness in detecting in vivo fluorescent signals in several reporter mouse strains. Comparative analyses against commercial instruments confirm BrightMice's sensitivity and underscore its potential to democratize in vivo fluorescence imaging. By providing a cost-effective and accessible solution, BrightMice stands to benefit diverse research environments.

Use of indocyanine green-human serum albumin complexes in fluorescence image-guided laparoscopic anatomical liver resection: a case series study (with video)

BackgroundThis study aimed to investigate the feasibility and efficacy of near-infrared fluorescence-guided laparoscopic anatomical hepatectomy (LAH) using a novel indocyanine green (ICG)-human serum albumin complex (HSA) in patients with hepatocellular carcinoma.MethodsClinical data of hepatocellular carcinoma patients who underwent ICG-HSA fluorescence-guided LAH at our center from January 2024 to April 2024 were prospectively collected and analyzed. Ultraviolet absorption spectroscopy was used to test the absorption and stability of ICG-HSA complex solutions under different conditions. After determining the optimal ratio, the complex was administered intravenously during surgery to perform negative staining via Glissonean pedicle isolation. LAH was performed along the fluorescence-demarcated boundaries.ResultsThirty-one patients were included (24 men; mean age, 54.61 ± 13.54 years). The median maximum tumor diameter was 2.80 (interquartile range [IQR], 2.00–4.00) cm. S8 segmentectomy (22.6%) and right posterior segmentectomy (19.4%) were the most common resections performed. Successful fluorescence negative staining was achieved in all patients using ICG and HSA at a 1:6 molar ratio at room temperature. Mean operation time was 297.58 ± 85.53 min, Median intraoperative blood loss was 100.0 mL (IQR, 50.0–200.0). The median surgical margin distance was 0.90 cm (IQR, 0.40–1.50). The postoperative complication rate was 45.2% (35.5% Clavien–Dindo grade I and 9.7% grade II). The median length of hospital stay was 5.0 days (IQR, 4.0–5.0).ConclusionICG-HSA-assisted LAH is safe and feasible. Compared with free ICG, the novel ICG-HSA complex exhibits better optical properties and in vivo stability, which can improve the accuracy of intraoperative liver segment localization and optimize the anatomical dissection plane. It has the potential to become an ideal fluorescent imaging agent for anatomical hepatectomy.Graphical abstractA In vitro binding experiments of indocyanine green (ICG) and human serum albumin (HSA) suggested that a 1:6 molar ratio and a configuration temperature of 37°C were optimal. B During surgery, after extrahepatic Glissonean isolation of the liver pedicle planned for resection, the ICG-HSA complex was administered through a peripheral vein using an infusion pump. C ICG-HSA metabolism comprises three stages: intravascular, liver, and systemic clearance. D The actual intraoperative fluorescence imaging and guidance effects of ICG-HSA in laparoscopic fluorescence negative-staining right hemihepatectomy.

Safety Evaluation of Carbon Dots in UM-UC-5 and A549 Cells for Biomedical Applications

Backgroung: The rising complexity and associated side effects of cancer treatments highlight the need for safer and more effective therapeutic agents. Carbon-based nanomaterials such as CDs have been gaining prominence for their unique characteristics, opening avenues for diverse applications such as fluorescence imaging, drug and gene transport, controlled drug delivery, medical diagnosis, and biosensing. Despite promising advancements in research, it remains imperative to scrutinize the properties and potential cytotoxicity of newly developed CDs, ensuring their viability for these applications. Methods: We synthesized four N-doped CDs through a hydrothermal method. Cell viability assays were conducted on A549 and UM-UC-5 cancer cells at a range of concentrations and incubation times, both individually and with the chemotherapeutic agent 5-fluorouracil (5-FU). Results: The obtained results suggest that the newly developed CDs exhibit suitability for applications such as bioimaging, as no significant impact on cell viability was observed for CDs alone.

Optimization of GEM detectors for applications in X-ray fluorescence imaging

In this work a set of simulations that aim at the optimization of Micropattern Gaseous Detectors (MPGD) for applications in X-ray fluorescence imaging in the energy range of 3–30keV is presented. By studying the statistical distribution of electrons from interactions of X-rays with gases, the energy resolution limits after charge multiplication for 6keV X-ray photons in Ar/CO2(70/30) and Kr/CO2(90/10) were calculated, resulting in energy resolutions of 15.4(4)% and 14.6(2)% respectively. These two mixtures were studied in simulations to evaluate the advantages of using krypton-based mixtures to reduce the presence of escape peaks in fluorescence spectra. A model to evaluate the X-ray fluorescence from the conductive materials inside the detectors was implemented, serving as a tool to estimate the extent of contamination of fluorescence spectra when using copper or aluminum layers in the material composition of MPGDs.

A ratiometric fluorescent probe based on phenothiazine and quinazolinone for rapid detection of ClO− and its application

A novel fluorescent probe (BPQ-1) with large conjugated system (phenothiazine was conjugated with quinazolinone) was synthesized. BPQ-1 showed a remarkable ratiometric fluorescence response to ClO− within 10 s, and achieved large stokes shift (168 nm), high sensitivity (LOD = 16.7 nM) and excellent selectivity. Among the analytes to be detected, there is a significant change from bright yellow fluorescence to cyan fluorescence only when ClO− is added to BPQ-1. Through density functional theory calculation and mass spectrometry experiments, the fluorescence response mechanisms of the BPQ-1 are given. In addition, BPQ-1 has been successfully applied to the detection of ClO− in real water samples and test strips for detecting ClO− with different concentrations have been prepared. In particular, the probe has been successfully applied to the fluorescence imaging of exogenous ClO− in living cells.

Hyaluronic acid-zein shell-core biopolymer nanoparticles enhance hepatocellular carcinoma therapy of celastrol via CD44-mediated cellular uptake

Low concentrations or limited residence times in tumor tissues, making celastrol (Cel) difficult to exert significant therapeutic effects. Thus, we developed Zein/hyaluronic acid core-shell nanoparticles (Cel/Zein@HA NPs) for active targeted delivery of Cel via CD44 receptor over-expression on cancer cells, which may strengthen the therapeutic efficacy of Cel and improve delivery targeting. Cel-loaded Zein nanoparticles (core), are elegantly enveloped by a hydrophilic HA coating that forms the shell, resulting in significantly improved encapsulation efficiency and ensured good stability. The cellular uptake of Cel/Zein@HA NPs in HepG2 cells was 1.57-fold higher than nontargeting Cel/Zein NPs. Near-infrared fluorescence imaging confirmed the accumulation of Cel/Zein@HA NPs in H22 liver cancer tumors in mice, resulting in effective antitumor effects and good biosafety. Besides, in vitro and in vivo experiments showed that compared with Cel/Zein NPs, Cel/Zein@HA NPs had more efficient inhibitory effect on tumor proliferation and lower systemic toxicity. Further studies revealed that Cel/Zein@HA NPs induced apoptosis in hepatocellular carcinoma cells by modulating Bax and Bcl-2 expression, while also inhibiting tumor angiogenesis by decreasing CD31 and VEGF levels. Overall, this study presents a promising strategy for enhancing targeted liver cancer therapy through the utilization of biopolymer nanoparticle-based nano-pharmaceuticals that facilitate CD44-mediated cellular uptake.

Evaluating waterlogging stress response and recovery in barley (Hordeum vulgare L.): an image-based phenotyping approach

Waterlogging is expected to become a more prominent yield restricting stress for barley as rainfall frequency is increasing in many regions due to climate change. The duration of waterlogging events in the field is highly variable throughout the season, and this variation is also observed in experimental waterlogging studies. Such variety of protocols make intricate physiological responses challenging to assess and quantify. To assess barley waterlogging tolerance in controlled conditions, we present an optimal duration and setup of simulated waterlogging stress using image-based phenotyping. Six protocols durations, 5, 10, and 14 days of stress with and without seven days of recovery, were tested. To quantify the physiological effects of waterlogging on growth and greenness, we used top down and side view RGB (Red-Green-Blue) images. These images were taken daily throughout each of the protocols using the PSI PlantScreen™ imaging platform. Two genotypes of two-row spring barley, grown in glasshouse conditions, were subjected to each of the six protocols, with stress being imposed at the three-leaf stage. Shoot biomass and root imaging data were analysed to determine the optimal stress protocol duration, as well as to quantify the growth and morphometric changes of barley in response to waterlogging stress. Our time-series results show a significant growth reduction and alteration of greenness, allowing us to determine an optimal protocol duration of 14 days of stress and seven days of recovery for controlled conditions. Moreover, to confirm the reproducibility of this protocol, we conducted the same experiment in a different facility equipped with RGB and chlorophyll fluorescence imaging sensors. Our results demonstrate that the selected protocol enables the assessment of genotypic differences, which allow us to further determine tolerance responses in a glasshouse environment. Altogether, this work presents a new and reproducible image-based protocol to assess early stage waterlogging tolerance, empowering a precise quantification of waterlogging stress relevant markers such as greenness, Fv/Fm and growth rates.

Immunohistochemical Evaluation of Cathepsin B, L, and S Expression in Breast Cancer Patients.

Cysteine cathepsins are proteases that play a role in normal cellular physiology and neoplastic transformation. Elevated expression and enzymatic activity of cathepsins in breast cancer (BCa) indicates their potential as a target for tumor imaging. In particular cathepsin B (CTSB), L (CTSL), and S (CTSS) are used as targets for near-infrared (NIR) fluorescence imaging (FI), a technique that allows real-time intraoperative tumor visualization and resection margin assessment. Therefore, this immunohistochemical study explores CTSB, CTSL, and CTSS expression levels in a large breast cancer patient cohort, to investigate in which BCa patients the use of cathepsin-targeted NIR FI may have added value. Protein expression was analyzed in tumor tissue microarrays (TMA) of BCa patients using immunohistochemistry and quantified as a total immunostaining score (TIS), ranging from 0-12. In total, the tissues of 557 BCa patients were included in the TMA. CTSB, CTSL, and CTSS were successfully scored in respectively 340, 373 and 252 tumors. All tumors showed CTSB, CTSL, and/or CTSS expression to some extent (TIS > 0). CTSB, CTSL, and CTSS expression was scored as high (TIS > 6) in respectively 28%, 80%, and 18% of tumors. In 89% of the tumors scored for all three cathepsins, the expression level of one or more of these proteases was scored as high (TIS > 6). Tumors showed significantly higher cathepsin expression levels with advancing Bloom-Richardson grade (p < 0.05). Cathepsin expression was highest in estrogen receptor (ER)-negative, human epidermal growth factor receptor 2(HER2)-positive and triple-negative (TN) tumors. There was no significant difference in cathepsin expression between tumors that were treated with neoadjuvant systemic therapy and tumors that were not. The expression of at least one of the cysteine cathepsins B, L and S in all breast tumor tissues tested suggests that cathepsin-activatable imaging agents with broad reactivity for these three proteases will likely be effective in the vast majority of breast cancer patients, regardless of molecular subtype and treatment status. Patients with high grade ER-negative, HER2-positive, or TN tumors might show higher imaging signals.

Indocyanine green and methylene blue dye guided sentinel lymph node biopsy in patients with penile cancer (PeCa): results of 50 inguinal basins assessed at a single institution in India.

The primary aim of this study was to validate the reliability, sensitivity and safety profile of novel combination of ICG- methylene blue dye as an SN tracer for PeCa. This is a validation and non-randomised prospective observational study involving 25 patients (50 inguinal basins) who underwent SLNB where in ICG and methylene blue were used for localisation. The patients with clinically node negative groins were recruited in the study. SNs were identified intraoperatively using near infrared fluorescence imaging (NIRF Imaging system, SPY-PHI, Stryker, Sweden) and blue dye. The numbers of SNs identified by each tracer and the rates of complications and nodal recurrence during the followup. Overall 137 SNs were identified intraoperatively. Among the 137 SNs excised fluorescence, blue dye and Combined (blue + green) identified 57(41.6%), 27 (19.7%), and 51 (37.2%), respectively. The average number of SLNs removed per patient was 5 (range, 1-11) with sentinel lymph nodes detection rate at 94% (47/50). Seven patients had malignancy on SLNB and underwent ipsilateral radical inguinal lymphadenectomy. One patient had false negative SN and positive node in modified inguinal lymphadenectomy specimen. No adverse events were observed in all cases. The Novel combination of ICG fluorescence-Methylene blue dye technique is simple, reliable and safe. Moreover, it demonstrates a high SLN detection rate with a low false-negative rate, and it avoids radiation exposure.

3-Benzylmenadiones and their Heteroaromatic Analogues Target the Apicoplast of Apicomplexa Parasites: Synthesis and Bioimaging Studies.

The apicoplast is an essential organelle for the viability of apicomplexan parasites Plasmodium falciparum or Toxoplasma gondii, which has been proposed as a suitable drug target for the development of new antiplasmodial drug-candidates. Plasmodione, an antimalarial redox-active lead drug is active at low nM concentrations on several blood stages of Plasmodiumsuch as early rings and gametocytes. Nevertheless, its precise biological targets remain unknown. Here, we described the synthesis and the evaluation of new heteroaromatic analogues of plasmodione, active on asexual blood P. falciparum stages and T. gondii tachyzoites. Using a bioimaging-based analysis, we followed the morphological alterations of T. gondii tachyzoites and revealed a specific loss of the apicoplast upon drug treatment. Lipidomic and fluxomic analyses determined that drug treatment severely impacts apicoplast-hosted FASII activity in T. gondii tachyzoites, further supporting that the apicoplast is a primary target of plasmodione analogues. To follow the drug localization, "clickable" analogues of plasmodione were designed as tools for fluorescence imaging through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Short-time incubation of two probes with P. falciparum trophozoites and T. gondii tachyzoites showed that the clicked products localize within, or in the vicinity of, the apicoplast of both Apicomplexa parasites. In P. falciparum, the fluorescence signal was also associated with the mitochondrion, suggesting that bioactivation and activity of plasmodione and related analogues are potentially associated with these two organelles in malaria parasites.

EDB-FN-targeted probes for near infrared fluorescent imaging and positron emission tomography imaging of breast cancer in mice.

The extra domain B splice variant of fibronectin (EDB-FN), which is overexpressed in several cancers, is an approved diagnostic and therapeutic target of cancers. The aim of this study was to evaluate the EDB-FN-targeting peptide EDBp as a noninvasive imaging modality for molecular imaging of breast cancer in mice. Western blot, flow cytometry and immunofluorescence were used to assess the expression level of EDB-FN and its binding to EDRp in MCF7, SKBR3, 4T1, EMT6, MDA-MB-231 and MDA-MB-453 cells. Establishment MDA-MB-231-luc cells-based subcutaneous tumor model mice or pulmonary metastasis model mice. The EDRp molecular probes to perform fluorescent probes for near-infrared fluorescence (NIRF)·and PET imaging of model mice. Our results demonstrate that EDBp-Cy5 had a strong binding ability to the MDA-MB-231 cells and exhibited specific tumor accumulation in MDA-MB-231 subcutaneous and pulmonary metastasis model mice. Importantly, the EDBp peptide-based radiotracer [18F]-AlF-NOTA-EDBp provided excellent diagnostic value for positron emission tomography (PET) imaging of breast cancer, especially in subcutaneous model mice. The uptake of [18F]-AlF-NOTA-EDBp in subcutaneous tumors (6.53 ± 0.89%, ID/g) was unexpectedly higher than that in the kidney (4.96 ± 0.20, %ID/g). The high tumor uptake of these probes in mice suggests their potential for application in imaging of EDB-FN-positive breast cancer for disease staging of regional and distant metastases.

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is, however, a growing gap between the state of the art and what is accessible to biologists, biochemists, medical researchers, or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed microspectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy, time-correlated single-photon counting and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines, are provided, to allow non-optics specialists to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and open-source Brick-MIC platform will be a valuable tool for many laboratories worldwide.