Fluorescence Intensity Curve Research Articles

Abstract PO4-07-07: The construction and clinical research value of the prediction model of sentinel lymph node metastasis based on indocyanine green fluorescence quantification in breast cancer

Abstract Objective: To establish a prediction model of sentinel lymph node metastasis by quantitative indocyanine green fluorescence analysis in breast cancer. Methods: From May 2023 to June 2023, 20 patients with breast cancer underwent modified radical mastectomy in the General Surgery Department of Jiangsu Provincial People's Hospital were chosen for research and analysis. Sentinel lymph node were taken from all the patients. The sentinel lymph nodes were located by using the double staining method of subcutaneous injection of Methylene blue and intravenous injection of indocyanine green. The postoperative pathology is used as the standard for evaluating the presence or absence of metastatic cancer in lymph nodes. At the same time, the transcutaneous fluorescence of sentinel lymph node was recorded and the peak time of fluorescence intensity curve was plotted. To establish a logistic diagnostic prediction model and fluorescence threshold, the sentinel lymph node was incised during operation and the fluorescence of sentinel lymph node was recorded again. The criteria for ICG positive determination are determined by the Youden index, which was used to judge the lymph nodes above the threshold in the Logistic diagnostic prediction model. Lymph nodes below the threshold determined in the Logistic diagnostic prediction model are the criteria for ICG negative determination. Cases with less than 3 sentinel lymph node were excluded. Results: 19 cases were ultimately selected. Methylene blue staining data was taking as the judgment standard of sentinel lymph node. Fluorescence imaging was 100% in 14 patients, 66.7% (2/3) in 1 patient, and 125%-150% (5/4-6/4) in 4 patients. Fluorescence quantification values below 2.91 are negative lymph nodes, and values above 4.69 are positive lymph nodes. Moreover, caution is required between 2.91 and 4.69. Combining with the fluorescence dynamic attenuation curve, the positive group has a plateau curve, while the negative group mostly has a decline curve. Combining fluorescence quantification with fluorescence dynamic attenuation curve, the diagnostic rate of positive lymph nodes has a lower false negative rate compared to simple fluorescence quantification and fluorescence dynamic attenuation curve. Conclusion: The quantitative difference value of indocyanine green fluorescence angiography can be used to predict the positive metastasis of sentinel lymph node in breast cancer, making it an attempt to exempt sentinel lymph node biopsy. Citation Format: Yang Wu, Wei Zhang, Jinhai Tang. The construction and clinical research value of the prediction model of sentinel lymph node metastasis based on indocyanine green fluorescence quantification in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-07-07.

Characteristics of the photosynthetic activity of corn using different drugs under stress

Goal. To study under controlled conditions the effect of hormonal preparations based on humus extracts and anti-stress stimulants with a high content of amino acids and biologically active substances on the development of corn under conditions of stress, as well as their effect on the photosynthetic activity of plants.
 Methods. The research was carried out in a climatic chamber (a room with adjustable control of temperature, daylight and air humidity) of the Ukrainian research plant quarantine station of the Institute of Plant Protection of the National Academy of Sciences. Photosynthetic activity was measured using a portable fluorometer «Floratest». During the growing season, two foliar feedings were carried out in the phase of development of corn VVSN 12, the first application of drugs during the growing season — VVSN 13, — the second application of drugs during the vegetation period — VVSN 14.
 Results. The analysis of the induction curves of chlorophyll fluorescence and the indicators of the maximum background fluorescence showed a difference between the plants that were under soil moisture of 70% and 50% RH. When corn plants were in a state close to stress for a long time, the difference between the curves of chlorophyll fluorescence intensity between the two controls increased. The use of the studied drugs provided the finding of the indicator of the maximum value of fluorescence induction above the dry control in all variants of the experiment. Best results provided: preparation B (high concentrated suspension for nutrition and plant stress decreasing based upon the humus extract. They contented N — 150 g/l, K2O — 300 g/l, Mg — 30 g/l, metal cations in В, Cu, Fe, Mn and Zn with additional adhesive effects and surfactant), at rate of application 0.5 and 0.25 l/ha; Axofol (high concentrated suspension from extract of brown algae Ascophyllum nodosum with microelements, B — 38.1 g/l, Mn — 10.2 g/l, Zn — 6.4 g/l) at rate of application 1.0 and 0.5 l/ha.
 Conclusions. As the time of corn plants being in a state close to stress increases, the difference between the curve of chlorophyll fluorescence intensity in the wet control and the dry control increases. The use of the studied drugs provided the finding of the indicator of the maximum value of fluorescence induction above the dry control in all variants of the experiment. The use of the researched preparations in conditions of soil moisture of 50% ensured the improvement of vegetation indicators of plants.

Intraoperative indocyanine green fluorescence imaging to predict early hepatic arterial complications after liver transplantation.

The purpose of this study was to propose an innovative intraoperative criterion in a liver transplantation setting that would judge arterial flow abnormality that may lead to early hepatic arterial occlusion, that is, thrombosis or stenosis, when left untreated and to carry out reanastomosis. After liver graft implantation, and after ensuring that there is no abnormality on the Doppler ultrasound (qualitative and quantitative assessment), we intraoperatively injected indocyanine green dye (0.01mg/Kg), and we quantified the fluorescence signal at the graft pedicle using ImageJ software. From the obtained images of 89 adult patients transplanted in our center between September 2017 and April 2019, we constructed fluorescence intensity curves of the hepatic arterial signal and examined their relationship with the occurrence of early hepatic arterial occlusion (thrombosis or stenosis). Early hepatic arterial occlusion occurred in 7 patients (7.8%), including 3 thrombosis and 4 stenosis. Among various parameters of the flow intensity curve analyzed, the ratio of peak to plateau fluorescence intensity and the jagged wave pattern at the plateau phase were closely associated with this dreaded event. By combining the ratio of peak to plateau at 0.275 and a jagged wave, we best predicted the occurrence of early hepatic arterial occlusion and thrombosis, with sensitivity/specificity of 0.86/0.98 and 1.00/0.94, respectively. Through a simple composite parameter, the indocyanine green fluorescence imaging system is an additional and promising intraoperative modality for identifying recipients of transplant at high risk of developing early hepatic arterial occlusion. This tool could assist the surgeon in the decision to redo the anastomosis despite normal Doppler ultrasonography.

Technical and functional design considerations for a real-world interpretable AI solution for NIR perfusion analysis (including cancer)

Near infrared (NIR) analysis of tissue perfusion via indocyanine green fluorescence assessment is performed clinically during surgery for a range of indications. Its usefulness can potentially be further enhanced through the application of interpretable artificial intelligence (AI) methods to improve dynamic interpretation accuracy in these and also open new applications. While its main use currently is for perfusion assessment as a tissue health check prior to performing an anastomosis, there is increasing interest in using fluorophores for cancer detection during surgical interventions with most research being based on the paradigm of static imaging for fluorophore uptake hours after preoperative dosing. Although some image boosting and relative estimation of fluorescence signals is already inbuilt into commercial NIR systems, fuller implementation of AI methods can enable actionable predictions especially when applied during the dynamic, early inflow-outflow phase that occurs seconds to minutes after ICG (or indeed other fluorophore) administration. Already research has shown that such methods can accurately differentiate cancer from benign tissue in the operating theatre in real time in principle based on their differential signalling and could be useful for tissue perfusion classification more generally. This can be achieved through the generation of fluorescence intensity curves from an intra-operative NIR video stream. These curves are processed to adjust for image disturbances and curve features known to be influential in tissue characterisation are extracted. Existing machine learning based classifiers can then use these features to classify the tissue in question according to prior training sets. The use of this interpretable methodology enables accurate classification algorithms to be built with modest training sets in comparison to those required for deep learning modelling in addition to achieving compliance with medical device regulations. Integration of the multiple algorithms required to achieve this classification into a desktop application or medical device could make the use of this method accessible and useful to (as well as useable by) surgeons without prior training in computer technology. This document details some technical and functional design considerations underlying such a novel recommender system to advance the foundational concept and methodology as software as medical device for in situ cancer characterisation with relevance more broadly also to other tissue perfusion applications.

Multi-parametric Retinal Microvascular Functional Perfusion Imaging Based on Dynamic Fundus Fluorescence Angiography.

Retinal microvascular disease has caused serious visual impairment widely in the world, which can be hopefully prevented via early and precision microvascular hemodynamic diagnosis. Due to artifacts from choroidal microvessels and tiny movements, current fundus microvascular imaging techniques including fundus fluorescein angiography (FFA) precisely identify retinal microvascular microstructural damage and abnormal hemodynamic changes difficulty, especially in the early stage. Therefore, this study proposes an FFA-based multi-parametric retinal microvascular functional perfusion imaging (RM-FPI) scheme to assess the microstructural damage and quantify its hemodynamic distribution precisely. Herein, a spatiotemporal filter based on singular value decomposition combined with a lognormal fitting model was used to remove the above artifacts. Dynamic FFAs of patients (n = 7) were collected first. The retinal time fluorescence intensity curves were extracted and the corresponding perfusion parameters were estimated after decomposition filtering and model fitting. Compared with in vivo results without filtering and fitting, the signal-to-clutter ratio of retinal perfusion curves, average contrast, and resolution of RM-FPI were up to 7.32 ± 0.43 dB, 14.34 ± 0.24 dB, and 11.0 ± 2.0 μm, respectively. RM-FPI imaged retinal microvascular distribution and quantified its spatial hemodynamic changes, which further characterized the parabolic distribution of local blood flow within diameters ranging from 9 to 400 μm. Finally, RM-FPI was used to quantify, visualize, and diagnose the retinal hemodynamics of retinal vein occlusion from mild to severe. Therefore, this study provided a scheme for early and precision diagnosis of retinal microvascular disease, which might be beneficial in preventing its development.

Combination of intraoperative indocyanine green video-angiography FLOW 800 and computed tomography perfusion to assess the risk of cerebral hyperperfusion syndrome in chronic internal carotid artery occlusion patients after revascularization surgery.

To study the changes of corticocerebral hemodynamics in surgical area and postoperative hyperperfusion syndrome in patients with chronic internal carotid artery occlusion (CICAO) by intraoperative indocyanine green videoangiography (ICGA)-FLOW 800 and CT perfusion after superficial temporal artery (STA)-middle cerebral artery (MCA) bypass surgery. From October 2019 to January 2021, 77 patients diagnosed with CICAO underwent direct bypass surgery at Huadong hospital (affiliated with Fudan University) were enrolled. Regions of interest (ROIs) at STA, proximal MCA (PMCA), distal MCA (DMCA), cortical blood capillary (CBC), and cortical vein (CV) were identified after anastomosis by ICGV-FLOW 800 including peak fluorescence intensity (PFI), time to peak (TTP), and area under the time curve (AUC) of fluorescence intensity. All patients underwent perfusion-weighted CT before bypass surgery and those patients with HPS were verified by CTP after bypass. 14 patients with HPS were verified by perfusion-weighted CT after bypass. In HPS group, the AUCTTP of DMCA was significantly larger (T = -3.301, p = 0.004) and TTP of CBC was shorter (T = -2.929, p = 0.005) than patients in non-HPS group. The larger AUCTTP of DMCA (OR = 3.024, 95%CI 1.390-6.578, p = 0.0050) was an independent risk factor by further multivariate logistic regression analysis. The hemodynamic changes of cortical vessels during STA-MCA bypass surgery could be recorded accurately by ICGV-FLOW 800. Furthermore, the increased AUCTTP of DMCA and shorter TTP of CBC may be potential risk factors of HPS.

Continuous Online Titer Monitoring in CHO Cell Culture Supernatant Using a Herringbone Nanofluidic Filter Array.

Online monitoring of monoclonal antibody product titers throughout biologics process development and production enables rapid bioprocess decision-making and process optimization. Conventional analytical methods, including high-performance liquid chromatography and turbidimetry, typically require interfacing with an automated sampling system capable of online sampling and fractionation, which suffers from increased cost, a higher risk of failure, and a higher mechanical complexity of the system. In this study, a novel nanofluidic system for continuous direct (no sample preparation) IgG titer measurements was investigated. Tumor necrosis factor α (TNF-α), conjugated with fluorophores, was utilized as a selective binder for adalimumab in the unprocessed cell culture supernatant. The nanofluidic device can separate the bound complex from unbound TNF-α and selectively concentrate the bound complex for high-sensitivity detection. Based on the fluorescence intensity from the concentrated bound complex, a fluorescence intensity versus titer curve can be generated, which was used to determine the titer of samples from filtered, unpurified Chinese hamster ovary cell cultures continuously. The system performed direct monitoring of IgG titers with nanomolar resolution and showed a good correlation with the biolayer interferometry assays. Furthermore, by variation of the concentration of the indicator (TNF-α), the dynamic range of the system can be tuned and further expanded.

Spatiotemporally quantitative in vivo imaging of mitochondrial fatty acid β-oxidation at cellular-level resolution in mice.

Mitochondrial fatty acid β-oxidation (FAO) plays a key role in energy homeostasis. Several FAO evaluation methods are currently available, but they are not necessarily suitable for capturing the dynamics of FAO in vivo at a cellular-level spatial resolution and seconds-level time resolution. FAOBlue is a coumarin-based probe that undergoes β-oxidation to produce a fluorescent substrate, 7-hydroxycoumarin-3-(N-(2-hydroxyethyl))-carboxamide (7-HC). After confirming that 7-HC could be specifically detected using multiphoton microscopy at excitation/emission wavelength = 820/415-485 nm, wild-type C57BL/6 mice were randomly divided into control, pemafibrate, fasting (24 or 72 h), and etomoxir groups. These mice received a single intravenous injection of FAOBlue. FAO activities in the liver of these mice were visualized using multiphoton microscopy at 4.2 s/frame. These approaches could visualize the difference in FAO activities between periportal and pericentral hepatocytes in the control, pemafibrate, and fasting groups. FAO velocity, which was expressed by the maximum slope of the fluorescence intensity curve, was accelerated in the pemafibrate and 72-h fasting groups both in the periportal and the pericentral hepatocytes in comparison with the control group. Our approach revealed differences in the FAO activation mode by the two stimuli, i.e., pemafibrate and fasting, with pemafibrate accelerating the time of first detection of FAO-derived fluorescence. No increase in the fluorescence was observed in etomoxir-pretreated mice, confirming that FAOBlue specifically detected FAO in vivo. Thus, FAOBlue is useful for visualizing in vivo liver FAO dynamics at the single-cell-level spatial resolution and seconds-level time resolution.NEW & NOTEWORTHY Fatty acid β-oxidation (FAO) plays a key role in energy homeostasis. Here, the authors established a strategy for visualizing FAO activity in vivo at the cellular-level spatial resolution and seconds-level time resolution in mice. Quantitative analysis revealed spatiotemporal heterogeneity in hepatic FAO dynamics. Our method is widely applicable because it is simple and uses a multiphoton microscope to observe the FAOBlue-injected mice.

Ex Vivo Thrombolysis to Salvage Free Flaps Using Machine Perfusion: A Pilot Study in a Porcine Model

Mechanical evacuation of capillary thrombi in free flaps is difficult, and often requires thrombolytic therapy. Utilizing machine perfusion systems, the possibility rises to salvage free flaps ex vivo by administering high doses of thrombolytic agents. The primary aim of this pilot study in a porcine model is to investigate the feasibility of ex vivo thrombolysis using an extracorporeal perfusion machine. A model of stasis-induced thrombosis was used in 12 free rectus abdominis flaps harvested from six Dutch Landrace pigs. Compromised flaps were ex vivo perfused with University of Wisconsin preservation solution and treated according to the following study groups: (1) 1 mg of tissue plasminogen activator (t-PA) as additive, (2) 3 mg of t-PA as an additive, and (3) no thrombolytic additive. Microcirculation was assessed using near-infrared fluorescence angiography. Pedicled abdominal flaps were created and thrombus formation was successfully induced. Eleven abdominal flaps were perfused using the modified heart-lung machine setup. Near-infrared fluorescence angiography showed delayed or no filling was noted in the control group. In comparison, the flaps which were perfused with 1 mg t-PA or 3 mg t-PA as additive showed increased fluorescence intensity curves. This pilot study in a porcine model presents a reliable and reproductive stasis-induced thrombosis model in free flaps. By adding t-PA to a custom-made extracorporeal perfusion system, the indocyanine green fluorescence intensity curves increased of all flaps that were perfused with different dosages of t-PA as additives, indicating restoration of capillary pressure and microcirculatory inflow.

An Image Analysis Pipeline for Quantifying the Features of Fluorescently-Labeled Biomolecular Condensates in Cells.

Biomolecular condensates are cellular organelles formed through liquid-liquid phase separation (LLPS) that play critical roles in cellular functions including signaling, transcription, translation, and stress response. Importantly, condensate misregulation is associated with human diseases, including neurodegeneration and cancer among others. When condensate-forming biomolecules are fluorescently-labeled and examined with fluorescence microscopy they appear as illuminated foci, or puncta, in cells. Puncta features such as number, volume, shape, location, and concentration of biomolecular species within them are influenced by the thermodynamics of biomolecular interactions that underlie LLPS. Quantification of puncta features enables evaluation of the thermodynamic driving force for LLPS and facilitates quantitative comparisons of puncta formed under different cellular conditions or by different biomolecules. Our work on nucleoporin 98 (NUP98) fusion oncoproteins (FOs) associated with pediatric leukemia inspired us to develop an objective and reliable computational approach for such analyses. The NUP98-HOXA9 FO forms hundreds of punctate transcriptional condensates in cells, leading to hematopoietic cell transformation and leukemogenesis. To quantify the features of these puncta and derive the associated thermodynamic parameters, we developed a live-cell fluorescence microscopy image processing pipeline based on existing methodologies and open-source tools. The pipeline quantifies the numbers and volumes of puncta and fluorescence intensities of the fluorescently-labeled biomolecule(s) within them and generates reports of their features for hundreds of cells. Using a standard curve of fluorescence intensity versus protein concentration, the pipeline determines the apparent molar concentration of fluorescently-labeled biomolecules within and outside of puncta and calculates the partition coefficient (Kp) and Gibbs free energy of transfer (ΔGTr), which quantify the favorability of a labeled biomolecule partitioning into puncta. In addition, we provide a library of R functions for statistical analysis of the extracted measurements for certain experimental designs. The source code, analysis notebooks, and test data for the Punctatools pipeline are available on GitHub: https://github.com/stjude/punctatools. Here, we provide a protocol for applying our Punctatools pipeline to extract puncta features from fluorescence microscopy images of cells.

Quantification of gastric tube perfusion following esophagectomy using fluorescence imaging with indocyanine green

IntroductionAnastomotic leakage (AL) remains a prevalent and life-threatening complication after esophagectomy. Gastric tube perfusion assessment using indocyanine green fluorescence imaging (ICG-FI) has been published in several studies and appears to be a promising tool to reduce AL rates by changing the surgical approach, namely by an intraoperative evaluation of the anastomosis localization.MethodsIn this study, gastric tube perfusion was quantified by using ICG-FI in 20 high-risk patients undergoing esophagectomy. From a time-dependent fluorescence intensity curve, the following three parameters were evaluated: slope of fluorescence intensity (SFI), background subtracted peak fluorescence intensity (BSFI), and time to slope (TTS).ResultsThe values between pyloric region and tip showed a similar downward trend and SFI and BSFI significantly correlated with the distance to the pyloric region. SFI and BSFI were significantly decreased at the tip of the gastric tube. The placement of anastomosis in an area with homogenous fluorescence pattern was correlated with no AL in 92.9% of cases. An inhomogeneous fluorescence pattern at anastomotic site was a risk factor for the occurrence of an AL (p < 0.05). Reduction of perfusion up to 32% using SFI and up to 23% using BSFI was not associated with AL.ConclusionICG-FI can be used to quantify the gastric tube perfusion by calculating SFI, BSFI, and TTS. The anastomosis should be created in areas with homogeneous fluorescence pattern. A reduction in blood flow of up to 32% can be accepted without causing an increased rate of insufficiency.

Near-infrared fluorescence imaging with indocyanine green for assessment of donor livers in a rat model of ischemia\u2013reperfusion

BackgroundAlthough marginal donor livers expand the donor pool, an ideal method for quantitatively evaluating the quality of donor livers has not been developed. This study aimed to explore the feasibility of indocyanine green (ICG) fluorescence imaging for estimating liver function in an ischemia–reperfusion model.MethodsForty-eight rats were randomly and evenly divided into 8 groups: the control group and the experimental groups (I-VII). The portal vein blocking period was 0 min, 10 min, 20 min, 30 min, 40 min, 50 min and 60 min. After blood flow was reestablished and the hemodynamics stabilized, ICG was injected through the dorsal penile vein as a bolus, and the fluorescence signal was recorded for 30 min in real time. The fluorescence intensity (FI) curve of the liver was fitted with an asymptotic regression model. Fresh liver tissues and serum were obtained from the middle lobe of the liver on postoperative day (POD) 1 and POD 7 for histopathological evaluation and liver function tests.ResultsThe growth rate of the FI curve, parameter b3, decreased from groups I to VII. According to the two sudden changes in b3 (20 min, 50 min), the experimental groups could be classified into 3 groups (A, B and C). Hepatocytes in groups I-II showed slight edema, group III began to show obvious hepatocyte edema and vacuolar degeneration, and in groups VI-VII, severe hepatocyte degeneration, necrosis and large inflammatory cell infiltration were observed. Suzuki’s scores in the 3 groups were also significantly different (P < 0.01). At the same time, the serum liver function in the experimental groups showed a significant increase on POD 1 and a decrease on POD 7. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TB) levels of groups A, B, and C were significantly different on POD 1 (P < 0.05), and the ALT and direct bilirubin (DB) levels were significantly different on POD 7 (P < 0.05); the lactic dehydrogenase (LDH) level of the group C was significantly higher than that of the groups A and B on POD 1 and POD 7. Meanwhile, the 7-day survival rate of the rats in group C was poor compared to that of the rats in groups A and B (58.3% vs. 100% vs. 100%).ConclusionICG fluorescence imaging is effective for estimating the degree of liver damage and grading in an ischemia–reperfusion model. It probably has the potential for use in assessing the quality of the donor liver in liver transplantation.

A novel fluorescent cardiac imaging system for preclinical intraoperative angiography

BackgroundIntraoperative coronary angiography can tremendously reduce early coronary bypass graft failures. Fluorescent cardiac imaging provides an advanced method for intraoperative observation and real-time quantitation of blood flow with high resolution.MethodsWe devised a system comprised of an LED light source, special filters, lenses and a detector for preclinical coronary artery angiography. The optical setup was implemented by using two achromatic doublet lenses, two positive meniscus lenses, a band-pass filter, a pinhole and a CCD sensor. The setup was optimized by Zemax software. Optical design was further challenged to obtain more parallel light beams, less diffusion and higher resolutions to levels as small as arterioles. Ex vivo rat hearts were prepared and coronary arteries were retrogradely perfused by indocyanine green (ICG). Video angiography was employed to assess blood flow and plot time-dependent fluorescence intensity curve (TIC). Quantitation of blood flow was performed by calculating either the gradient of TIC or area under curve. The correlation between blood flow and each calculated parameters was assessed and used to evaluate the quality of flow.ResultsHigh-resolution images of flow in coronary arteries were obtained as precise as 62 µm vessel diameter, by our custom-made ICG angiography system. The gradient of TIC was 3.4–6.3 s−1, while the area under curve indicated 712–1282 s values which ultimately gained correlation coefficients of 0.9938 and 0.9951 with relative blood flow, respectively.ConclusionThe present ICG angiography system may facilitate evaluation of blood flow in animal studies of myocardial infarction and coronary artery grafts intraoperatively.

Indocyanine Green Fluorescence Imaging to Predict Graft Survival After Orthotopic Liver Transplantation: A Pilot Study

The incidence of primary nonfunction (PNF) after liver transplantation (LT) remains a major concern with the increasing use of marginal grafts. Indocyanine green (ICG) fluorescence is an imaging technique used in hepatobiliary surgery and LT. Because few early predictors are available, we aimed to quantify in real time the fluorescence of grafts during LT to predict 3-month survival. After graft revascularization, ICG was intravenously injected, and then the fluorescence of the graft was captured with a near infrared camera and postoperatively quantified. A multiparametric modeling of the parenchymal fluorescence intensity (FI) curve was proposed, and a predictive model of graft survival was tested. Between July 2017 and May 2019, 76 LTs were performed, among which 6 recipients underwent retransplantation. No adverse effects of ICG injection were observed. The parameter a150 (temporal course of FI) was significantly higher in the re-LT group (0.022seconds-1 (0.0011-0.059) versus 0.012seconds-1 (0.0001-0.054); P=0.01). This parameter was the only independent predictive factor of graft survival at 3 months (OR,2.4; 95% CI, 1.05-5.50; P=0.04). The best cutoff for the parameter a150 (0.0155seconds-1 ) predicted the graft survival at 3 months with a sensitivity (Se) of 83.3% and a specificity (Spe) of 78.6% (area under the curve,0.82; 95% CI, 0.67-0.98; P=0.01). Quantitative assessment of intraoperative ICG fluorescence on the graft was feasible to predict graft survival at 3months with a good Se and Spe. Further prospective studies should be undertaken to validate these results over larger cohorts and evaluate the clinical impact of this tool.

Noncontact temperature‐dependent fluorescence depicting phase transition in Nd3+‐doped (K0.5Na0.5)NbO3 ceramics

AbstractA noncontact temperature measurement technique based on fluorescence variation was used to depict the temperature‐dependent evolution of phase transition of a ferroelectric material, that is, Nd3+‐doped (K0.5Na0.5)NbO3 ceramics. The slope of the fluorescence intensity curve changes dramatically in the two temperature regions of 450‐475 K and 650‐675 K, which correspond to orthorhombic‐tetragonal and tetragonal‐cubic transitions as confirmed by the temperature dependence of dielectric constant. Furthermore, the small deviations in δTO‐T and δTc indicate the good accuracy of this noncontact method. This work can guide other ergodic ferroelectrics to describe phase experience by the noncontact fluorescence method.

A Study on the Fluorescence Properties of New Laser Material-n-B18H22 in SDS Aqueous Solution

The effect of anionic surfactant sodium dodecyl sulfate (SDS) on the fluorescence of n-B18H22 was studied. The experimental result revealed that the fluorescence of n-B18H22 was obviously enhanced with the increase of the concentration of SDS, mainly due to the hydrophobic protection of n-B18H22 by SDS. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of n-B18H22 in the range of 0-8.0×10−3 mol·L−1 and 8.0×10−3 mol·L−1-3.3×10−2 mol·L−1, respectively. It was observed that a break appeared in the curve of fluorescence intensity with surfactant concentration. The critical micelle concentration (CMC, 7.9×10−3 mol·L−1) of SDS was obtained from this break point in a good agreement with the reported value. The fluorescence intensity increased initially with time and gradually stabilized. The results form the basis for further study of the properties and application of n-B18H22 in SDS solutions.

Quantitative microinjection using fluorescence calibration of streaming microdroplets on a superhydrophobic surface

A simple and reproducible procedure was developed to measure the volume of liquid microinjected into cells. A calibration curve of droplet fluorescence intensity versus volume was constructed by injecting a fluorescent dextran solution through a 125–150 µm diameter micropipette into an oil-filled culture dish to create a spray of varied-sized droplets. The droplets retained a spherical shape because they were in an oil medium and they settled onto a glass surface coated with a superhydrophobic surface. Fluorescent micrographs of the droplets were obtained and analyzed with Image-J software to quantify the fluorescence intensity and radius of each spherical droplet to produce the calibration curve. Subsequently, Dut-145 human prostate carcinoma cells were microinjected with the same fluorescent dextran solution and fluorescent micrographs of the cells were obtained using the identical exposure conditions used to photograph the droplets. The measured fluorescence intensity of the microinjected cells was entered into the formula for the regression line that was fit to the calibration curve allowing determination of the volume of solution injected into each cell. Thus, a mixture consisting of known concentrations of a test material of test material (macromolecules, drugs, etc.) and a fluorescent dextran, volumetric, tracer can be used to quantify the relationship between the amount of a microinjected material and subsequent effects on cells.

Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes.

We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand-receptor (antigen-antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct ( (5.6 ± 0.2) × 107 M-1 min-1 ) and reverse ( (1.3 ± 0.2) × 10-2 min-1 ) rate constants of ligand-receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.

Selective detection of Mg2+ ions via enhanced fluorescence emission using Au-DNA nanocomposites.

The biophysical properties of DNA-modified Au nanoparticles (AuNPs) have attracted a great deal of research interest for various applications in biosensing. AuNPs have strong binding capability to the phosphate and sugar groups in DNA, rendering unique physicochemical properties for detection of metal ions. The formation of Au–DNA nanocomposites is evident from the observed changes in the optical absorption, plasmon band, zeta potential, DLS particle size distribution, as well as TEM and AFM surface morphology analysis. Circular dichroism studies also revealed that DNA-functionalized AuNP binding caused a conformational change in the DNA structure. Due to the size and shape dependent plasmonic interactions of AuNPs (33–78 nm) with DNA, the resultant Au–DNA nanocomposites (NCs) exhibit superior fluorescence emission due to chemical binding with Ca2+, Fe2+ and Mg2+ ions. A significant increase in fluorescence emission (λex = 260 nm) of Au–DNA NCs was observed after selectively binding with Mg2+ ions (20–800 ppm) in an aqueous solution where a minimum of 100 ppm Mg2+ ions was detected based on the linearity of concentration versus fluorescence intensity curve (λem = 400 nm). The effectiveness of Au–DNA nanocomposites was further verified by comparing the known concentration (50–120 ppm) of Mg2+ ions in synthetic tap water and a real life sample of Gelusil (300–360 ppm Mg2+), a widely used antacid medicine. Therefore, this method could be a sensitive tool for the estimation of water hardness after careful preparation of a suitably designed Au–DNA nanostructure.

New Quantitative Analysis Method Using HyperEye Medical System Image for Coronary Artery Bypass Grafting

Purpose: The current evaluation by indocyanine green angiography in coronary artery bypass patients has been performed by qualitative analysis. We devised an original quantitative analysis method using Image J. Methods: Indocyanine green angiography movie was converted to spatiotemporal images. Using the spatiotemporal image, an indocyanine green fluorescence intensity curve was drawn and converted to a firstderivative (acceleration) curve. Indocyanine green angiography evaluations were classified into four types. We collected the peak indocyanine green fluorescence intensity value (peak-I) and both time interval and ratio of maximum indocyanine green acceleration value (max dI/dt) between the mid and distal portions of the graft. Results: In 61 left internal thoracic artery grafts, 49 were patent and 12 were abnormal on CAG. There were significant differences between peak-I at the mid portion of the graft (221.47 ± 39.33 vs. 184.82 ± 40.15 gray scale) and time delay with max dI/dt (0.45 ± 1.28 vs. −1.00 ± 1.25 s), but there was no significant difference in the ratio of max dI/dt between patent and abnormal grafts. Conclusions: The spatiotemporal image method may become a model of the analysis software and the time interval of max dI/dt may become a predictor for future graft failure.