Journal articles on the topic 'FLIMM method'
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1
Zhang, Beini, Liping Li, Yetao Lyu, Shuguang Chen, Lin Xu, and Guanhua Chen. "A New Instrument Monitoring Method Based on Few-Shot Learning." Applied Sciences 13, no. 8 (April 21, 2023): 5185. http://dx.doi.org/10.3390/app13085185.
Abstract:
As an important part of the industrialization process, fully automated instrument monitoring and identification are experiencing an increasingly wide range of applications in industrial production, autonomous driving, and medical experimentation. However, digital instruments usually have multi-digit features, meaning that the numeric information on the screen is usually a multi-digit number greater than 10. Therefore, the accuracy of recognition with traditional algorithms such as threshold segmentation and template matching is low, and thus instrument monitoring still relies heavily on human labor at present. However, manual monitoring is costly and not suitable for risky experimental environments such as those involving radiation and contamination. The development of deep neural networks has opened up new possibilities for fully automated instrument monitoring; however, neural networks generally require large training datasets, costly data collection, and annotation. To solve the above problems, this paper proposes a new instrument monitoring method based on few-shot learning (FLIMM). FLIMM improves the average accuracy (ACC) of the model to 99% with only 16 original images via effective data augmentation method. Meanwhile, due to the controllability of simulated image generation, FLIMM can automatically generate annotation information for simulated numbers, which greatly reduces the cost of data collection and annotation.
2
Wang, Quan, Yahui Li, Dong Xiao, Zhenya Zang, Zi’ao Jiao, Yu Chen, and David Day Uei Li. "Simple and Robust Deep Learning Approach for Fast Fluorescence Lifetime Imaging." Sensors 22, no. 19 (September 26, 2022): 7293. http://dx.doi.org/10.3390/s22197293.
Abstract:
Fluorescence lifetime imaging (FLIM) is a powerful tool that provides unique quantitative information for biomedical research. In this study, we propose a multi-layer-perceptron-based mixer (MLP-Mixer) deep learning (DL) algorithm named FLIM-MLP-Mixer for fast and robust FLIM analysis. The FLIM-MLP-Mixer has a simple network architecture yet a powerful learning ability from data. Compared with the traditional fitting and previously reported DL methods, the FLIM-MLP-Mixer shows superior performance in terms of accuracy and calculation speed, which has been validated using both synthetic and experimental data. All results indicate that our proposed method is well suited for accurately estimating lifetime parameters from measured fluorescence histograms, and it has great potential in various real-time FLIM applications.
3
Needham, Sarah R., Laura C. Zanetti-Domingues, Kathrin M. Scherer, Michael Hirsch, Daniel J. Rolfe, Selene K. Roberts, Marisa L. Martin-Fernandez, David T. Clarke, and Christopher J. Tynan. "Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution." Biochemical Society Transactions 43, no. 3 (June 1, 2015): 309–14. http://dx.doi.org/10.1042/bst20140318.
Abstract:
There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5–50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm.
4
Ji, Mingmei, Jiahui Zhong, Runzhe Xue, Wenhua Su, Yawei Kong, Yiyan Fei, Jiong Ma, Yulan Wang, and Lan Mi. "Early Detection of Cervical Cancer by Fluorescence Lifetime Imaging Microscopy Combined with Unsupervised Machine Learning." International Journal of Molecular Sciences 23, no. 19 (September 29, 2022): 11476. http://dx.doi.org/10.3390/ijms231911476.
Abstract:
Cervical cancer has high morbidity and mortality rates, affecting hundreds of thousands of women worldwide and requiring more accurate screening for early intervention and follow-up treatment. Cytology is the current dominant clinical screening approach, and though it has been used for decades, it has unsatisfactory sensitivity and specificity. In this work, fluorescence lifetime imaging microscopy (FLIM) was used for the imaging of exfoliated cervical cells in which an endogenous coenzyme involved in metabolism, namely, reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H], was detected to evaluate the metabolic status of cells. FLIM images from 71 participants were analyzed by the unsupervised machine learning method to build a prediction model for cervical cancer risk. The FLIM method combined with unsupervised machine learning (FLIM-ML) had a sensitivity and specificity of 90.9% and 100%, respectively, significantly higher than those of the cytology approach. One cancer recurrence case was predicted as high-risk several months earlier using this method as compared to using current clinical methods, implying that FLIM-ML may be very helpful for follow-up cancer care. This study illustrates the clinical applicability of FLIM-ML as a detection method for cervical cancer screening and a convenient tool for follow-up cancer care.
5
Pande, P., C. A. Trivedi, and J. A. Jo. "Analysis of Fluorescence Lifetime Imaging Microscopy (FLIM) Data." Methods of Information in Medicine 49, no. 05 (2010): 531–36. http://dx.doi.org/10.3414/me09-02-0046.
Abstract:
Summary Objectives: A novel Fluorescence Lifetime Imaging Microscopy (FLIM) deconvolution method based on the linear expansion of fluorescence decays on a set of orthonormal Laguerre functions was recently proposed. The Laguerre deconvolution method applies linear least-square estimation to estimate the expansion coefficients of all pixel decays simultaneously, performing at least two orders of magnitude faster than the other algorithms. In the original Laguerre FLIM deconvolution implementation, however, the Laguerre parameter α is selected using a heuristic approach, making it unsuitable for online applications. Methods: In this study, we present a fully automated implementation of the Laguerre FLIM deconvolution, whereby the Laguerre parameter α is treated as a free parameter within a nonlinear least-squares optimization scheme. Results: The performance of this method has been successfully validated on simulated data, and experimental FLIM images of standard fluorescent dyes and endogenous tissue fluorescence. Conclusions: The main advantage of the proposed method is that it does not require any user intervention for tuning up the deconvolution process. Thus, we believe this method will facilitate the translation of FLIM to online applications, including real-time clinical diagnosis.
6
Liu, Guoying, Pengwei Li, and Yun Zhang. "A Color Texture Image Segmentation Method Based on Fuzzy c-Means Clustering and Region-Level Markov Random Field Model." Mathematical Problems in Engineering 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/240354.
Abstract:
This paper presents a variation of the fuzzy local information c-means clustering (FLICM) algorithm that provides color texture image clustering. The proposed algorithm incorporates region-level spatial, spectral, and structural information in a novel fuzzy way. The new algorithm, called RFLICM, combines FLICM and region-level Markov random field model (RMRF) together to make use of large scale interactions between image patches instead of pixels. RFLICM can overcome the weakness of FLICM when dealing with textured images and at the same time enhances the clustering performance. The major characteristic of RFLICM is the use of a region-level fuzzy factor, aiming to guarantee texture homogeneity and preserve region boundaries. Experiments performed on synthetic and remote sensing images show that RFLICM is effective in providing accuracy to color texture images.
7
XU, LINGLING, ZHONG-CHAO WEI, SHAOQUN ZENG, and ZHEN-LI HUANG. "QUANTIFYING THE SHORT LIFETIME WITH TCSPC-FLIM: FIRST MOMENT VERSUS FITTING METHODS." Journal of Innovative Optical Health Sciences 06, no. 04 (October 2013): 1350030. http://dx.doi.org/10.1142/s1793545813500302.
Abstract:
Combing the time-correlated single photon counting (TCSPC) with fluorescence lifetime imaging microscopy (FLIM) provides promising opportunities in revealing important information on the microenvironment of cells and tissues, but the applications are thus far mainly limited by the accuracy and precision of the TCSPC-FLIM technique. Here we present a comprehensive investigation on the performance of two data analysis methods, the first moment (M1) method and the conventional least squares (Fitting) method, in quantifying fluorescence lifetime. We found that the M1 method is more superior than the Fitting method when the lifetime is short (70 ~ 400 ps) or the signal intensity is weak (<103 photons).
8
Ji, Chao, Xing Wang, Kai He, Yanhua Xue, Yahui Li, Liwei Xin, Wei Zhao, Jinshou Tian, and Liang Sheng. "Compressed fluorescence lifetime imaging via combined TV-based and deep priors." PLOS ONE 17, no. 8 (August 12, 2022): e0271441. http://dx.doi.org/10.1371/journal.pone.0271441.
Abstract:
Compressed fluorescence lifetime imaging (Compressed-FLIM) is a novel Snapshot compressive imaging (SCI) method for single-shot widefield FLIM. This approach has the advantages of high temporal resolution and deep frame sequences, allowing for the analysis of FLIM signals that follow complex decay models. However, the precision of Compressed-FLIM is limited by reconstruction algorithms. To improve the reconstruction accuracy of Compressed-FLIM in dealing with large-scale FLIM problem, we developed a more effective combined prior model 3DTGp V_net, based on the Plug and Play (PnP) framework. Extensive numerical simulations indicate the proposed method eliminates reconstruction artifacts caused by the Deep denoiser networks. Moreover, it improves the reconstructed accuracy by around 4dB (peak signal-to-noise ratio; PSNR) over the state-of-the-art TV+FFDNet in test data sets. We conducted the single-shot FLIM experiment with different Rhodamine reagents and the results show that in practice, the proposed algorithm has promising reconstruction performance and more negligible lifetime bias.
9
Mikicin, Mirosław. "Relationships of attention and arousal are responsible for action in sports." Biomedical Human Kinetics 14, no. 1 (January 1, 2022): 229–35. http://dx.doi.org/10.2478/bhk-2022-0028.
Abstract:
Abstract Study aim: The most important psychological mechanisms that are responsible for sports activity are arousal and attention. Study aim the relationships between the level of arousal and the level of attention, an attempt has been made to explain the mechanisms responsible for sportsman activity. Material and method: The study was conducted in a group of sportsman-students (68 individuals) using the Vienna Test System: FLIM, a test of flicker/fusion frequency, which is a measurement procedure allowing to determine the functional readiness of the central nervous system in terms of arousal and COG (Cognitron), which is a test measuring the level of attention. Results: The following in statistical analysis of the data were observed: inversely proportional relationships of image fusion frequency (FLIM1) during the recording of the level of arousal with: the correct acceptance of stimuli (COG1, r = –0.287), the correct rejection of stimuli (COG2, r = –0.320), the time of correctly accepted stimuli (COG3, r = –0.299), and with the time of correctly rejected stimuli (COG4, r = –0.317) in the attentional activity. Conclusion: Fusion frequency indicates the level of fatigue and is inversely proportional to the correctly accepted stimuli in attentional activity, the correctly rejected stimuli in attentional activities, and the duration of the attentional actions.
10
Adhikari, Mou, Rola Houhou, Julian Hniopek, and Thomas Bocklitz. "Review of Fluorescence Lifetime Imaging Microscopy (FLIM) Data Analysis Using Machine Learning." Journal of Experimental and Theoretical Analyses 1, no. 1 (September 21, 2023): 44–63. http://dx.doi.org/10.3390/jeta1010004.
Abstract:
Fluorescence lifetime imaging microscopy (FLIM) has emerged as a promising tool for all scientific studies in recent years. However, the utilization of FLIM data requires complex data modeling techniques, such as curve-fitting procedures. These conventional curve-fitting procedures are not only computationally intensive but also time-consuming. To address this limitation, machine learning (ML), particularly deep learning (DL), can be employed. This review aims to focus on the ML and DL methods for FLIM data analysis. Subsequently, ML and DL strategies for evaluating FLIM data are discussed, consisting of preprocessing, data modeling, and inverse modeling. Additionally, the advantages of the reviewed methods are deliberated alongside future implications. Furthermore, several freely available software packages for analyzing the FLIM data are highlighted.
11
Liu, Chao, Xinwei Wang, Yan Zhou, and Yuliang Liu. "Timing and Operating Mode Design for Time-Gated Fluorescence Lifetime Imaging Microscopy." Scientific World Journal 2013 (2013): 1–5. http://dx.doi.org/10.1155/2013/801901.
Abstract:
Steady-state fluorence imaging and time-resolved fluorescence imaging are two important areas in fluorescence imaging research. Fluorescence lifetime imaging is an absolute measurement method which is independent of excitation laser intensity, fluorophore concentration, and photobleaching compared to fluorescence intensity imaging techniques. Time-gated fluorescence lifetime imaging microscopy (FLIM) can provide high resolution and high imaging frame during mature FLIM methods. An abstract time-gated FLIM model was given, and important temporal parameters are shown as well. Aiming at different applications of steady and transient fluorescence processes, two different operation modes, timing and lifetime computing algorithm are designed. High resolution and high frame can be achieved by one-excitation one-sampling mode and least square algorithm for steady imaging applications. Correspondingly, one-excitation two-sampling mode and rapid lifetime determination algorithm contribute to transient fluorescence situations.
12
Mandrou, Elena, Peter A. Thomason, Peggy I. Paschke, Nikki R. Paul, Luke Tweedy, and Robert H. Insall. "A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells." Cells 13, no. 13 (June 27, 2024): 1114. http://dx.doi.org/10.3390/cells13131114.
Abstract:
Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.
13
Tan, M., and M. Hao. "CHANGE DETECTION BY FUSING ADVANTAGES OF THRESHOLD AND CLUSTERING METHODS." ISPRS - International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences XLII-2/W7 (September 13, 2017): 897–901. http://dx.doi.org/10.5194/isprs-archives-xlii-2-w7-897-2017.
Abstract:
In change detection (CD) of medium-resolution remote sensing images, the threshold and clustering methods are two kinds of the most popular ones. It is found that the threshold method of the expectation maximum (EM) algorithm usually generates a CD map including many false alarms but almost detecting all changes, and the fuzzy local information c-means algorithm (FLICM) obtains a homogeneous CD map but with some missed detections. Therefore, we aim to design a framework to improve CD results by fusing the advantages of threshold and clustering methods. Experimental results indicate the effectiveness of the proposed method.
14
Yamazaki, Takeru, Xiao Liu, Young-Tae Chang, and Satoshi Arai. "Applicability and Limitations of Fluorescence Intensity-Based Thermometry Using a Palette of Organelle Thermometers." Chemosensors 11, no. 7 (July 4, 2023): 375. http://dx.doi.org/10.3390/chemosensors11070375.
Abstract:
Fluorescence thermometry is a microscopy technique in which a fluorescent temperature sensor records temperature changes as alterations of fluorescence signals. Fluorescence lifetime imaging (FLIM) is a promising method for quantitative analysis of intracellular temperature. Recently, we developed small-molecule thermometers, termed Organelle Thermo Greens, that target various organelles and achieved quantitative temperature mapping using FLIM. Despite its highly quantitative nature, FLIM-based thermometry cannot be used widely due to expensive instrumentation. Here, we investigated the applicability and limitations of fluorescence intensity (FI)-based analysis, which is more commonly used than FLIM-based thermometry. Temperature gradients generated by artificial heat sources and physiological heat produced by brown adipocytes were visualized using FI- and FLIM-based thermometry. By comparing the two thermometry techniques, we examined how the shapes of organelles and cells affect the accuracy of the temperature measurements. Based on the results, we concluded that FI-based thermometry could be used for “qualitative”, rather than quantitative, thermometry under the limited condition that the shape change and the dye leakage from the target organelle were not critical.
15
Leiter, Nina, Maximilian Wohlschläger, Martin Versen, and Christian Laforsch. "An algorithmic method for the identification of wood species and the classification of post-consumer wood using fluorescence lifetime imaging microscopy." Journal of Sensors and Sensor Systems 11, no. 1 (May 3, 2022): 129–36. http://dx.doi.org/10.5194/jsss-11-129-2022.
Abstract:
Abstract. In this contribution the frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) technique is evaluated for post-consumer wood sorting. The fluorescence characteristics of several wood samples were determined, whereby two excitation wavelengths (405 and 488 nm) were used. The measured data were processed using algorithmic methods to identify the wood species and post-consumer wood category. With the excitation wavelength of 405 nm, 16 out of 19 samples could be correctly assigned to the corresponding post-consumer wood category by means of the fluorescence lifetimes. Thus, the experimental results revealed the high potential of the FD-FLIM technique for automated post-consumer wood sorting.
16
Huang, Maojia, Xinyue Liang, Zixiao Zhang, Jing Wang, Yiyan Fei, Jiong Ma, Songnan Qu, and Lan Mi. "Carbon Dots for Intracellular pH Sensing with Fluorescence Lifetime Imaging Microscopy." Nanomaterials 10, no. 4 (March 25, 2020): 604. http://dx.doi.org/10.3390/nano10040604.
Abstract:
The monitoring of intracellular pH is of great importance for understanding intracellular trafficking and functions. It has various limitations for biosensing based on the fluorescence intensity or spectra study. In this research, pH-sensitive carbon dots (CDs) were employed for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time. FLIM is a highly sensitive method that is used to detect a microenvironment and it can overcome the limitations of biosensing methods based on fluorescence intensity. The different groups on the CDs surfaces changing with pH environments led to different fluorescence lifetime values. The CDs aqueous solution had a gradual change from 1.6 ns to 3.7 ns in the fluorescence lifetime with a pH range of 2.6–8.6. Similar fluorescence lifetime changes were found in pH buffer-treated living cells. The detection of lysosomes, cytoplasm, and nuclei in living cells was achieved by measuring the fluorescence lifetime of CDs. In particular, a phasor FLIM analysis was used to improve the pH imaging. Moreover, the effects of the coenzymes, amino acids, and proteins on the fluorescence lifetime of CDs were examined in order to mimic the complex microenvironment inside the cells.
17
Zhou, Li, Yawei Kong, Junxin Wu, Xingzhi Li, Yiyan Fei, Jiong Ma, Yulan Wang, and Lan Mi. "Metabolic Changes in Maternal and Cord Blood in One Case of Pregnancy-Associated Breast Cancer Seen by Fluorescence Lifetime Imaging Microscopy." Diagnostics 11, no. 8 (August 19, 2021): 1494. http://dx.doi.org/10.3390/diagnostics11081494.
Abstract:
Pregnancy-associated breast cancer (PABC) is a rare disease, which is frequently diagnosed at an advanced stage due to limitations in current diagnostic methods. In this study, fluorescence lifetime imaging microscopy (FLIM) was used to study the metabolic changes by measuring maternal blood and umbilical cord blood via the autofluorescence of coenzymes, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), and flavin adenine dinucleotide (FAD). The NAD(P)H data showed that a PABC case had significant differences compared with normal cases, which may indicate increased glycolysis. The FAD data showed that both maternal and cord blood of PABC had shorter mean lifetimes and higher bound-FAD ratios. The significant differences suggested that FLIM testing of blood samples may be a potential method to assist in PABC non-radiative screening.
18
Liu, Lixin, Qianqian Yang, Meiling Zhang, Zhaoqing Wu, and Ping Xue. "Fluorescence lifetime imaging microscopy and its applications in skin cancer diagnosis." Journal of Innovative Optical Health Sciences 12, no. 05 (September 2019): 1930004. http://dx.doi.org/10.1142/s1793545819300040.
Abstract:
Fluorescence lifetime (FLT) of fluorophores is sensitive to the changes in their surrounding microenvironment, and hence it can quantitatively reveal the physiological characterization of the tissue under investigation. Fluorescence lifetime imaging microscopy (FLIM) provides not only morphological but also functional information of the tissue by producing spatially resolved image of fluorophore lifetime, which can be used as a signature of disorder and/or malignancy in diseased tissues. In this paper, we begin by introducing the basic principle and common detection methods of FLIM. Then the recent advances in the FLIM-based diagnosis of three different skin cancers, including basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant melanoma (MM) are reviewed. Furthermore, the potential advantages of FLIM in skin cancer diagnosis and the challenges that may be faced in the future are prospected.
19
Pena, Ana-Maria, Shosuke Ito, Thomas Bornschlögl, Sébastien Brizion, Kazumasa Wakamatsu, and Sandra Del Bino. "Multiphoton FLIM Analyses of Native and UVA-Modified Synthetic Melanins." International Journal of Molecular Sciences 24, no. 5 (February 24, 2023): 4517. http://dx.doi.org/10.3390/ijms24054517.
Abstract:
To better understand the impact of solar light exposure on human skin, the chemical characterization of native melanins and their structural photo-modifications is of central interest. As the methods used today are invasive, we investigated the possibility of using multiphoton fluorescence lifetime (FLIM) imaging, along with phasor and bi-exponential fitting analyses, as a non-invasive alternative method for the chemical analysis of native and UVA-exposed melanins. We demonstrated that multiphoton FLIM allows the discrimination between native DHI, DHICA, Dopa eumelanins, pheomelanin, and mixed eu-/pheo-melanin polymers. We exposed melanin samples to high UVA doses to maximize their structural modifications. The UVA-induced oxidative, photo-degradation, and crosslinking changes were evidenced via an increase in fluorescence lifetimes along with a decrease in their relative contributions. Moreover, we introduced a new phasor parameter of a relative fraction of a UVA-modified species and provided evidence for its sensitivity in assessing the UVA effects. Globally, the fluorescence lifetime properties were modulated in a melanin-dependent and UVA dose-dependent manner, with the strongest modifications being observed for DHICA eumelanin and the weakest for pheomelanin. Multiphoton FLIM phasor and bi-exponential analyses hold promising perspectives for in vivo human skin mixed melanins characterization under UVA or other sunlight exposure conditions.
20
Ngeh-Nkeng, Alembong Eleanor, Forchap Nkemanjong Milton, Nkengfua Samuel, Elong Felix Adolphe, and Egbe Obinchemti Thomas. "Outcome of Trial of Labor after Cesarean Delivery: Evaluating the Flamm Scoring System in Two Hospitals in Cameroon: A 5-year Retrospective Study." Journal of Integrated Health Sciences 12, no. 1 (January 2024): 21–27. http://dx.doi.org/10.4103/jihs.jihs_5_24.
Abstract:
Abstract Background: Outcomes of trial of scar (TOS) correlate well with predictions provided by vaginal birth after cesarean scoring systems. In Cameroon, there is limited data on the implementation and effectiveness of these scoring systems, as well as the materno-fetal outcomes of TOS. Objectives: The objective was to utilize the Flamm score in predicting the success of TOS, and to evaluate the maternal and fetal outcomes observed following TOS in Yaounde and Douala Gyneco-Obstetric and Pediatric Hospitals from 2017 to 2021. Material and Methods: We included files of 310 parturient women with single transverse lower uterine segment cesarean section, who had TOS. Data on sociodemographic, reproductive health, Flamm scoring, and labor outcomes were documented using a structured data extraction form and analyzed using the Statistical Package for the Social Sciences version 25. Results: Of the 310 patients, 73.9% had successful vaginal deliveries (VDs) and 26.1% had repeated emergency cesarean. Flamm score predictions were associated with TOS outcomes (P = 0.0001), with mean score of 5.87 ± 0.01 for successful VD compared to 3.72 ± 0.126 for repeat cesarean. Probability of successful VD increased with Flamm score, ranging from 0% for scores of two to 100% for scores > 7, with a cutoff score of 4.5 (Se = 75.0%, Sp = 75.3%). The main maternal complication was one uterine rupture (0.32%) and neonatal complications were three stillbirths (0.98%) and one early neonatal death (0.32%) after repeat cesarean. Conclusions: TOS is associated with high success rates in tertiary hospitals in Cameroon. Applying Flamm score will improve the chances of a successful VD following TOS, and should be encouraged.
21
Dvornikov, Alexander, Suman Ranjit, and Enrico Gratton. "Application of the Diver Detection Method to Multiphoton Microscopy and Flim." Biophysical Journal 110, no. 3 (February 2016): 482a. http://dx.doi.org/10.1016/j.bpj.2015.11.2577.
22
Garsha, Karl. "A Comment on using FLIM with FRET." Microscopy Today 14, no. 3 (May 2006): 52–53. http://dx.doi.org/10.1017/s1551929500057709.
Abstract:
Depending on the nature of the study and what sort of information one is trying to gather through the use of FRET, FLIM has some compelling advantages in certain situations, and can provide a quantitative evaluation of the donor, acceptor and FRET pair stoichiometry. It does require access to specialized equipment and software. Different approaches to FLIM data acquisition have different strengths and weaknesses. For dynamic studies requiring high time resolution, FLIM acquisition times can fall well short of ideal.If a yes/no answer to whether FRET is occurring is all that is required, then the polarization anisotropy of the acceptor can be used to determine FRET between fluorescent proteins (Rizzo and Piston, 2005). This is a relatively simple and robust method for confirming the presence/absence of FRET.
23
Lee, Jiung-De, Ping-Chun Huang, Yi-Cheng Lin, Lung-Sen Kao, Chien-Chang Huang, Fu-Jen Kao, Chung-Chih Lin, and De-Ming Yang. "In-Depth Fluorescence Lifetime Imaging Analysis Revealing SNAP25A-Rabphilin 3A Interactions." Microscopy and Microanalysis 14, no. 6 (November 6, 2008): 507–18. http://dx.doi.org/10.1017/s1431927608080628.
Abstract:
AbstractThe high sensitivity and spatial resolution enabled by two-photon excitation fluorescence lifetime imaging microscopy/fluorescence resonance energy transfer (2PE-FLIM/FRET) provide an effective approach that reveals protein-protein interactions in a single cell during stimulated exocytosis. Enhanced green fluorescence protein (EGFP)–labeled synaptosomal associated protein of 25 kDa (SNAP25A) and red fluorescence protein (mRFP)–labeled Rabphillin 3A (RPH3A) were co-expressed in PC12 cells as the FRET donor and acceptor, respectively. The FLIM images of EGFP-SNAP25A suggested that SNAP25A/RPH3A interaction was increased during exocytosis. In addition, the multidimensional (three-dimensional with time) nature of the 2PE-FLIM image datasets can also resolve the protein interactions in the z direction, and we have compared several image analysis methods to extract more accurate and detailed information from the FLIM images. Fluorescence lifetime was fitted by using one and two component analysis. The lifetime FRET efficiency was calculated by the peak lifetime (τpeak) and the left side of the half-peak width (τ1/2), respectively. The results show that FRET efficiency increased at cell surface, which suggests that SNAP25A/RPH3A interactions take place at cell surface during stimulated exocytosis. In summary, we have demonstrated that the 2PE-FLIM/FRET technique is a powerful tool to reveal dynamic SNAP25A/RPH3A interactions in single neuroendocrine cells.
24
Ermakova, Polina, Alena Kashirina, Irina Kornilova, Aleksandra Bogomolova, Darya Myalik, Nasipbek Naraliev, Denis Kuchin, et al. "Contrast-Free FLIM Reveals Metabolic Changes in Pathological Islets of Langerhans." International Journal of Molecular Sciences 23, no. 22 (November 8, 2022): 13728. http://dx.doi.org/10.3390/ijms232213728.
Abstract:
FLIM (Fluorescence Lifetime Imaging Microscopy) is a powerful tool that could be used in the future to diagnose islet cell recovery after therapy. The identification of appropriate FLIM parameters is required to determine islet quality and islet cell metabolism throughout the organ under various conditions of insulin deficiency. The aim of the work was to identify key FLIM parameters, changes of which are characteristic of pancreatic pathologies. The τm, τ1, τ2, α1, α2 and α1/α2 of free and bound forms of NAD(P)H of the islet cells of animals (rats and pigs) and of humans with and without pathologies were measured and analyzed. The data were confirmed by IHC and histological studies. We identified three FLIM parameters in islet cells from animals with streptozotocin (STZ)-induced diabetes mellitus (DM) and from humans with chronic pancreatitis + type 2 diabetes (T2D), which differ in the same way: τm and α2 take lower values compared to the nonpathological islet cells, while α1/α2 takes higher values. In islet cells from patients with adenocarcinoma (PDAC) and chronic pancreatitis, these parameters had reverse tendency relative to the norm or did not differ. Thus, minimally invasive and non-contrast FLIM methods may, in the future, be used to diagnose pathological islet cells.
25
Ouellette, Jonathan, Ellen Wargowski, Eric Wait, Chris Zahm, Scott Johnson, and Jon Oliner. "Abstract 2386: Label free imaging for rapid assessment of tumor viability in live tumor fragments." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2386. http://dx.doi.org/10.1158/1538-7445.am2023-2386.
Abstract:
Abstract Live tumor fragments (LTF) that maintain a patient's relevant tumor microenvironment provides an opportunity to study treatment response. To verify viability of the fragments over time, we have devised and applied a label-free quantitative microscopy-based approach to monitor LTF health. This method directly assesses intrinsic fluorescence from metabolic co-factors and allows the spatial and temporal visualization of cell status. Using the label-free method of multiphoton fluorescence lifetime microscopy (MP-FLIM), we can assess excised tumor samples over 48 hours to determine the health of individual cells. Specifically, with MP-FLIM, we can analyze signals generated by the intrinsically fluorescent metabolic co-factor NAD(P)H which correlates with mitochondrial outer membrane permeabilization and the irreversible cascades leading to cell death. Our findings using MP-FLIM were confirmed using a standard caspase 3/7 live apoptosis assay. These data demonstrate MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on LTF. Citation Format: Jonathan Ouellette, Ellen Wargowski, Eric Wait, Chris Zahm, Scott Johnson, Jon Oliner. Label free imaging for rapid assessment of tumor viability in live tumor fragments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2386.
26
Todaro, Biagio, Luca Pesce, Francesco Cardarelli, and Stefano Luin. "Pioglitazone Phases and Metabolic Effects in Nanoparticle-Treated Cells Analyzed via Rapid Visualization of FLIM Images." Molecules 29, no. 9 (May 4, 2024): 2137. http://dx.doi.org/10.3390/molecules29092137.
Abstract:
Fluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools.
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Li, Shuai, Jianhua Zhang, Bei Liu, Chengzhi Jiang, Lanxu Ren, Jingjing Xue, and Yansong Song. "An Algorithm to Extract the Boundary and Center of EUV Solar Image Based on Sobel Operator and FLICM." Photonics 9, no. 12 (November 22, 2022): 889. http://dx.doi.org/10.3390/photonics9120889.
Abstract:
An algorithm to extract the disk boundary and center of EUV solar image using the Sobel operator, Fuzzy Local Information C-Means Clustering algorithm (FLICM), and the least square circle fitting method is proposed in this paper. The Sobel operator can determine the solar disk boundary preliminarily, and then the image is processed further using the FLICM algorithm. After the background is removed based on the clustered image and the boundary points can be highlighted, these points are fitted using the least square circle fitting method as the final boundary circle. The solar data used in this paper was from the observation of the Solar Dynamics Observatory Atmospheric Imaging Assembly (SDO/AIA) instrument. The 2523 19.3 nm solar images covering solar minimum, moderate solar activity, and more active suns were calculated using the proposed algorithm to analyze the accuracy statistically. The statistical comparison results demonstrate that the method is accurate and effective. This method can support the processing of solar EUV images and serve the operational system of a space weather forecast.
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Ma, Chunli, Hong Li, Kui Zhang, Yuzhu Gao, and Lei Yang. "Risk Factors of Restroke in Patients with Lacunar Cerebral Infarction Using Magnetic Resonance Imaging Image Features under Deep Learning Algorithm." Contrast Media & Molecular Imaging 2021 (November 18, 2021): 1–11. http://dx.doi.org/10.1155/2021/2527595.
Abstract:
This study was aimed to explore the magnetic resonance imaging (MRI) image features based on the fuzzy local information C-means clustering (FLICM) image segmentation method to analyze the risk factors of restroke in patients with lacunar infarction. In this study, based on the FLICM algorithm, the Canny edge detection algorithm and the Fourier shape descriptor were introduced to optimize the algorithm. The difference of Jaccard coefficient, Dice coefficient, peak signal-to-noise ratio (PSNR), structural similarity index measure (SSIM), running time, and segmentation accuracy of the optimized FLICM algorithm and other algorithms when the brain tissue MRI images were segmented was studied. 36 patients with lacunar infarction were selected as the research objects, and they were divided into a control group (no restroke, 20 cases) and a stroke group (restroke, 16 cases) according to whether the patients had restroke. The differences in MRI imaging characteristics of the two groups of patients were compared, and the risk factors for restroke in lacunar infarction were analyzed by logistic multivariate regression. The results showed that the Jaccard coefficient, Dice coefficient, PSNR value, and SSIM value of the optimized FLICM algorithm for segmenting brain tissue were all higher than those of other algorithms. The shortest running time was 26 s, and the highest accuracy rate was 97.86%. The proportion of patients with a history of hypertension, the proportion of patients with paraventricular white matter lesion (WML) score greater than 2 in the stroke group, the proportion of patients with a deep WML score of 2, and the average age of patients in the stroke group were much higher than those in the control group ( P < 0.05 ). Logistic multivariate regression showed that age and history of hypertension were risk factors for restroke after lacunar infarction ( P < 0.05 ). It showed that the optimized FLICM algorithm can effectively segment brain MRI images, and the risk factors for restroke in patients with lacunar infarction were age and hypertension history. This study could provide a reference for the diagnosis and prognosis of lacunar infarction.
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Gratton, Enrico, Ning Ma, Michelle A. Digman, and Leonel Malacrida. "Of Absolute Concentrations of NADH in Cells using the Phasor Flim Method." Biophysical Journal 112, no. 3 (February 2017): 581a. http://dx.doi.org/10.1016/j.bpj.2016.11.3128.
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Rasha H. Ahmed, Abdul Majeed E. Ibrahim, and Kadhim A. Aadem. "Study of the optical proprieties of copper oxide nanoparticles prepared by PLD method." Tikrit Journal of Pure Science 23, no. 10 (January 20, 2019): 72–75. http://dx.doi.org/10.25130/tjps.v23i10.566.
Abstract:
Nano CuO thin films on glass substrates were prepared at a constant temperature of (300°C), by pulsed laser deposition (PLD) using Nd:YAG laser at 1064 nm wavelength and five deposition energies (400, 500, 600, 700 and 800 mJ) with fixed pulses (300 pulse and 6 Hz) was used on the properties of CuO films. CuO nanoparticles were deposited on glass substrates to study optical properties and formed thin films of thickness (200 nm).CuO thin flims were characterized by X-ray diffraction (XRD) measurements have shown that the polycrystalline CuO prepared at laser energies , includes optical transmittance and absorption measurements and energy gap of these films.
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Bloch, Orin, Alba Alfonso Garcia, Silvia Noble Anbunesan, Roberto Frusciante, Julien Bec, and Laura Marcu. "FLGS-04. Fluorescence lifetime imaging (FLIm) is a dye-free, high sensitivity approach for fluorescence guided surgery in high-grade and low-grade gliomas." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi226. http://dx.doi.org/10.1093/neuonc/noab196.908.
Abstract:
Abstract INTRODUCTION Fluorescence-guided surgery can improve tumor identification and extent of surgical resection. 5-ALA is the standard for GBM, but is limited by lack of quantitative fluorescence, a need to work in the dark, and a lack of sensitivity for low grade tumors. We have developed a novel instrument for dye-free tissue autofluorescence lifetime imaging (FLIm) to identify glioma tissue during resection. This approach utilizes time-resolved autofluorescence measurements in narrow-band channels to assess markers of tissue metabolism. Compared to intensity-based imaging of exogenous fluorophores, FLIm has greater sensitivity without dependence on background lighting. The advantages of FLIm include quantitative tissue analysis, the ability to work under full light conditions, sensitivity for high and low grade gliomas, and the potential ability to identify IDH mutational status. In this study, we validated the use of FLIm for identification of glioma tissue at tumor resection margins. METHODS FLIm was used to image tissue margins during glioma resections and compared to microbiopsies from imaged regions to correlate fluorescence with histopathology. RESULTS FLIm was applied intraoperatively to 11 GBM and 5 LGG patients (7 imaged biopsies per patient). In GBM, tumor infiltration of cortex was associated with significantly decreased fluorescence lifetime (FL) in channels 2 (470/28nm;p<0.05) and 3 (542/50nm;p<0.002). In subcortical margins, FL was inversely proportional to the density of tumor in channels 2,3 (p<0.05). When IDH wild-type GBMs were compared to IDH1-mutant tumors, FL was noted to be significantly longer in channel 1 (390/40nm;p<0.05), and trended towards longer FL in channel 2, shorter FL in channel 3. In LGG, FL was significantly correlated with tumor density in channel 2 (p<0.01). CONCLUSIONS FLIm is a dye-free, quantitative alternative to 5-ALA for fluorescence guided glioma resections with sensitivity to high and low-grade tumors, and the ability to predict IDH mutations in GBM. Further validation studies are on-going.
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Li, Liangliang, Hongbing Ma, and Zhenhong Jia. "Multiscale Geometric Analysis Fusion-Based Unsupervised Change Detection in Remote Sensing Images via FLICM Model." Entropy 24, no. 2 (February 18, 2022): 291. http://dx.doi.org/10.3390/e24020291.
Abstract:
Remote sensing image change detection is widely used in land use and natural disaster detection. In order to improve the accuracy of change detection, a robust change detection method based on nonsubsampled contourlet transform (NSCT) fusion and fuzzy local information C-means clustering (FLICM) model is introduced in this paper. Firstly, the log-ratio and mean-ratio operators are used to generate the difference image (DI), respectively; then, the NSCT fusion model is utilized to fuse the two difference images, and one new DI is obtained. The fused DI can not only reflect the real change trend but also suppress the background. The FLICM is performed on the new DI to obtain the final change detection map. Four groups of homogeneous remote sensing images are selected for simulation experiments, and the experimental results demonstrate that the proposed homogeneous change detection method has a superior performance than other state-of-the-art algorithms.
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Phillips, David. "A lifetime in photochemistry; some ultrafast measurements on singlet states." Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences 472, no. 2190 (June 2016): 20160102. http://dx.doi.org/10.1098/rspa.2016.0102.
Abstract:
We describe here the development of time-correlated single-photon counting techniques from the early use of spark discharge lamps as light sources through to the use of femtosecond mode-locked lasers through the personal work of the author. We used laser-excited fluorescence in studies on energy migration and rotational relaxation in synthetic polymer solutions, in biological probe molecules and in supersonic jet expansions. Time-correlated single-photon counting was the first method used in early fluorescence lifetime imaging microscopy (FLIM), and we outline the development of this powerful technique, with a comparison of techniques including wide-field microscopy. We employed these modern forms of FLIM to study single biological cells, and applied FLIM also to gain an understanding the distribution in tissue, and fates of photosensitizer molecules used in photodynamic therapy. We also describe the uses and instrumental design of laser systems for the study of ultrafast time-resolved vibrational spectroscopy.
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Yuzhakova, Diana V., Daria A. Sachkova, Marina V. Shirmanova, Artem M. Mozherov, Anna V. Izosimova, Anna S. Zolotova, and Konstantin S. Yashin. "Measurement of Patient-Derived Glioblastoma Cell Response to Temozolomide Using Fluorescence Lifetime Imaging of NAD(P)H." Pharmaceuticals 16, no. 6 (May 26, 2023): 796. http://dx.doi.org/10.3390/ph16060796.
Abstract:
Personalized strategies in glioblastoma treatment are highly necessary. One of the possible approaches is drug screening using patient-derived tumor cells. However, this requires reliable methods for assessment of the response of tumor cells to treatment. Fluorescence lifetime imaging microscopy (FLIM) is a promising instrument to detect early cellular response to chemotherapy using the autofluorescence of metabolic cofactors. Here, we explored FLIM of NAD(P)H to evaluate the sensitivity of patient-derived glioma cells to temozolomide (TMZ) in vitro. Our results demonstrate that the more-responsive cell cultures displayed the longest mean fluorescence lifetime τm after TMZ treatment due to an increase in the protein-bound NAD(P)H fraction α2 associated with a shift to oxidative phosphorylation. The cell cultures that responded poorly to TMZ had generally shorter τm, i.e., were more glycolytic, and showed no or insignificant changes after treatment. The FLIM data correlate well with standard measurements of cellular drug response—cell viability and proliferation index and clinical response in patients. Therefore, FLIM of NAD(P)H provides a highly sensitive, label-free assay of treatment response directly on patient-derived glioblastoma cells and can become an innovative platform for individual drug screening for patients.
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Trujillo, Justin, Aliyah S. Khan, Dhruba P. Adhikari, Michael R. Stoneman, Jenu V. Chacko, Kevin W. Eliceiri, and Valerica Raicu. "Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study." International Journal of Molecular Sciences 25, no. 9 (April 26, 2024): 4706. http://dx.doi.org/10.3390/ijms25094706.
Abstract:
Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.
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Tans-Kersten, Julie, Huayu Huang, and Caitilyn Allen. "Ralstonia solanacearum Needs Motility for Invasive Virulence on Tomato." Journal of Bacteriology 183, no. 12 (June 15, 2001): 3597–605. http://dx.doi.org/10.1128/jb.183.12.3597-3605.2001.
Abstract:
ABSTRACT Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found thatR. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lackingfliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphAcassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.
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H. Ahmed1, Rasha, ,. Abdul Majeed E. Ibrahim1, and Kadhim A. Aadem2. "Study of the optical proprieties of copper oxide nanoparticles prepared by PLD method." Tikrit Journal of Pure Science 23, no. 10 (January 20, 2019): 72. http://dx.doi.org/10.25130/j.v23i10.760.
Abstract:
Nano CuO thin films on glass substrates were prepared at a constant temperature of (300°C), by pulsed laser deposition (PLD) using Nd:YAG laser at 1064 nm wavelength and five deposition energies (400, 500, 600, 700 and 800 mJ) with fixed pulses (300 pulse and 6 Hz) was used on the properties of CuO films. CuO nanoparticles were deposited on glass substrates to study optical properties and formed thin films of thickness (200 nm).CuO thin flims were characterized by X-ray diffraction (XRD) measurements have shown that the polycrystalline CuO prepared at laser energies , includes optical transmittance and absorption measurements and energy gap of these films.
http://dx.doi.org/10.25130/tjps.23.2018.172
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Alhibah, Mohammad, Marius Kröger, Sabine Schanzer, Loris Busch, Jürgen Lademann, Ingeborg Beckers, Martina C. Meinke, and Maxim E. Darvin. "Penetration Depth of Propylene Glycol, Sodium Fluorescein and Nile Red into the Skin Using Non-Invasive Two-Photon Excited FLIM." Pharmaceutics 14, no. 9 (August 26, 2022): 1790. http://dx.doi.org/10.3390/pharmaceutics14091790.
Abstract:
The stratum corneum (SC) forms a strong barrier against topical drug delivery. Therefore, understanding the penetration depth and pathways into the SC is important for the efficiency of drug delivery and cosmetic safety. In this study, TPT-FLIM (two-photon tomography combined with fluorescence lifetime imaging) was applied as a non-invasive optical method for the visualization of skin structure and components to study penetration depths of exemplary substances, like hydrophilic propylene glycol (PG), sodium fluorescein (NaFl) and lipophilic Nile red (NR) into porcine ear skin ex vivo. Non-fluorescent PG was detected indirectly based on the pH-dependent increase in the fluorescence lifetime of SC components. The pH similarity between PG and viable epidermis limited the detection of PG. NaFl reached the viable epidermis, which was also proved by laser scanning microscopy. Tape stripping and confocal Raman micro-spectroscopy were performed additionally to study NaFl, which revealed penetration depths of ≈5 and ≈8 μm, respectively. Lastly, NR did not permeate the SC. We concluded that the amplitude-weighted mean fluorescence lifetime is the most appropriate FLIM parameter to build up penetration profiles. This work is anticipated to provide a non-invasive TPT-FLIM method for studying the penetration of topically applied drugs and cosmetics into the skin.
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Alfonso-Garcia, Alba, Stephanie A. Cevallos, Jee-Yon Lee, Cai Li, Julien Bec, Andreas J. Bäumler, and Laura Marcu. "Assessment of Murine Colon Inflammation Using Intraluminal Fluorescence Lifetime Imaging." Molecules 27, no. 4 (February 15, 2022): 1317. http://dx.doi.org/10.3390/molecules27041317.
Abstract:
Inflammatory bowel disease (IBD) is typically diagnosed by exclusion years after its onset. Current diagnostic methods are indirect, destructive, or target overt disease. Screening strategies that can detect low-grade inflammation in the colon would improve patient prognosis and alleviate associated healthcare costs. Here, we test the feasibility of fluorescence lifetime imaging (FLIm) to detect inflammation from thick tissue in a non-destructive and label-free approach based on tissue autofluorescence. A pulse sampling FLIm instrument with 355 nm excitation was coupled to a rotating side-viewing endoscopic probe for high speed (10 mm/s) intraluminal imaging of the entire mucosal surface (50–80 mm) of freshly excised mice colons. Current results demonstrate that tissue autofluorescence lifetime was sensitive to the colon anatomy and the colonocyte layer. Moreover, mice under DSS-induced colitis and 5-ASA treatments showed changes in lifetime values that were qualitatively related to inflammatory markers consistent with alterations in epithelial bioenergetics (switch between β-oxidation and aerobic glycolysis) and physical structure (colon length). This study demonstrates the ability of intraluminal FLIm to image mucosal lifetime changes in response to inflammatory treatments and supports the development of FLIm as an in vivo imaging technique for monitoring the onset, progression, and treatment of inflammatory diseases.
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Wang, Shiqi, Binglin Shen, Sheng Ren, Yihua Zhao, Silu Zhang, Junle Qu, and Liwei Liu. "Implementation and application of FRET–FLIM technology." Journal of Innovative Optical Health Sciences 12, no. 05 (September 2019): 1930010. http://dx.doi.org/10.1142/s1793545819300106.
Abstract:
With the development of the new detection methods and the function of fluorescent molecule, researchers hope to further explore the internal mechanisms of organisms, monitor changes in the intracellular microenvironment, and dynamic processes of molecular interactions in cells. Fluorescence resonance energy transfer (FRET) describes the energy transfer process between donor fluorescent molecules and acceptor fluorescent molecules. It is an important means to detect protein–protein interactions and protein conformation changes in cells. Fluorescence lifetime imaging microscopy (FLIM) enables noninvasive measurement of the fluorescence lifetime of fluorescent particles in vivo. The FRET–FLIM technology, which is use FLIM to quantify and analyze FRET, enables real-time monitoring of dynamic changes of proteins in biological cells and analysis of protein interaction mechanisms. The distance between donor and acceptor and their respective fluorescent lifetime, which are of great importance for studying the mechanism of intracellular activity can be obtained by data analysis and algorithm fitting.
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Venturas, Marta, Jaimin S. Shah, Xingbo Yang, Tim H. Sanchez, William Conway, Denny Sakkas, and Dan J. Needleman. "Metabolic state of human blastocysts measured by fluorescence lifetime imaging microscopy." Human Reproduction 37, no. 3 (January 6, 2022): 411–27. http://dx.doi.org/10.1093/humrep/deab283.
Abstract:
Abstract STUDY QUESTION Can non-invasive metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) detect variations in metabolic profiles between discarded human blastocysts? SUMMARY ANSWER FLIM revealed extensive variations in the metabolic state of discarded human blastocysts associated with blastocyst development over 36 h, the day after fertilization and blastocyst developmental stage, as well as metabolic heterogeneity within individual blastocysts. WHAT IS KNOWN ALREADY Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in ART to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state. STUDY DESIGN, SIZE, DURATION We conducted a prospective observational study. We used 215 morphologically normal human embryos from 137 patients that were discarded and donated for research under an approved institutional review board protocol. These embryos were imaged using metabolic imaging via FLIM to measure the autofluorescence of two central coenzymes, nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD+), which are essential for cellular respiration and glycolysis. PARTICIPANTS/MATERIALS, SETTING, METHODS Here, we used non-invasive FLIM to measure the metabolic state of human blastocysts. We first studied spatial patterns in the metabolic state within human blastocysts and the association of the metabolic state of the whole blastocysts with stage of expansion, day of development since fertilization and morphology. We explored the sensitivity of this technique in detecting metabolic variations between blastocysts from the same patient and between patients. Next, we explored whether FLIM can quantitatively measure metabolic changes through human blastocyst expansion and hatching via time-lapse imaging. For all test conditions, the level of significance was set at P < 0.05 after correction for multiple comparisons using Benjamini–Hochberg’s false discovery rate. MAIN RESULTS AND THE ROLE OF CHANCE We found that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We found that metabolic variations between blastocyst are partially explained by both the time since fertilization and their developmental expansion stage (P < 0.05), but not their morphological grade. Substantial metabolic variations between blastocysts from the same patients remain, even after controlling for these factors. We also observe significant metabolic heterogeneity within individual blastocysts, including between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida (P < 0.05). And finally, we observed that the metabolic state of human blastocysts continuously varies over time. LIMITATIONS, REASONS FOR CAUTION Although we observed significant variations in metabolic parameters, our data are taken from human blastocysts that were discarded and donated for research and we do not know their clinical outcome. Moreover, the embryos used in this study are a mixture of aneuploid, euploid and embryos of unknown ploidy. WIDER IMPLICATIONS OF THE FINDINGS This work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism. These results further demonstrate that FLIM can provide biologically relevant information that may be valuable for the assessment of embryo quality. STUDY FUNDING/COMPETING INTEREST(S) Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. TRIAL REGISTRATION NUMBER N/A.
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Zhou, Fang, Xin Wang, Guangxin Wang, and Yanxia Zuo. "A Rapid Method for Detecting Microplastics Based on Fluorescence Lifetime Imaging Technology (FLIM)." Toxics 10, no. 3 (March 2, 2022): 118. http://dx.doi.org/10.3390/toxics10030118.
Abstract:
With the increasing use and release of plastic products, microplastics have rapidly accumulated in ecological environments. When microplastics enter the food chain, they cause serious harm to organisms and humans. Microplastics pollution has become a growing concern worldwide; however, there is still no standardized method for rapidly and accurately detecting microplastics. In this work, we used fluorescence lifetime imaging technology to detect four kinds of Nile red-stained and unstained microplastics, and the unique phasor fingerprints of different microplastics were obtained by phasor analysis. Tracing the corresponding pixels of the “fingerprint” in the fluorescence lifetime image allowed for the quick and intuitive identification of different microplastics and their location distributions in a mixed sample. In our work, compared with staining the four microplastics with a fluorescent dye, using the phasor “fingerprint library” formed by the autofluorescence lifetimes of the microplastics was more easily distinguished than microplastics in the mixed samples. The feasibility of this method was further tested by adding three single substances—SiO2, chitin and decabromodiphenyl ethane (DBDPE), and surface sediments to simulate interferent in the environment, and the results providing potential applications for the identification and analysis of microplastics in complex environments.
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Ma, Ning, Michelle A. Digman, Leonel Malacrida, and Enrico Gratton. "Measurements of absolute concentrations of NADH in cells using the phasor FLIM method." Biomedical Optics Express 7, no. 7 (June 1, 2016): 2441. http://dx.doi.org/10.1364/boe.7.002441.
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Ma, Ning, Hui Ren, Naveen Ramalingam, David King, Banafshé Larijani, and Stefanie S. Jeffrey. "Abstract 2337: Label-free single-cell drug response determined by fluorescence lifetime imaging microscopy (FLIM) and RNA sequencing." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2337. http://dx.doi.org/10.1158/1538-7445.am2022-2337.
Abstract:
Abstract Colorectal cancer (CRC) is the second-leading cause of cancer-related death worldwide, and treatment failure in patients with metastatic CRC portends a poor prognosis. Early detection of chemoresistance would help reduce the usage of ineffective and possibly toxic therapies and facilitate alternative treatment strategies. We present a new technique combining phasor-fluorescence lifetime imaging microscopy (phasor-FLIM) and a machine learning algorithm that quantitatively identifies chemoresistant single cells, which may also be potentially applicable for evaluating heterogeneous circulating tumor cells (CTCs) obtained by liquid biopsy. We used parental HCT116 CRC cells sensitive to 5-fluorouracil (5-FU), a common chemotherapeutic agent used in CRC, to generate a cell line resistant to 5-FU; we confirmed resistance by a cell proliferation assay (MTS). We then analyzed phasor-FLIM patterns in sensitive and resistant cells before and after treatment with 5-FU of two endogenously fluorescent biomarkers, NADH and NADPH, which are key co-enzymes involved in energy metabolism. This was performed at 21% and 1% oxygen, which simulates both standard cell culture and tumor hypoxic conditions. Differences in phasor-FLIM signatures were found in resistant cells that suggested upregulation of the electron transport chain under normoxia (21% oxygen) and reprogrammed glucose metabolism under hypoxia (1% oxygen). Such changes were confirmed using NADH, lactate, and pyruvate production colorimetric tests, which are readouts of energy metabolism. To improve the quantification of changes in phasor-FLIM patterns, a new drug resistance indexing algorithm was developed to separate sensitive and resistant cells based on fluorescence lifetime multiparametric analysis of the intrinsic fluorescent biomarkers at the single-cell level. We then used single-cell total RNA sequencing (scRNA-Seq) to explore gene expression differences between sensitive and resistant HCT116 cells. Principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) clustering showed a clear separation of 5-FU-sensitive and -resistant cells. There was increased expression of proliferation markers in resistant cells and upregulation of genes involved in electron transport from NADPH to ferredoxin, consistent with our single-cell phasor-FLIM findings. We demonstrate proof of concept that the phasor-FLIM method combined with our new drug resistance indexing algorithm provides a marker-free technology for quantitatively assaying drug response and discriminating drug resistance at single-cell resolution. Citation Format: Ning Ma, Hui Ren, Naveen Ramalingam, David King, Banafshé Larijani, Stefanie S. Jeffrey. Label-free single-cell drug response determined by fluorescence lifetime imaging microscopy (FLIM) and RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2337.
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Abdollahi, Elham, Gisela Taucher-Scholz, and Burkhard Jakob. "Application of fluorescence lifetime imaging microscopy of DNA binding dyes to assess radiation-induced chromatin compaction changes." International Journal of Molecular Sciences 19, no. 8 (August 14, 2018): 2399. http://dx.doi.org/10.3390/ijms19082399.
Abstract:
In recent years several approaches have been developed to address the chromatin status and its changes in eukaryotic cells under different conditions—but only few are applicable in living cells. Fluorescence lifetime imaging microscopy (FLIM) is a functional tool that can be used for the inspection of the molecular environment of fluorophores in living cells. Here, we present the use of single organic minor groove DNA binder dyes in FLIM for measuring chromatin changes following modulation of chromatin structure in living cells. Treatment with histone deacetylase inhibitors led to an increased fluorescence lifetime indicating global chromatin decompaction, whereas hyperosmolarity decreased the lifetime of the used dyes, thus reflecting the expected compaction. In addition, we demonstrate that time domain FLIM data based on single photon counting should be optimized using pile-up and counting loss correction, which affect the readout even at moderate average detector count rates in inhomogeneous samples. Using these corrections and utilizing Hoechst 34580 as chromatin compaction probe, we measured a pan nuclear increase in the lifetime following irradiation with X-rays in living NIH/3T3 cells thus providing a method to measure radiation-induced chromatin decompaction.
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Lou, Xuemei, Zhenhong Jia, Jie Yang, and Nikola Kasabov. "Change Detection in SAR Images Based on the ROF Model Semi-Implicit Denoising Method." Sensors 19, no. 5 (March 7, 2019): 1179. http://dx.doi.org/10.3390/s19051179.
Abstract:
The explicit solution of the traditional ROF model in image denoising has the disadvantages of unstable results and requiring many iterations. To solve the problem, a new method, ROF model semi-implicit denoising, is proposed in this paper and applied to change detections of synthetic aperture radar (SAR) images. All remote sensing images used in this article have been calibrated by ENVI software. First, the ROF model semi-implicit denoising method is used to denoise the remote sensing images. Second, for the denoised images, difference images are obtained by the logarithmic ratio and mean ratio methods. The final difference image is obtained by principal component analysis fusion (PCA fusion) of the two difference images. Finally, the final difference image is clustered by fuzzy local information C-means clustering (FLICM) to obtain the change regions. The research results show that the proposed method has high detection accuracy and time operation efficiency.
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Ren, Hang, and Taotao Hu. "A Local Neighborhood Robust Fuzzy Clustering Image Segmentation Algorithm Based on an Adaptive Feature Selection Gaussian Mixture Model." Sensors 20, no. 8 (April 22, 2020): 2391. http://dx.doi.org/10.3390/s20082391.
Abstract:
Since the fuzzy local information C-means (FLICM) segmentation algorithm cannot take into account the impact of different features on clustering segmentation results, a local fuzzy clustering segmentation algorithm based on a feature selection Gaussian mixture model was proposed. First, the constraints of the membership degree on the spatial distance were added to the local information function. Second, the feature saliency was introduced into the objective function. By using the Lagrange multiplier method, the optimal expression of the objective function was solved. Neighborhood weighting information was added to the iteration expression of the classification membership degree to obtain a local feature selection based on feature selection. Each of the improved FLICM algorithm, the fuzzy C-means with spatial constraints (FCM_S) algorithm, and the original FLICM algorithm were then used to cluster and segment the interference images of Gaussian noise, salt-and-pepper noise, multiplicative noise, and mixed noise. The performances of the peak signal-to-noise ratio and error rate of the segmentation results were compared with each other. At the same time, the iteration time and number of iterations used to converge the objective function of the algorithm were compared. In summary, the improved algorithm significantly improved the ability of image noise suppression under strong noise interference, improved the efficiency of operation, facilitated remote sensing image capture under strong noise interference, and promoted the development of a robust anti-noise fuzzy clustering algorithm.
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Sanchez, Tim, Marta Venturas, S. Ali Aghvami, Xingbo Yang, Seth Fraden, Denny Sakkas, and Daniel J. Needleman. "Combined noninvasive metabolic and spindle imaging as potential tools for embryo and oocyte assessment." Human Reproduction 34, no. 12 (December 1, 2019): 2349–61. http://dx.doi.org/10.1093/humrep/dez210.
Abstract:
Abstract STUDY QUESTION Is the combined use of fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging and second harmonic generation (SHG) spindle imaging a feasible and safe approach for noninvasive embryo assessment? SUMMARY ANSWER Metabolic imaging can sensitively detect meaningful metabolic changes in embryos, SHG produces high-quality images of spindles and the methods do not significantly impair embryo viability. WHAT IS KNOWN ALREADY Proper metabolism is essential for embryo viability. Metabolic imaging is a well-tested method for measuring metabolism of cells and tissues, but it is unclear if it is sensitive enough and safe enough for use in embryo assessment. STUDY DESIGN, SIZE, DURATION This study consisted of time-course experiments and control versus treatment experiments. We monitored the metabolism of 25 mouse oocytes with a noninvasive metabolic imaging system while exposing them to oxamate (cytoplasmic lactate dehydrogenase inhibitor) and rotenone (mitochondrial oxidative phosphorylation inhibitor) in series. Mouse embryos (n = 39) were measured every 2 h from the one-cell stage to blastocyst in order to characterize metabolic changes occurring during pre-implantation development. To assess the safety of FLIM illumination, n = 144 illuminated embryos were implanted into n = 12 mice, and n = 108 nonilluminated embryos were implanted into n = 9 mice. PARTICIPANTS/MATERIALS, SETTING, METHODS Experiments were performed in mouse embryos and oocytes. Samples were monitored with noninvasive, FLIM-based metabolic imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Between NADH cytoplasm, NADH mitochondria and FAD mitochondria, a single metabolic measurement produces up to 12 quantitative parameters for characterizing the metabolic state of an embryo. For safety experiments, live birth rates and pup weights (mean ± SEM) were used as endpoints. For all test conditions, the level of significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE Measured FLIM parameters were highly sensitive to metabolic changes due to both metabolic perturbations and embryo development. For oocytes, metabolic parameter values were compared before and after exposure to oxamate and rotenone. The metabolic measurements provided a basis for complete separation of the data sets. For embryos, metabolic parameter values were compared between the first division and morula stages, morula and blastocyst and first division and blastocyst. The metabolic measurements again completely separated the data sets. Exposure of embryos to excessive illumination dosages (24 measurements) had no significant effect on live birth rate (5.1 ± 0.94 pups/mouse for illuminated group; 5.7 ± 1.74 pups/mouse for control group) or pup weights (1.88 ± 0.10 g for illuminated group; 1.89 ± 0.11 g for control group). LIMITATIONS, REASONS FOR CAUTION The study was performed using a mouse model, so conclusions concerning sensitivity and safety may not generalize to human embryos. A limitation of the live birth data is also that although cages were routinely monitored, we could not preclude that some runt pups may have been eaten. WIDER IMPLICATIONS OF THE FINDINGS Promising proof-of-concept results demonstrate that FLIM with SHG provide detailed biological information that may be valuable for the assessment of embryo and oocyte quality. Live birth experiments support the method’s safety, arguing for further studies of the clinical utility of these techniques. STUDY FUNDING/COMPETING INTEREST(S) Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University and by the Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Institutes of Health Award UL1 TR001102), by NSF grants DMR-0820484 and PFI-TT-1827309 and by NIH grant R01HD092550-01. T.S. was supported by a National Science Foundation Postdoctoral Research Fellowship in Biology grant (1308878). S.F. and S.A. were supported by NSF MRSEC DMR-1420382. Becker and Hickl GmbH sponsored the research with the loaning of equipment for FLIM. T.S. and D.N. are cofounders and shareholders of LuminOva, Inc., and co-hold patents (US20150346100A1 and US20170039415A1) for metabolic imaging methods. D.S. is on the scientific advisory board for Cooper Surgical and has stock options with LuminOva, Inc.
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Koenig, Marcelle, Sandra Orthaus-Mueller, Rhys Dowler, Benedikt Kraemer, Astrid Tannert, Olaf Schulz, Tino Roehlicke, et al. "Rapid Flim: The New and Innovative Method for Ultra-Fast Imaging of Biological Processes." Biophysical Journal 112, no. 3 (February 2017): 298a. http://dx.doi.org/10.1016/j.bpj.2016.11.1614.
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Leray, A., C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot. "Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis." Cytometry Part A 79A, no. 2 (December 2, 2010): 149–58. http://dx.doi.org/10.1002/cyto.a.20996.