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KNIME GmbH data processing workflow
Data Processing Workflow, supplied by KNIME GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/workflow+process/data+processing+workflow/pmc12172290-492-4-9
Average 90 stars, based on 1 article reviews
data processing workflow - by Bioz Stars, 2026-10
90/100 stars

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Sequencing:

Article Title: Comparative Studies on Resampling Techniques in Machine Learning and Deep Learning Models for Drug-Target Interaction Prediction
Article Snippet: the fingerprints were written into a new CSV file for further processing. .. The overall process is shown in the KNIME workflow in . .. After successfully converting from SMILES to ECFP4, the inactive data for each of

Article Title: Comparison of Descriptor- and Fingerprint Sets in Machine Learning Models for ADME-Tox Targets
Article Snippet: is important to note that there was no overlap in one iteration circle between the cross-validation and internal validation set. .. The complete process can be followed in detail in our KNIME workflow in the . .. The predicted class probabilities with each algorithm were grouped by the validation sets (cross-validation, internal, and external

Article Title: Comparison of Descriptor- and Fingerprint Sets in Machine Learning Models for ADME-Tox Targets.
Article Snippet: is important to note that there was no overlap in one iteration circle between the cross-validation and internal validation set. .. The complete process can be followed in detail in our KNIME workflow in the Supplementary material. .. The predicted class probabilities with each algorithm were grouped by the validation sets (cross-validation, internal, and external

Article Title: Five Years of the KNIME Vernalis Cheminformatics Community Contribution
Article Snippet: checked by inspecting a raw text file written out on a nightly basis by the instrument control software, and if necessary, a helium fill was booked. .. This process has been replaced by a KNIME workflow which runs on a nightly basis. .. On Mondays, an email is sent to the spectroscopists showing the nightly helium level for the last 2 weeks graphically (Fig. ).

Article Title: Rapid optimisation of fragments and hits to lead compounds from screening of crude reaction mixtures
Article Snippet: of results may take a number of days, and at an early stage in a project is likely to be based on limited structural information availability. .. This process can take as little as 15 minutes using the KNIME workflow. .. Reagent picking and plating 25 | P a g e If a set of library reagents is ordered in a 96-well format, the time spent on this step is the time awaiting

Article Title: A case study evaluating the portability of an executable computable phenotype algorithm across multiple institutions and electronic health record environments
Article Snippet: the actual service was available, but may be responding in this way to the frequency of the requests from KNIME. , This has not yet been resolved.. .. KNIME workflow would not complete execution against the Mayo i2b2 instance. , The i2b2 instance was the largest of the sites involved with this study, at over 9 million patients. We confirmed that this was not a limitation of the i2b2 platform, by manually creating and executing the BPH algorithm using the i2b2 web interface. Instead, we identified scalability issues with our KNIME workflow construction process, which led to computational bottlenecks, especially when processing queries against a repository of this size. , This has not yet been resolved.. .. Execution speed of the workflow is slow. , Given the decompositional nature of the query, for which individual criteria are each queried and then late

Article Title: A computationally driven analysis of the polyphenol-protein interactome
Article Snippet: mainly alkaloids), and steroids (80) were removed to yield 36,064 unique polyphenols present in DNP. .. The process was designed as a KNIME workflow . .. Chemical Entities of Biological Interest (CHEBI) structural entries (90,129) were identified in the DNP bu

Article Title: Structure-based prediction of Wnt binding affinities for Frizzled-type cysteine-rich domains
Article Snippet: generated complexes were subject to refinement using Prime 4.1. .. The refinement process was facilitated through the use of a KNIME workflow ( supplemental Fig. S4 ). .. In each complex, non-template residues and residues within 6.0 Å of

Extraction:

Article Title: Comparative Studies on Resampling Techniques in Machine Learning and Deep Learning Models for Drug-Target Interaction Prediction
Article Snippet: the fingerprints were written into a new CSV file for further processing. .. The overall process is shown in the KNIME workflow in . .. After successfully converting from SMILES to ECFP4, the inactive data for each of

Article Title: Comparison of Descriptor- and Fingerprint Sets in Machine Learning Models for ADME-Tox Targets
Article Snippet: is important to note that there was no overlap in one iteration circle between the cross-validation and internal validation set. .. The complete process can be followed in detail in our KNIME workflow in the . .. The predicted class probabilities with each algorithm were grouped by the validation sets (cross-validation, internal, and external

Article Title: Comparison of Descriptor- and Fingerprint Sets in Machine Learning Models for ADME-Tox Targets.
Article Snippet: is important to note that there was no overlap in one iteration circle between the cross-validation and internal validation set. .. The complete process can be followed in detail in our KNIME workflow in the Supplementary material. .. The predicted class probabilities with each algorithm were grouped by the validation sets (cross-validation, internal, and external

Article Title: Five Years of the KNIME Vernalis Cheminformatics Community Contribution
Article Snippet: checked by inspecting a raw text file written out on a nightly basis by the instrument control software, and if necessary, a helium fill was booked. .. This process has been replaced by a KNIME workflow which runs on a nightly basis. .. On Mondays, an email is sent to the spectroscopists showing the nightly helium level for the last 2 weeks graphically (Fig. ).

Article Title: Rapid optimisation of fragments and hits to lead compounds from screening of crude reaction mixtures
Article Snippet: of results may take a number of days, and at an early stage in a project is likely to be based on limited structural information availability. .. This process can take as little as 15 minutes using the KNIME workflow. .. Reagent picking and plating 25 | P a g e If a set of library reagents is ordered in a 96-well format, the time spent on this step is the time awaiting

Article Title: A case study evaluating the portability of an executable computable phenotype algorithm across multiple institutions and electronic health record environments
Article Snippet: the actual service was available, but may be responding in this way to the frequency of the requests from KNIME. , This has not yet been resolved.. .. KNIME workflow would not complete execution against the Mayo i2b2 instance. , The i2b2 instance was the largest of the sites involved with this study, at over 9 million patients. We confirmed that this was not a limitation of the i2b2 platform, by manually creating and executing the BPH algorithm using the i2b2 web interface. Instead, we identified scalability issues with our KNIME workflow construction process, which led to computational bottlenecks, especially when processing queries against a repository of this size. , This has not yet been resolved.. .. Execution speed of the workflow is slow. , Given the decompositional nature of the query, for which individual criteria are each queried and then late

Article Title: A computationally driven analysis of the polyphenol-protein interactome
Article Snippet: mainly alkaloids), and steroids (80) were removed to yield 36,064 unique polyphenols present in DNP. .. The process was designed as a KNIME workflow . .. Chemical Entities of Biological Interest (CHEBI) structural entries (90,129) were identified in the DNP bu

Article Title: Structure-based prediction of Wnt binding affinities for Frizzled-type cysteine-rich domains
Article Snippet: generated complexes were subject to refinement using Prime 4.1. .. The refinement process was facilitated through the use of a KNIME workflow ( supplemental Fig. S4 ). .. In each complex, non-template residues and residues within 6.0 Å of

Modification:

Article Title: Comparative Studies on Resampling Techniques in Machine Learning and Deep Learning Models for Drug-Target Interaction Prediction
Article Snippet: the fingerprints were written into a new CSV file for further processing. .. The overall process is shown in the KNIME workflow in . .. After successfully converting from SMILES to ECFP4, the inactive data for each of

Article Title: Comparison of Descriptor- and Fingerprint Sets in Machine Learning Models for ADME-Tox Targets
Article Snippet: is important to note that there was no overlap in one iteration circle between the cross-validation and internal validation set. .. The complete process can be followed in detail in our KNIME workflow in the . .. The predicted class probabilities with each algorithm were grouped by the validation sets (cross-validation, internal, and external

Article Title: Comparison of Descriptor- and Fingerprint Sets in Machine Learning Models for ADME-Tox Targets.
Article Snippet: is important to note that there was no overlap in one iteration circle between the cross-validation and internal validation set. .. The complete process can be followed in detail in our KNIME workflow in the Supplementary material. .. The predicted class probabilities with each algorithm were grouped by the validation sets (cross-validation, internal, and external

Article Title: Five Years of the KNIME Vernalis Cheminformatics Community Contribution
Article Snippet: checked by inspecting a raw text file written out on a nightly basis by the instrument control software, and if necessary, a helium fill was booked. .. This process has been replaced by a KNIME workflow which runs on a nightly basis. .. On Mondays, an email is sent to the spectroscopists showing the nightly helium level for the last 2 weeks graphically (Fig. ).

Article Title: Rapid optimisation of fragments and hits to lead compounds from screening of crude reaction mixtures
Article Snippet: of results may take a number of days, and at an early stage in a project is likely to be based on limited structural information availability. .. This process can take as little as 15 minutes using the KNIME workflow. .. Reagent picking and plating 25 | P a g e If a set of library reagents is ordered in a 96-well format, the time spent on this step is the time awaiting

Article Title: A case study evaluating the portability of an executable computable phenotype algorithm across multiple institutions and electronic health record environments
Article Snippet: the actual service was available, but may be responding in this way to the frequency of the requests from KNIME. , This has not yet been resolved.. .. KNIME workflow would not complete execution against the Mayo i2b2 instance. , The i2b2 instance was the largest of the sites involved with this study, at over 9 million patients. We confirmed that this was not a limitation of the i2b2 platform, by manually creating and executing the BPH algorithm using the i2b2 web interface. Instead, we identified scalability issues with our KNIME workflow construction process, which led to computational bottlenecks, especially when processing queries against a repository of this size. , This has not yet been resolved.. .. Execution speed of the workflow is slow. , Given the decompositional nature of the query, for which individual criteria are each queried and then late

Article Title: A computationally driven analysis of the polyphenol-protein interactome
Article Snippet: mainly alkaloids), and steroids (80) were removed to yield 36,064 unique polyphenols present in DNP. .. The process was designed as a KNIME workflow . .. Chemical Entities of Biological Interest (CHEBI) structural entries (90,129) were identified in the DNP bu

Article Title: Structure-based prediction of Wnt binding affinities for Frizzled-type cysteine-rich domains
Article Snippet: generated complexes were subject to refinement using Prime 4.1. .. The refinement process was facilitated through the use of a KNIME workflow ( supplemental Fig. S4 ). .. In each complex, non-template residues and residues within 6.0 Å of



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KNIME GmbH data processing workflow
TDM imaging of mammalian cells. (a) <t>Workflow</t> of temporal domain multiplexing, (i) Multi-frame time series acquisition of Hella cell co-transfect with two FPs expressed in the nucleus and cytoplasm, the Raw data set is processed by background subtraction and denoising to produce the input dataset, the input dataset is uploaded to the Temporal domain <t>multiplexing</t> <t>MATLAB</t> script for unmixing to produced output images of individual channels. (b) Phasor plot and lifetime for Mito-mCardinal (green) (1.593ns) and H2B-mfRFP (red) (0.922ns) (c) Reference photobleaching curve trace for Mito-mCardinal (green) and H2B-mfRFP (red). (d) Unmixing results of FLIM microscopy (top), The raw image was unmix using phasor plot analysis resulting in two different FPs Mito-mCardinal (green) and H2B-mfRFP (red) TDM (bottom) unmixing result from the raw image (gray) was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging containing two different but spectrally similar (far-red) fluorescent proteins. the output image is a composite of Mito-mCardinal (green) and H2B-mfRFP (red). Scale bar 50µm. (e) Cross-talk plot showing the fluorescence intensity contribution of mito (magenta) and H2B (red) in FLIM mito-mCardinal channel (above) and FLIM mfRFP channel (below), in each plot, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (f) Per pixel Line profile analysis of FLIM raw image (grey) and TDM raw image (orange) Statistics Spearman correlation (0.95859) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length (g) Per pixel Line profile analysis of FLIM mito-mCardinal image (grey) and TDM mito-mCardinal image (orange) Statistical Spearman correlation (0.88849) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length. (h) Per pixel Line profile analysis of FLIM H2B-mCardinal image (grey) and TDM H2B-mCardinal image (orange) Statistics Spearman correlation (0.881771) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length. (i) Cross-talk plot showing the fluorescence intensity contribution of mito (magenta) and H2B (red) in TDM mito-mCardinal channel (above) and TDM mfRFP channel (below), in each plot, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (j) Simulation of Temporal domain multiplexing with two far-red fluorescent proteins, A 21-frame line plot obtained from previously acquired HeLa cell ROI illustrating the unique photobleaching curve of mfRFP and mfRFP-A that contribute to the overall signals, Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time. The line plot is subsequently used as reference trace for the unmixing algorithms (k)Acquired brief movie (Time series image) of HeLa cell expressing H2B-mfRFP and mfRFP-A-cytoplasm acquired using 594nmSoRA excitation laser channel. The unmixing result (MATLAB) the signal contribution of each FPS (OUTPUT) to their respective corresponding structures and the merged image depicting the complete HeLa cells structure (H2B and cytoplasm). Note that the image colors used above red-H2B and green-cytoplasm are pseudo colors to enhance visualization and both H2B and the cytoplasm were indeed red. Scale bar 25μm. (l) crosstalk plot showing the fluorescence intensity contributions of mfRFP and mfRFP-A in the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (m) Acquired photobleaching reference traces of mCardinal and mfRFP-A in life HeLa-cell, n=5 cells from 2 briefed movies, 41 frames long, from 1 cell culture batch. Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time (n), The input image was first refined via background subtraction (ImageJ) followed by denoising (Nikon Element AR software), the input image was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging(red), containing two different but spectrally similar (far-red) fluorescent proteins. the output image is a composite of mfRFP-H2B and Mito-mCardinal. (Scale-Bar 100um). (o) crosstalk plot showing the fluorescence intensity contributions of mCardinal and mfRFP-A in the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (p) Simulation of TDM using Confocal microscope acquired photobleaching reference traces of H2B-mCardinal, H2B-mRuby2 and H2B-mfRFP in fixed HeLa-cell, 25 frames long (q), the input image was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging(red), containing three different but spectrally similar (far-red) fluorescent proteins of H2B-mCardina, H2B-mruby2 and H2B-mfRFP, and overlay image is a composite. (Scale-Bar 100um). (r) crosstalk plot showing the fluorescence intensity contributions of H2B-mCardinal, H2B-mRuby2 and H2B-mfRFPin the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (s) Temporal domain multiplexing of six z-plain projection of two red fluorescent proteins(FPs). (A) Acquired photobleaching reference traces of mfRFP and mCardinal in life HeLa-cell, n=10 cells from 2 briefed movies, 41 frames long, from 1 cell culture batch. Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time). (t) Demultiplexing of co-expressed Keratin-targeted mCardinal and H2B-targeted mfRFP in HeLa cell. the raw image (top) was supplied to MATLAB script for linear unmixing of each reference at each pixel, the Merge TDM image (bottom) is a composite of mfRFP-H2B and mCardinal-Keratin. The output image mfRFP-H2B and mCardinal-Keratin are products of linear un-mixing (Scale-Bar 100um) (u) 3D image obtained from the z-stack of the unmixed z-planes images.
Data Processing Workflow, supplied by KNIME GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/workflow+process/data+processing+workflow/pmc12172290-492-4-9
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Image Search Results


Putative producers of compounds of interest are prioritized through cross-validation of the top ranked strains via Protein Language Model (PLM) embeddings and via our in-house developed Workflow for Intelligent Structural Elucidation (WISE).

Journal: npj Antimicrobials and Resistance

Article Title: Accelerating natural product discovery with linked MS-genomics and language/transformer-based models

doi: 10.1038/s44259-026-00206-7

Figure Lengend Snippet: Putative producers of compounds of interest are prioritized through cross-validation of the top ranked strains via Protein Language Model (PLM) embeddings and via our in-house developed Workflow for Intelligent Structural Elucidation (WISE).

Article Snippet: The data processing Workflow for Intelligent Structural Elucidation (WISE) is partially based on Mestrenova v15.0.1-35756 which is commercially available from Mestrelab Research S. L. ( www.mestrelab.com ).

Techniques: Biomarker Discovery

A Overview of the cheminformatics analysis workflow (WISE) to annotate individual molecules from raw liquid chromatography-tandem mass spectral (LC-MS/MS) data of fermentation extracts. B Distribution of WISE scores for correct structures (Real) versus isobaric decoy structures (within 0.005 m/z) with different molecular formulae (Decoy) from the Critical Assessment for Small Molecule Identification (CASMI2022) dataset. Low and high WISE score thresholds of 0.35 and 0.65 respectively are annotated for reference. C Receiver Operating Characteristic (ROC) curve demonstrating the discrimination of correct structures from decoys with differing molecular formulae within a 0.005 m/z window. AUC = Area Under Curve. CI = Confidence Interval. Detailed data in Table .

Journal: npj Antimicrobials and Resistance

Article Title: Accelerating natural product discovery with linked MS-genomics and language/transformer-based models

doi: 10.1038/s44259-026-00206-7

Figure Lengend Snippet: A Overview of the cheminformatics analysis workflow (WISE) to annotate individual molecules from raw liquid chromatography-tandem mass spectral (LC-MS/MS) data of fermentation extracts. B Distribution of WISE scores for correct structures (Real) versus isobaric decoy structures (within 0.005 m/z) with different molecular formulae (Decoy) from the Critical Assessment for Small Molecule Identification (CASMI2022) dataset. Low and high WISE score thresholds of 0.35 and 0.65 respectively are annotated for reference. C Receiver Operating Characteristic (ROC) curve demonstrating the discrimination of correct structures from decoys with differing molecular formulae within a 0.005 m/z window. AUC = Area Under Curve. CI = Confidence Interval. Detailed data in Table .

Article Snippet: The data processing Workflow for Intelligent Structural Elucidation (WISE) is partially based on Mestrenova v15.0.1-35756 which is commercially available from Mestrelab Research S. L. ( www.mestrelab.com ).

Techniques: Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy

A Elucidated biosynthetic gene cluster BGC0000709, responsible for neomycin production. B Similarity (%) of each embedded protein and sequence similarity (%) of the top-scoring protein matches. C Predicted protein structures of the top matches for Fe-S oxidoreductase ( CAF33317 ) from neomycin BGC. D Heatmap of WISE scores for strain-media combinations derived from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) data of fermentation extracts. Only strains with at least 1 media combination with non-zero WISE score are shown. Activated strains (if any) are referred to by their parent strain instead of individually (details of the activated strains are in Table ). E Structure of neomycin B. F Workflow of the co-occurrence strategy and matrix ranking table with 0.35 WISE score and 0.75 PLM score cutoff. Strains that have been cross validated are highlighted in green or yellow, indicating whether these compounds were present (green) or absent (yellow). Lines indicating higher confidence thresholds of 0.65 WISE score (in red) and 2.5 PLM score (in blue) are also included.

Journal: npj Antimicrobials and Resistance

Article Title: Accelerating natural product discovery with linked MS-genomics and language/transformer-based models

doi: 10.1038/s44259-026-00206-7

Figure Lengend Snippet: A Elucidated biosynthetic gene cluster BGC0000709, responsible for neomycin production. B Similarity (%) of each embedded protein and sequence similarity (%) of the top-scoring protein matches. C Predicted protein structures of the top matches for Fe-S oxidoreductase ( CAF33317 ) from neomycin BGC. D Heatmap of WISE scores for strain-media combinations derived from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) data of fermentation extracts. Only strains with at least 1 media combination with non-zero WISE score are shown. Activated strains (if any) are referred to by their parent strain instead of individually (details of the activated strains are in Table ). E Structure of neomycin B. F Workflow of the co-occurrence strategy and matrix ranking table with 0.35 WISE score and 0.75 PLM score cutoff. Strains that have been cross validated are highlighted in green or yellow, indicating whether these compounds were present (green) or absent (yellow). Lines indicating higher confidence thresholds of 0.65 WISE score (in red) and 2.5 PLM score (in blue) are also included.

Article Snippet: The data processing Workflow for Intelligent Structural Elucidation (WISE) is partially based on Mestrenova v15.0.1-35756 which is commercially available from Mestrelab Research S. L. ( www.mestrelab.com ).

Techniques: Sequencing, Derivative Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

TDM imaging of mammalian cells. (a) Workflow of temporal domain multiplexing, (i) Multi-frame time series acquisition of Hella cell co-transfect with two FPs expressed in the nucleus and cytoplasm, the Raw data set is processed by background subtraction and denoising to produce the input dataset, the input dataset is uploaded to the Temporal domain multiplexing MATLAB script for unmixing to produced output images of individual channels. (b) Phasor plot and lifetime for Mito-mCardinal (green) (1.593ns) and H2B-mfRFP (red) (0.922ns) (c) Reference photobleaching curve trace for Mito-mCardinal (green) and H2B-mfRFP (red). (d) Unmixing results of FLIM microscopy (top), The raw image was unmix using phasor plot analysis resulting in two different FPs Mito-mCardinal (green) and H2B-mfRFP (red) TDM (bottom) unmixing result from the raw image (gray) was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging containing two different but spectrally similar (far-red) fluorescent proteins. the output image is a composite of Mito-mCardinal (green) and H2B-mfRFP (red). Scale bar 50µm. (e) Cross-talk plot showing the fluorescence intensity contribution of mito (magenta) and H2B (red) in FLIM mito-mCardinal channel (above) and FLIM mfRFP channel (below), in each plot, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (f) Per pixel Line profile analysis of FLIM raw image (grey) and TDM raw image (orange) Statistics Spearman correlation (0.95859) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length (g) Per pixel Line profile analysis of FLIM mito-mCardinal image (grey) and TDM mito-mCardinal image (orange) Statistical Spearman correlation (0.88849) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length. (h) Per pixel Line profile analysis of FLIM H2B-mCardinal image (grey) and TDM H2B-mCardinal image (orange) Statistics Spearman correlation (0.881771) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length. (i) Cross-talk plot showing the fluorescence intensity contribution of mito (magenta) and H2B (red) in TDM mito-mCardinal channel (above) and TDM mfRFP channel (below), in each plot, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (j) Simulation of Temporal domain multiplexing with two far-red fluorescent proteins, A 21-frame line plot obtained from previously acquired HeLa cell ROI illustrating the unique photobleaching curve of mfRFP and mfRFP-A that contribute to the overall signals, Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time. The line plot is subsequently used as reference trace for the unmixing algorithms (k)Acquired brief movie (Time series image) of HeLa cell expressing H2B-mfRFP and mfRFP-A-cytoplasm acquired using 594nmSoRA excitation laser channel. The unmixing result (MATLAB) the signal contribution of each FPS (OUTPUT) to their respective corresponding structures and the merged image depicting the complete HeLa cells structure (H2B and cytoplasm). Note that the image colors used above red-H2B and green-cytoplasm are pseudo colors to enhance visualization and both H2B and the cytoplasm were indeed red. Scale bar 25μm. (l) crosstalk plot showing the fluorescence intensity contributions of mfRFP and mfRFP-A in the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (m) Acquired photobleaching reference traces of mCardinal and mfRFP-A in life HeLa-cell, n=5 cells from 2 briefed movies, 41 frames long, from 1 cell culture batch. Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time (n), The input image was first refined via background subtraction (ImageJ) followed by denoising (Nikon Element AR software), the input image was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging(red), containing two different but spectrally similar (far-red) fluorescent proteins. the output image is a composite of mfRFP-H2B and Mito-mCardinal. (Scale-Bar 100um). (o) crosstalk plot showing the fluorescence intensity contributions of mCardinal and mfRFP-A in the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (p) Simulation of TDM using Confocal microscope acquired photobleaching reference traces of H2B-mCardinal, H2B-mRuby2 and H2B-mfRFP in fixed HeLa-cell, 25 frames long (q), the input image was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging(red), containing three different but spectrally similar (far-red) fluorescent proteins of H2B-mCardina, H2B-mruby2 and H2B-mfRFP, and overlay image is a composite. (Scale-Bar 100um). (r) crosstalk plot showing the fluorescence intensity contributions of H2B-mCardinal, H2B-mRuby2 and H2B-mfRFPin the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (s) Temporal domain multiplexing of six z-plain projection of two red fluorescent proteins(FPs). (A) Acquired photobleaching reference traces of mfRFP and mCardinal in life HeLa-cell, n=10 cells from 2 briefed movies, 41 frames long, from 1 cell culture batch. Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time). (t) Demultiplexing of co-expressed Keratin-targeted mCardinal and H2B-targeted mfRFP in HeLa cell. the raw image (top) was supplied to MATLAB script for linear unmixing of each reference at each pixel, the Merge TDM image (bottom) is a composite of mfRFP-H2B and mCardinal-Keratin. The output image mfRFP-H2B and mCardinal-Keratin are products of linear un-mixing (Scale-Bar 100um) (u) 3D image obtained from the z-stack of the unmixed z-planes images.

Journal: bioRxiv

Article Title: A Set of Novel Far-Red Fluorescent Proteins for Temporal Domain Multiplexing and Super-Resolution Imaging

doi: 10.1101/2025.06.01.657196

Figure Lengend Snippet: TDM imaging of mammalian cells. (a) Workflow of temporal domain multiplexing, (i) Multi-frame time series acquisition of Hella cell co-transfect with two FPs expressed in the nucleus and cytoplasm, the Raw data set is processed by background subtraction and denoising to produce the input dataset, the input dataset is uploaded to the Temporal domain multiplexing MATLAB script for unmixing to produced output images of individual channels. (b) Phasor plot and lifetime for Mito-mCardinal (green) (1.593ns) and H2B-mfRFP (red) (0.922ns) (c) Reference photobleaching curve trace for Mito-mCardinal (green) and H2B-mfRFP (red). (d) Unmixing results of FLIM microscopy (top), The raw image was unmix using phasor plot analysis resulting in two different FPs Mito-mCardinal (green) and H2B-mfRFP (red) TDM (bottom) unmixing result from the raw image (gray) was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging containing two different but spectrally similar (far-red) fluorescent proteins. the output image is a composite of Mito-mCardinal (green) and H2B-mfRFP (red). Scale bar 50µm. (e) Cross-talk plot showing the fluorescence intensity contribution of mito (magenta) and H2B (red) in FLIM mito-mCardinal channel (above) and FLIM mfRFP channel (below), in each plot, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (f) Per pixel Line profile analysis of FLIM raw image (grey) and TDM raw image (orange) Statistics Spearman correlation (0.95859) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length (g) Per pixel Line profile analysis of FLIM mito-mCardinal image (grey) and TDM mito-mCardinal image (orange) Statistical Spearman correlation (0.88849) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length. (h) Per pixel Line profile analysis of FLIM H2B-mCardinal image (grey) and TDM H2B-mCardinal image (orange) Statistics Spearman correlation (0.881771) the y-axis represents the normalized fluorescence intensity, and the x-axis represent the length. (i) Cross-talk plot showing the fluorescence intensity contribution of mito (magenta) and H2B (red) in TDM mito-mCardinal channel (above) and TDM mfRFP channel (below), in each plot, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (j) Simulation of Temporal domain multiplexing with two far-red fluorescent proteins, A 21-frame line plot obtained from previously acquired HeLa cell ROI illustrating the unique photobleaching curve of mfRFP and mfRFP-A that contribute to the overall signals, Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time. The line plot is subsequently used as reference trace for the unmixing algorithms (k)Acquired brief movie (Time series image) of HeLa cell expressing H2B-mfRFP and mfRFP-A-cytoplasm acquired using 594nmSoRA excitation laser channel. The unmixing result (MATLAB) the signal contribution of each FPS (OUTPUT) to their respective corresponding structures and the merged image depicting the complete HeLa cells structure (H2B and cytoplasm). Note that the image colors used above red-H2B and green-cytoplasm are pseudo colors to enhance visualization and both H2B and the cytoplasm were indeed red. Scale bar 25μm. (l) crosstalk plot showing the fluorescence intensity contributions of mfRFP and mfRFP-A in the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (m) Acquired photobleaching reference traces of mCardinal and mfRFP-A in life HeLa-cell, n=5 cells from 2 briefed movies, 41 frames long, from 1 cell culture batch. Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time (n), The input image was first refined via background subtraction (ImageJ) followed by denoising (Nikon Element AR software), the input image was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging(red), containing two different but spectrally similar (far-red) fluorescent proteins. the output image is a composite of mfRFP-H2B and Mito-mCardinal. (Scale-Bar 100um). (o) crosstalk plot showing the fluorescence intensity contributions of mCardinal and mfRFP-A in the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (p) Simulation of TDM using Confocal microscope acquired photobleaching reference traces of H2B-mCardinal, H2B-mRuby2 and H2B-mfRFP in fixed HeLa-cell, 25 frames long (q), the input image was supplied to MATLAB script for linear unmixing of each reference at each pixel (the input images are brief movies obtained from single channel imaging(red), containing three different but spectrally similar (far-red) fluorescent proteins of H2B-mCardina, H2B-mruby2 and H2B-mfRFP, and overlay image is a composite. (Scale-Bar 100um). (r) crosstalk plot showing the fluorescence intensity contributions of H2B-mCardinal, H2B-mRuby2 and H2B-mfRFPin the TDM output channels, the y-axis represents the fluorescence intensity of selected pixel, and the x-axis represent the fluorophore in each channel. (s) Temporal domain multiplexing of six z-plain projection of two red fluorescent proteins(FPs). (A) Acquired photobleaching reference traces of mfRFP and mCardinal in life HeLa-cell, n=10 cells from 2 briefed movies, 41 frames long, from 1 cell culture batch. Data are shown as ± mean standard deviation SD. Illumination(594nm at 100% laser power, 500ms Exposure time). (t) Demultiplexing of co-expressed Keratin-targeted mCardinal and H2B-targeted mfRFP in HeLa cell. the raw image (top) was supplied to MATLAB script for linear unmixing of each reference at each pixel, the Merge TDM image (bottom) is a composite of mfRFP-H2B and mCardinal-Keratin. The output image mfRFP-H2B and mCardinal-Keratin are products of linear un-mixing (Scale-Bar 100um) (u) 3D image obtained from the z-stack of the unmixed z-planes images.

Article Snippet: The workflow integrated raw image preprocessing, computational decomposition, and validation using MATLAB (MathWorks R2020b), Nikon NIS-Elements AR (version 5.30.00 64-bit), and Leica STELLARIS FLIM microscopy.

Techniques: Imaging, Multiplexing, Produced, Microscopy, Fluorescence, Standard Deviation, Expressing, Cell Culture, Software