It is well-known that turbo equalization with the max-log-map （MLM） rather than the log-map （LM） algorithm is insensitive to signal to noise ratio （SNR） mismatch. As our first contribution, an improved MLM algor...It is well-known that turbo equalization with the max-log-map （MLM） rather than the log-map （LM） algorithm is insensitive to signal to noise ratio （SNR） mismatch. As our first contribution, an improved MLM algorithm called scaled max-log-map （SMLM） algorithm is presented. Simulation results show that the SMLM scheme can dramatically outperform the MLM without sacrificing the robustness against SNR mismatch. Unfortunately, its performance is still inferior to that of the LM algorithm with exact SNR knowledge over the class of high-loss channels. As our second contribution, a switching turbo equalization scheme, which switches between the SMLM and LM schemes, is proposed to practically close the performance gap. It is based on a novel way to estimate the SNR from the reliability values of the extrinsic information of the SMLM algorithm.展开更多
Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets...Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin.Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales,from nanometers to micrometers.Single molecule localization microscopy(SMLM)methods,such as STORM,are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization.Multi-label imaging of chromatin is necessary to unpack its structural complexity.However,these efforts are challenged by the high-density nuclear environment,which can affect antibody binding affinities,diffusivity and non-specific interactions.Optimizing buffer conditions,fluorophore stability,and antibody specificity is crucial for achieving effective antibody conjugates.Here,we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment.This protocol couples multiplexed localization datasets with a robust analysis algorithm,which utilizes localizations from one target as seed points for distance,density and multi-label joint affinity measurements to explore complex organization of all three targets.Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories,but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters.This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.展开更多
Multidimensional single-molecule localization microscopy(mSMLM)represents a paradigm shift in the realm of super-resolution microscopy techniques.It affords the simultaneous detection of singlemolecule spatial locatio...Multidimensional single-molecule localization microscopy(mSMLM)represents a paradigm shift in the realm of super-resolution microscopy techniques.It affords the simultaneous detection of singlemolecule spatial locations at the nanoscale and functional information by interrogating the emission properties of switchable fluorophores.The latter is finely tuned to report its local environment through carefully manipulated laser illumination and single-molecule detection strategies.This Perspective highlights recent strides in mSMLM with a focus on fluorophore designs and their integration into mSMLM imaging systems.Particular interests are the accomplishments in simultaneous multiplexed super-resolution imaging,nanoscale polarity and hydrophobicity mapping,and single-molecule orientational imaging.Challenges and prospects in mSMLM are also discussed,which include the development of more vibrant and functional fluorescent probes,the optimization of optical implementation to judiciously utilize the photon budget,and the advancement of imaging analysis and machine learning techniques.展开更多
基金This work was supported by the National Nature Science Foundation of China under Grant No.60496313, 60502010, and 60602008.
文摘It is well-known that turbo equalization with the max-log-map （MLM） rather than the log-map （LM） algorithm is insensitive to signal to noise ratio （SNR） mismatch. As our first contribution, an improved MLM algorithm called scaled max-log-map （SMLM） algorithm is presented. Simulation results show that the SMLM scheme can dramatically outperform the MLM without sacrificing the robustness against SNR mismatch. Unfortunately, its performance is still inferior to that of the LM algorithm with exact SNR knowledge over the class of high-loss channels. As our second contribution, a switching turbo equalization scheme, which switches between the SMLM and LM schemes, is proposed to practically close the performance gap. It is based on a novel way to estimate the SNR from the reliability values of the extrinsic information of the SMLM algorithm.
基金supported by NIH grants U54CA268084,U54CA261694,and R01CA228272National Science Foundation grants EFMA-1830961 and CBET-2430743+1 种基金philanthropic support from Rob and Kristin Goldman,Mr.David Sachsthe Christina Carinato Charitable Foundation.
文摘Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin.Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales,from nanometers to micrometers.Single molecule localization microscopy(SMLM)methods,such as STORM,are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization.Multi-label imaging of chromatin is necessary to unpack its structural complexity.However,these efforts are challenged by the high-density nuclear environment,which can affect antibody binding affinities,diffusivity and non-specific interactions.Optimizing buffer conditions,fluorophore stability,and antibody specificity is crucial for achieving effective antibody conjugates.Here,we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment.This protocol couples multiplexed localization datasets with a robust analysis algorithm,which utilizes localizations from one target as seed points for distance,density and multi-label joint affinity measurements to explore complex organization of all three targets.Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories,but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters.This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.
基金supported by the National Institutes of Health grants R21GM141675 and R01GM143397the National Science Foundation grant CHM-1954430 and CHM-2246548the National Research Foundation of Korea grant No.NRF-2022R1C1C1002850.
文摘Multidimensional single-molecule localization microscopy(mSMLM)represents a paradigm shift in the realm of super-resolution microscopy techniques.It affords the simultaneous detection of singlemolecule spatial locations at the nanoscale and functional information by interrogating the emission properties of switchable fluorophores.The latter is finely tuned to report its local environment through carefully manipulated laser illumination and single-molecule detection strategies.This Perspective highlights recent strides in mSMLM with a focus on fluorophore designs and their integration into mSMLM imaging systems.Particular interests are the accomplishments in simultaneous multiplexed super-resolution imaging,nanoscale polarity and hydrophobicity mapping,and single-molecule orientational imaging.Challenges and prospects in mSMLM are also discussed,which include the development of more vibrant and functional fluorescent probes,the optimization of optical implementation to judiciously utilize the photon budget,and the advancement of imaging analysis and machine learning techniques.
