M. Schmitt, S. Heuke, T. Meyer, O. Chernavskaia, T. Bocklitz, J. Popp
The realization of label-free molecule specific imaging of morphology and chemical composition of tissue at subcellular spatial resolution in real time is crucial for many envisioned applications in medicine, e.g., precise surgical guidance and non-invasive histopathologic examination of tissue. Thus, new approaches for a fast and reliable in vivo and near in vivo (ex corpore in vivo) tissue characterization to supplement routine pathological diagnostics is needed. Spectroscopic imaging approaches are particularly important since they have the potential to provide a pathologist with adequate support in the form of clinically-relevant information under both ex vivo and in vivo conditions. In this contribution it is demonstrated, that multimodal nonlinear microscopy combining coherent anti-Stokes Raman scattering (CARS), two photon excited fluorescence (TPEF) and second harmonic generation (SHG) enables the detection of characteristic structures and the accompanying molecular changes of widespread diseases, particularly of cancer and atherosclerosis. The detailed images enable an objective evaluation of the tissue samples for an early diagnosis of the disease status. Increasing the spectral resolution and analyzing CARS images at multiple Raman resonances improves the chemical specificity. To facilitate handling and interpretation of the image data characteristic properties can be automatically extracted by advanced image processing algorithms, e.g., for tissue classification. Overall, the presented examples show the great potential of multimodal imaging to augment standard intraoperative clinical assessment with functional multimodal CARS/SHG/TPEF images to highlight functional activity and tumor boundaries. It ensures fast, label-free and non-invasive intraoperative tissue classification paving the way towards in vivo optical pathology.
{"title":"Multimodal nonlinear microscopy of biopsy specimen: towards intraoperative diagnostics (Conference Presentation)","authors":"M. Schmitt, S. Heuke, T. Meyer, O. Chernavskaia, T. Bocklitz, J. Popp","doi":"10.1117/12.2216043","DOIUrl":"https://doi.org/10.1117/12.2216043","url":null,"abstract":"The realization of label-free molecule specific imaging of morphology and chemical composition of tissue at subcellular spatial resolution in real time is crucial for many envisioned applications in medicine, e.g., precise surgical guidance and non-invasive histopathologic examination of tissue. Thus, new approaches for a fast and reliable in vivo and near in vivo (ex corpore in vivo) tissue characterization to supplement routine pathological diagnostics is needed. Spectroscopic imaging approaches are particularly important since they have the potential to provide a pathologist with adequate support in the form of clinically-relevant information under both ex vivo and in vivo conditions. In this contribution it is demonstrated, that multimodal nonlinear microscopy combining coherent anti-Stokes Raman scattering (CARS), two photon excited fluorescence (TPEF) and second harmonic generation (SHG) enables the detection of characteristic structures and the accompanying molecular changes of widespread diseases, particularly of cancer and atherosclerosis. The detailed images enable an objective evaluation of the tissue samples for an early diagnosis of the disease status. Increasing the spectral resolution and analyzing CARS images at multiple Raman resonances improves the chemical specificity. To facilitate handling and interpretation of the image data characteristic properties can be automatically extracted by advanced image processing algorithms, e.g., for tissue classification. Overall, the presented examples show the great potential of multimodal imaging to augment standard intraoperative clinical assessment with functional multimodal CARS/SHG/TPEF images to highlight functional activity and tumor boundaries. It ensures fast, label-free and non-invasive intraoperative tissue classification paving the way towards in vivo optical pathology.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"16 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116026587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yibo Zhang, Seung Yoon Celine Lee, Yun Zhang, D. Furst, John Fitzgerald, A. Ozcan
Gout and pseudogout are forms of crystal arthropathy caused by monosodium urate (MSU) and calcium pyrophosphate dehydrate (CPPD) crystals in the joint, respectively, that can result in painful joints. Detecting the unique-shaped, birefringent MSU/CPPD crystals in a synovial fluid sample using a compensated polarizing microscope has been the gold-standard for diagnosis since the 1960’s. However, this can be time-consuming and inaccurate, especially if there are only few crystals in the fluid. The high-cost and bulkiness of conventional microscopes can also be limiting for point-of-care diagnosis. Lens-free on-chip microscopy based on digital holography routinely achieves high-throughput and high-resolution imaging in a cost-effective and field-portable design. Here we demonstrate, for the first time, polarized lens-free on-chip imaging of MSU and CPPD crystals over a wide field-of-view (FOV ~ 20.5 mm2, i.e., <20-fold larger compared a typical 20X objective-lens FOV) for point-of-care diagnostics of gout and pseudogout. Circularly polarizer partially-coherent light is used to illuminate the synovial fluid sample on a glass slide, after which a quarter-wave-plate and an angle-mismatched linear polarizer are used to analyze the transmitted light. Two lens-free holograms of the MSU/CPPD sample are taken, with the sample rotated by 90, to rule out any non-birefringent objects within the specimen. A phase-recovery algorithm is also used to improve the reconstruction quality, and digital pseudo-coloring is utilized to match the color and contrast of the lens-free image to that of a gold-standard microscope image to ease the examination by a rheumatologist or a laboratory technician, and to facilitate computerized analysis.
{"title":"Wide-field synovial fluid imaging using polarized lens-free on-chip microscopy for point-of-care diagnostics of gout (Conference Presentation)","authors":"Yibo Zhang, Seung Yoon Celine Lee, Yun Zhang, D. Furst, John Fitzgerald, A. Ozcan","doi":"10.1117/12.2209715","DOIUrl":"https://doi.org/10.1117/12.2209715","url":null,"abstract":"Gout and pseudogout are forms of crystal arthropathy caused by monosodium urate (MSU) and calcium pyrophosphate dehydrate (CPPD) crystals in the joint, respectively, that can result in painful joints. Detecting the unique-shaped, birefringent MSU/CPPD crystals in a synovial fluid sample using a compensated polarizing microscope has been the gold-standard for diagnosis since the 1960’s. However, this can be time-consuming and inaccurate, especially if there are only few crystals in the fluid. The high-cost and bulkiness of conventional microscopes can also be limiting for point-of-care diagnosis. Lens-free on-chip microscopy based on digital holography routinely achieves high-throughput and high-resolution imaging in a cost-effective and field-portable design. Here we demonstrate, for the first time, polarized lens-free on-chip imaging of MSU and CPPD crystals over a wide field-of-view (FOV ~ 20.5 mm2, i.e., <20-fold larger compared a typical 20X objective-lens FOV) for point-of-care diagnostics of gout and pseudogout. Circularly polarizer partially-coherent light is used to illuminate the synovial fluid sample on a glass slide, after which a quarter-wave-plate and an angle-mismatched linear polarizer are used to analyze the transmitted light. Two lens-free holograms of the MSU/CPPD sample are taken, with the sample rotated by 90, to rule out any non-birefringent objects within the specimen. A phase-recovery algorithm is also used to improve the reconstruction quality, and digital pseudo-coloring is utilized to match the color and contrast of the lens-free image to that of a gold-standard microscope image to ease the examination by a rheumatologist or a laboratory technician, and to facilitate computerized analysis.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"9699 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"129241438","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mechanical properties of tissues play an important role in biological development. However, the current elasticity-specific imaging techniques are either destructive / invasive, or have a limited spatial and/or temporal resolution. Recently, we introduced Brillouin microscopy imaging as a local non-invasive probe of microscopic viscoelasticity in cells and tissues. In this study, by taking advantage of Brillouin spectroscopy, we imaged the viscoelasticity properties of different compartments of living zebrafish embryos, including yolk-sac, skin, spine and heart. Brillouin and Raman spectra were collected simultaneously at each location using a recently developed Brillouin/Raman microscope.
{"title":"Watching embryonic development in a new light: elasticity specific imaging with dual Brillouin/Raman microspectroscopy","authors":"Zhaokai Meng, Jessica Hanson, V. Yakovlev","doi":"10.1117/12.2213978","DOIUrl":"https://doi.org/10.1117/12.2213978","url":null,"abstract":"Mechanical properties of tissues play an important role in biological development. However, the current elasticity-specific imaging techniques are either destructive / invasive, or have a limited spatial and/or temporal resolution. Recently, we introduced Brillouin microscopy imaging as a local non-invasive probe of microscopic viscoelasticity in cells and tissues. In this study, by taking advantage of Brillouin spectroscopy, we imaged the viscoelasticity properties of different compartments of living zebrafish embryos, including yolk-sac, skin, spine and heart. Brillouin and Raman spectra were collected simultaneously at each location using a recently developed Brillouin/Raman microscope.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"8 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"128828915","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. Marquet, K. Rothenfusser, B. Rappaz, C. Depeursinge, P. Jourdain, P. Magistretti
Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.
{"title":"Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases","authors":"P. Marquet, K. Rothenfusser, B. Rappaz, C. Depeursinge, P. Jourdain, P. Magistretti","doi":"10.1117/12.2213454","DOIUrl":"https://doi.org/10.1117/12.2213454","url":null,"abstract":"Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"73 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133079594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mayank Goswami, Pengfei Zhang, E. Pugh, R. Zawadzki
Chorioretinal blood vessel morphology in mice is of great interest to researchers studying eye disease mechanisms in animal models. Two leading retinal imaging modalities -- Optical Coherence Tomography (OCT) and Scanning Laser Ophthalmoscopy (SLO) -- have offered much insight into vascular morphology and blood flow. OCT “flow-contrast” methods have provided detailed mapping of vascular morphology with micrometer depth resolution, while OCT Doppler methods have enabled the measurement of local flow velocities. SLO remains indispensable in studying blood leakage, microaneurysms, and the clearance time of contrast agents of different sizes. In this manuscript we present results obtained with a custom OCT/SLO system applied to visualize the chorioretinal vascular morphology of pigmented C57Bl/6J and albino nude (Nu/Nu) mice. Blood perfusion maps of choroidal vessels and choricapillaris created by OCT and SLO are presented, along with detailed evaluation of different OCT imaging parameters, including the use of the scattering contrast agent Intralipid. Future applications are discussed.
{"title":"Visualization of chorioretinal vasculature in mice in vivo using a combined OCT/SLO imaging system","authors":"Mayank Goswami, Pengfei Zhang, E. Pugh, R. Zawadzki","doi":"10.1117/12.2213609","DOIUrl":"https://doi.org/10.1117/12.2213609","url":null,"abstract":"Chorioretinal blood vessel morphology in mice is of great interest to researchers studying eye disease mechanisms in animal models. Two leading retinal imaging modalities -- Optical Coherence Tomography (OCT) and Scanning Laser Ophthalmoscopy (SLO) -- have offered much insight into vascular morphology and blood flow. OCT “flow-contrast” methods have provided detailed mapping of vascular morphology with micrometer depth resolution, while OCT Doppler methods have enabled the measurement of local flow velocities. SLO remains indispensable in studying blood leakage, microaneurysms, and the clearance time of contrast agents of different sizes. In this manuscript we present results obtained with a custom OCT/SLO system applied to visualize the chorioretinal vascular morphology of pigmented C57Bl/6J and albino nude (Nu/Nu) mice. Blood perfusion maps of choroidal vessels and choricapillaris created by OCT and SLO are presented, along with detailed evaluation of different OCT imaging parameters, including the use of the scattering contrast agent Intralipid. Future applications are discussed.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"101 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124709546","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. Rechmann, B. Rechmann, W. H. Groves, C. Le, Marcia L. Rapozo-Hilo, J. Featherstone
The objective of this laboratory study was to test whether irradiation with a new 9.3µm microsecond short-pulsed CO2-laser enhances enamel caries resistance with and without additional fluoride applications. 101 human enamel samples were divided into 7 groups. Each group was treated with different laser parameters (Carbon-dioxide laser, wavelength 9.3µm, 43Hz pulse-repetition rate, pulse duration between 3μs to 7μs (1.5mJ/pulse to 2.9mJ/pulse). Using a pH-cycling model and cross-sectional microhardness testing determined the mean relative mineral loss delta Z (∆Z) for each group. The pH-cycling was performed with or without additional fluoride. The CO2 9.3μm short-pulsed laser energy rendered enamel caries resistant with and without additional fluoride use.
{"title":"Enhancing caries resistance with a short-pulsed CO2 9.3-μm laser: a laboratory study (Conference Presentation)","authors":"P. Rechmann, B. Rechmann, W. H. Groves, C. Le, Marcia L. Rapozo-Hilo, J. Featherstone","doi":"10.1117/12.2224641","DOIUrl":"https://doi.org/10.1117/12.2224641","url":null,"abstract":"The objective of this laboratory study was to test whether irradiation with a new 9.3µm microsecond short-pulsed CO2-laser enhances enamel caries resistance with and without additional fluoride applications. 101 human enamel samples were divided into 7 groups. Each group was treated with different laser parameters (Carbon-dioxide laser, wavelength 9.3µm, 43Hz pulse-repetition rate, pulse duration between 3μs to 7μs (1.5mJ/pulse to 2.9mJ/pulse). Using a pH-cycling model and cross-sectional microhardness testing determined the mean relative mineral loss delta Z (∆Z) for each group. The pH-cycling was performed with or without additional fluoride. The CO2 9.3μm short-pulsed laser energy rendered enamel caries resistant with and without additional fluoride use.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"139 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"113968846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
U. Resch‐Genger, C. Würth, J. Pauli, Soheil Hatami, Martin Kaiser
There is an increasing interest in optical reporters like semiconductor quantum dots and upconversion nanophosphors with emission < 1000 nm for bioanalysis, medical diagnostics, and safety barcodes and hence, in reliable fluorescence measurements in this wavelength region, e.g., for the comparison of material performance and the rational design of new nanomaterials with improved properties [1-4]. The performance of fluorescence measurements < 800 nm and especially < 1000 nm is currently hampered by the lack of suitable methods and standards for the simple determination of the wavelength-dependent spectral responsivity of fluorescence measuring systems and the control of measured emission spectra and intensities [3-5]. This is of special relevance for nanocrystalline emitters like quantum dots and rods as well as for upconversion nanocrystals, where surface states and the accessibility of emissive states by quenchers largely control accomplishable quantum yields and hence, signal sizes and detection sensitivities from the reporter side. Here, we present the design of an integrating sphere setup for the absolute measurement of emission spectra and quantum yields in the wavelength region of 650 to 1600 nm and its calibration as well as examples for potential fluorescence standards from different reporter classes for the control of the reliability of such measurements [5]. This includes new spectral fluorescence standards for the wavelength region of 650 nm to 1000 nm as well as a set of quantum yield standards covering the wavelength region from 400 nm to 1000 nm.
{"title":"Absolute fluorescence measurements > 1000 nm: setup design, calibration and standards (Conference Presentation)","authors":"U. Resch‐Genger, C. Würth, J. Pauli, Soheil Hatami, Martin Kaiser","doi":"10.1117/12.2213033","DOIUrl":"https://doi.org/10.1117/12.2213033","url":null,"abstract":"There is an increasing interest in optical reporters like semiconductor quantum dots and upconversion nanophosphors with emission < 1000 nm for bioanalysis, medical diagnostics, and safety barcodes and hence, in reliable fluorescence measurements in this wavelength region, e.g., for the comparison of material performance and the rational design of new nanomaterials with improved properties [1-4]. The performance of fluorescence measurements < 800 nm and especially < 1000 nm is currently hampered by the lack of suitable methods and standards for the simple determination of the wavelength-dependent spectral responsivity of fluorescence measuring systems and the control of measured emission spectra and intensities [3-5]. This is of special relevance for nanocrystalline emitters like quantum dots and rods as well as for upconversion nanocrystals, where surface states and the accessibility of emissive states by quenchers largely control accomplishable quantum yields and hence, signal sizes and detection sensitivities from the reporter side. Here, we present the design of an integrating sphere setup for the absolute measurement of emission spectra and quantum yields in the wavelength region of 650 to 1600 nm and its calibration as well as examples for potential fluorescence standards from different reporter classes for the control of the reliability of such measurements [5]. This includes new spectral fluorescence standards for the wavelength region of 650 nm to 1000 nm as well as a set of quantum yield standards covering the wavelength region from 400 nm to 1000 nm.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"103 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132922761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
We present a technique that records transient changes in the concentration of free calcium in live neurons by TCSPC FLIM. The sample is incubated with a calcium-sensitive dye. To measure the temporal change in the calcium-ion concentration the sample is periodically stimulated by an electrical signal and scanned at high image rate with a high-frequency pulsed laser beam. Single photons of the fluorescence light are detected, and a photon distribution over the coordinates of the scan, the arrival times of the photons after the excitation pulses, and the time after the stimulation pulses is built up. The result can be interpreted as a sequence of FLIM images for different times after the stimulation pulses. The signal-to-noise ratio only depends on the available photon rate and the total acquisition time, not on the speed of the sequence. The maximum resolution at which lifetime changes can be recorded is given by the frame rate of the scanner which is currently 38 ms. Faster changes can be recorded by line scanning. Transient lifetime effects can then be resolved at a resolution of about one millisecond.
{"title":"Imaging of Ca2+ transients in cultured neurons by temporal mosaic FLIM (Conference Presentation)","authors":"W. Becker, Samuel Frere","doi":"10.1117/12.2214083","DOIUrl":"https://doi.org/10.1117/12.2214083","url":null,"abstract":"We present a technique that records transient changes in the concentration of free calcium in live neurons by TCSPC FLIM. The sample is incubated with a calcium-sensitive dye. To measure the temporal change in the calcium-ion concentration the sample is periodically stimulated by an electrical signal and scanned at high image rate with a high-frequency pulsed laser beam. Single photons of the fluorescence light are detected, and a photon distribution over the coordinates of the scan, the arrival times of the photons after the excitation pulses, and the time after the stimulation pulses is built up. The result can be interpreted as a sequence of FLIM images for different times after the stimulation pulses. The signal-to-noise ratio only depends on the available photon rate and the total acquisition time, not on the speed of the sequence. The maximum resolution at which lifetime changes can be recorded is given by the frame rate of the scanner which is currently 38 ms. Faster changes can be recorded by line scanning. Transient lifetime effects can then be resolved at a resolution of about one millisecond.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"27 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130062138","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The importance of microRNA (miRNA) dysregulation in the development and progression of diseases has made these short-length nucleic acids to next generation biomarkers. Tb-to-QD Förster resonance energy transfer (FRET) has several unique advantages over organic dye-based FRET systems for biomolecular sensing. Large Förster distances (6-11 nm) offer much high FRET efficiencies, exceptionally long Tb excited-state lifetimes (ms) enable time-gated detection void of autofluorecence background, and the narrow, symmetric, and tunable emission bands of QDs provide unrivaled potential for multiplexing. Here we report a rapid and homogeneous method to sensitively detect three different miRNAs (hsa-miR-20a-5p, hsa-miR-20b-5p, and hsa-miR-21-5p) from a single 150 µL sample based on multiplexed FRET between a luminescent Lumi4-Tb complex and three different QDs. The biosensing approach exploits both base pairing and stacking. Careful design and optimization of sequence lengths and orientations of the QD and Tb-DNA conjugates was performed to provide maximum selectivity and sensitivity for all three miRNA biomarkers. The assays work at room temperature and were designed for their application on a KRYPTOR diagnostic plate reader system.Only 30 min of sample incubation and 7.5 s of measurement are required to obtain ca. 1 nM (subpicomol) detection limits. We also demonstrate precise multiplexed measurements of these miRNAs at different and varying concentrations and the feasibility of adapting the technology to point-of-care testing (POCT) in buffer containing 10% serum. Our assay does not only demonstrate an important milestone for the integration of quantum dots to multiplexed clinical diagnostics but also a unique rapid miRNA detection technology that is complimentary to the rather complicated high-throughput and high-sensitivity approaches that are established today.
{"title":"Terbium complex to quantum dot Förster resonance energy transfer for homogeneous and multiplexed microRNA assay (Conference Presentation)","authors":"X. Qiu, N. Hildebrandt","doi":"10.1117/12.2209674","DOIUrl":"https://doi.org/10.1117/12.2209674","url":null,"abstract":"The importance of microRNA (miRNA) dysregulation in the development and progression of diseases has made these short-length nucleic acids to next generation biomarkers. Tb-to-QD Förster resonance energy transfer (FRET) has several unique advantages over organic dye-based FRET systems for biomolecular sensing. Large Förster distances (6-11 nm) offer much high FRET efficiencies, exceptionally long Tb excited-state lifetimes (ms) enable time-gated detection void of autofluorecence background, and the narrow, symmetric, and tunable emission bands of QDs provide unrivaled potential for multiplexing. Here we report a rapid and homogeneous method to sensitively detect three different miRNAs (hsa-miR-20a-5p, hsa-miR-20b-5p, and hsa-miR-21-5p) from a single 150 µL sample based on multiplexed FRET between a luminescent Lumi4-Tb complex and three different QDs. The biosensing approach exploits both base pairing and stacking. Careful design and optimization of sequence lengths and orientations of the QD and Tb-DNA conjugates was performed to provide maximum selectivity and sensitivity for all three miRNA biomarkers. The assays work at room temperature and were designed for their application on a KRYPTOR diagnostic plate reader system.Only 30 min of sample incubation and 7.5 s of measurement are required to obtain ca. 1 nM (subpicomol) detection limits. We also demonstrate precise multiplexed measurements of these miRNAs at different and varying concentrations and the feasibility of adapting the technology to point-of-care testing (POCT) in buffer containing 10% serum. Our assay does not only demonstrate an important milestone for the integration of quantum dots to multiplexed clinical diagnostics but also a unique rapid miRNA detection technology that is complimentary to the rather complicated high-throughput and high-sensitivity approaches that are established today.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"9722 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130074882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
It is difficult to overstate the physiological importance of potassium for life as its indispensable roles in a variety of biological processes are widely known. As a result, efficient methods for determining physiological levels of potassium are of paramount importance. Despite this, relatively few K+ fluorescence sensors have been reported, with only one being commercially available. A new two-photon excited fluorescent K+ sensor is reported. The sensor is comprised of three moieties, a highly selective K+ chelator as the K+ recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (<52-fold) in detecting K+ over other physiological metal cations. Upon binding K+, the sensor switches from non-fluorescent to highly fluorescent, emitting red to near-IR (NIR) fluorescence. The sensor exhibited a good two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K+ sensing in living cells.
{"title":"Two-photon fluorescent sensor for K+ imaging in live cells (Conference Presentation)","authors":"Binglin Sui, Xiling Yue, Bosung Kim, K. Belfield","doi":"10.1117/12.2211202","DOIUrl":"https://doi.org/10.1117/12.2211202","url":null,"abstract":"It is difficult to overstate the physiological importance of potassium for life as its indispensable roles in a variety of biological processes are widely known. As a result, efficient methods for determining physiological levels of potassium are of paramount importance. Despite this, relatively few K+ fluorescence sensors have been reported, with only one being commercially available. A new two-photon excited fluorescent K+ sensor is reported. The sensor is comprised of three moieties, a highly selective K+ chelator as the K+ recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (<52-fold) in detecting K+ over other physiological metal cations. Upon binding K+, the sensor switches from non-fluorescent to highly fluorescent, emitting red to near-IR (NIR) fluorescence. The sensor exhibited a good two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K+ sensing in living cells.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"144 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125418793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：