This document presents a technique called K-factor image deshadowing that can improve the localization accuracy of single fluorescent particles in stochastic super-resolution fluorescence microscopy. K-factor decomposes an image into a nonlinear set of contrast-ordered images whose product reassembles the original. Applying K-factor to raw fluorescence data prior to localization can improve localization precision by up to 85% compared to single fitting, enabling the localization of overlapping particles and faster data collection. Implementing this on experimental cellular data yielded a 37% improvement in resolution for the same acquisition time, or a 42% decrease in time needed for the same resolution.
This document summarizes a study on errors in particle tracking microrheology. It separates errors into static errors from position measurements of immobilized particles due to limited spatial resolution, and dynamic errors from particle motion during the finite exposure time required for visualization. The authors develop models to calculate how these errors propagate to measurements of mean-squared displacement. They verify the models using simulations and experiments on viscous fluids, showing static errors can be corrected using static experiments at similar noise levels. This increases accuracy for microrheology studies.
Percolation of light through whispering gallery modes in 3D lattices of coupl...Shashaanka Ashili
This document summarizes research on 3D lattices of coupled microspheres with whispering gallery modes (WGMs). Key points:
1) The researchers synthesized 3D lattices of dye-doped fluorescent polystyrene spheres with controlled thickness from one to 43 monolayers using flow-assisted self-assembly.
2) Optical transmission spectra of the lattices showed signatures of coupling between spheres, including WGM peak splitting and anomalously high transmission at peak wavelengths.
3) The observed WGM transport is interpreted using an analogy to percolation theory, where optical "bonds" connect lattice sites depending on size dispersion. Near-perfect lattices could enable resonant sensing and light emission applications
This document describes a new technique for wide-field background-free fluorescence imaging in vivo using magnetic modulation of fluorescent nanodiamond emission. Fluorescent nanodiamonds are promising probes for in vivo imaging but are limited by autofluorescence. The technique uses a rotating magnetic field to selectively modulate nanodiamond fluorescence, which is then detected using phase-sensitive lock-in detection to improve signal-to-background ratio up to 100-fold. This overcomes autofluorescence and improves nanodiamond imaging capabilities for in vivo applications.
Improved two-photon imaging of living neurons in brain tissue through tempora...julian choy
This document describes a study that optimized two-photon imaging of living neurons in brain tissue by temporally gating the incident laser to reduce photon flux while maximizing fluorescence signal. The study found that gating the laser at the sampling frequency compromised cell viability despite high fluorescence. An optimum gating frequency range was identified that maintained cell viability while preserving fluorescence levels in two-photon images. Cell viability was monitored by measuring changes in membrane input resistance during whole-cell patch recording of neurons.
Part 2 of 2 of lecture series introducing undergraduate neuroscience students to the core electrophysiological and imaging techniques used to study neuronal activity.
This document discusses a new design of plasmonic nanoantenna with a slant gap that can enhance optical chirality. The slant gap provides an enhanced electric field parallel to an external magnetic field with a phase delay of π/2, resulting in enhanced optical chirality in the near field. Numerical simulations show this nanoantenna design can generate a near field with enhanced optical chirality when excited by linearly polarized light. This enhanced optical chirality could allow for circular dichroism analysis using linearly polarized light and may find applications in analyzing the chirality of surface-bound matter.
This document proposes using a thin layer of chalcogenide phase change material like Ge2Sb2Te5 to optically tune the EIT-like response of an all-dielectric metasurface. The metasurface consists of silicon nanoresonators that support an EIT-like resonant response at 1550nm when the phase change material is amorphous. Switching the material to crystalline shifts the EIT response to higher wavelengths due to increased losses. This provides a reversible tuning mechanism for the metasurface response with nanosecond laser pulses, allowing dynamic control over transmission and dispersion properties.
Neurotar Ltd provides in vivo two-photon microscopy services to help clients in drug discovery and development. Their services include obtaining high-resolution, high-relevance imaging of biological processes in living animals to facilitate better informed preclinical decisions, improve predictive power in clinical trials, and accelerate time to market for new drugs.
This document summarizes a study on errors in particle tracking microrheology. It separates errors into static errors from position measurements of immobilized particles due to limited spatial resolution, and dynamic errors from particle motion during the finite exposure time required for visualization. The authors develop models to calculate how these errors propagate to measurements of mean-squared displacement. They verify the models using simulations and experiments on viscous fluids, showing static errors can be corrected using static experiments at similar noise levels. This increases accuracy for microrheology studies.
Percolation of light through whispering gallery modes in 3D lattices of coupl...Shashaanka Ashili
This document summarizes research on 3D lattices of coupled microspheres with whispering gallery modes (WGMs). Key points:
1) The researchers synthesized 3D lattices of dye-doped fluorescent polystyrene spheres with controlled thickness from one to 43 monolayers using flow-assisted self-assembly.
2) Optical transmission spectra of the lattices showed signatures of coupling between spheres, including WGM peak splitting and anomalously high transmission at peak wavelengths.
3) The observed WGM transport is interpreted using an analogy to percolation theory, where optical "bonds" connect lattice sites depending on size dispersion. Near-perfect lattices could enable resonant sensing and light emission applications
This document describes a new technique for wide-field background-free fluorescence imaging in vivo using magnetic modulation of fluorescent nanodiamond emission. Fluorescent nanodiamonds are promising probes for in vivo imaging but are limited by autofluorescence. The technique uses a rotating magnetic field to selectively modulate nanodiamond fluorescence, which is then detected using phase-sensitive lock-in detection to improve signal-to-background ratio up to 100-fold. This overcomes autofluorescence and improves nanodiamond imaging capabilities for in vivo applications.
Improved two-photon imaging of living neurons in brain tissue through tempora...julian choy
This document describes a study that optimized two-photon imaging of living neurons in brain tissue by temporally gating the incident laser to reduce photon flux while maximizing fluorescence signal. The study found that gating the laser at the sampling frequency compromised cell viability despite high fluorescence. An optimum gating frequency range was identified that maintained cell viability while preserving fluorescence levels in two-photon images. Cell viability was monitored by measuring changes in membrane input resistance during whole-cell patch recording of neurons.
Part 2 of 2 of lecture series introducing undergraduate neuroscience students to the core electrophysiological and imaging techniques used to study neuronal activity.
This document discusses a new design of plasmonic nanoantenna with a slant gap that can enhance optical chirality. The slant gap provides an enhanced electric field parallel to an external magnetic field with a phase delay of π/2, resulting in enhanced optical chirality in the near field. Numerical simulations show this nanoantenna design can generate a near field with enhanced optical chirality when excited by linearly polarized light. This enhanced optical chirality could allow for circular dichroism analysis using linearly polarized light and may find applications in analyzing the chirality of surface-bound matter.
This document proposes using a thin layer of chalcogenide phase change material like Ge2Sb2Te5 to optically tune the EIT-like response of an all-dielectric metasurface. The metasurface consists of silicon nanoresonators that support an EIT-like resonant response at 1550nm when the phase change material is amorphous. Switching the material to crystalline shifts the EIT response to higher wavelengths due to increased losses. This provides a reversible tuning mechanism for the metasurface response with nanosecond laser pulses, allowing dynamic control over transmission and dispersion properties.
Neurotar Ltd provides in vivo two-photon microscopy services to help clients in drug discovery and development. Their services include obtaining high-resolution, high-relevance imaging of biological processes in living animals to facilitate better informed preclinical decisions, improve predictive power in clinical trials, and accelerate time to market for new drugs.
The document discusses plasmonic nanoantennas and their use in controlling the radiative properties of nanoemitters. It begins with a brief history of plasmonics and defines key concepts like localized surface plasmon resonances and nanoantennas. It then covers topics like the effects of nanoantenna shape and fabrication techniques. Applications discussed include surface-enhanced spectroscopy, fluorescence, solar cells, and nanomedicine. In conclusion, nanoantennas can modify emission properties and provide field enhancement and confinement, enabling applications while fabrication challenges at the nanoscale remain.
Wei Li has published extensively in international scientific journals and books as well as international conference proceedings. Some of his notable publications include papers on spectral characterization of monolithic mode-locked lasers, superluminescent diodes at 1.55 μm, characterization of 1550 nm Fabry-Perot laser structures, and invited chapters in books on dilute nitride semiconductors and GaInNAs quantum well lasers. He has also published work on extending the emission wavelength of GaInNAs/GaAs quantum well lasers beyond 1300 nm and high performance 1.32 μm GaInNAs/GaAs single-quantum-well lasers.
Study of Mitotic Index and DNA profile when exposure to He-Ne laser and UVC r...IOSR Journals
This study examined the effects of He-Ne laser radiation and UVC radiation on mitotic index and DNA profiles in mice. 100 mice were divided into groups exposed to laser radiation at different time periods, UVC radiation for 1 hour, laser pre-exposure followed by UVC, and UVC pre-exposure followed by laser. Mitotic index, which measures the percentage of cells undergoing cell division, and DNA electrophoresis were analyzed at various time points after exposure. Results showed that UVC radiation decreased mitotic index and increased DNA damage over time, while laser pre-exposure protected against these effects of subsequent UVC exposure by inducing antioxidant defenses and DNA repair mechanisms. Laser exposure alone had minor effects on mitotic index.
NANOPARTICLES IN CANCER DIAGNOSIS AND TREATMENTKeshav Das Sahu
This document discusses the use of nanoparticles in cancer diagnosis and treatment. It introduces several types of nanoparticles that can be used, including nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanowires, nanodiamonds, and nanosponges. Nanoshells and dendrimers are highlighted as promising for targeted drug delivery. The document also discusses magnetic resonance imaging contrast agents, including both paramagnetic gadolinium agents and superparamagnetic iron oxide nanoparticles, which can enhance MRI images and improve cancer diagnosis.
The document discusses small animal imaging techniques. It provides an overview of various imaging modalities including their resolutions and applications. Specific techniques covered include bioluminescence imaging, fluorescence imaging, computed tomography, magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography. The document also discusses fluorescent probes and reagents used for small animal imaging including near-infrared dyes, quantum dots, fluorescent microspheres, and protease-activatable probes. Potential applications highlighted include tumor detection, vascular imaging, and monitoring of enzyme activity.
Use of nanotechnology in medical science (pros and cons)Vikram Kataria
here in this presentation I had shared the basic information regarding use of nanotechnology in medical science and what wonders and improvements that nano technology did in the field of medical science.
This document reviews the use of quantum dots for bioimaging applications. It discusses:
1) The synthesis of quantum dots, particularly CdSe/ZnS core/shell structures, and methods to tune their optical properties.
2) Modification of quantum dot surfaces with ligands to make them water soluble and biocompatible while maintaining fluorescence. Common surface modifications include adding carboxylic acids, silica shells, and encapsulation in micelles.
3) Applications of quantum dots in biology, including labeling of proteins and targeting of specific cell surface receptors due to their photostability and ability to detect multiple signals simultaneously.
Nanoparticles show promise for biomedical imaging and diagnosis due to their large size and multifunctionality compared to small molecules. Magnetic iron oxide nanoparticles are commonly used in MRI because they shorten T2 relaxation times, allowing hydrogen protons to move closer to the magnet and produce clearer images. Various types of functionalized magnetic nanoparticles including amine, carboxyl, epoxy and IDA functionalized nanoparticles are used for applications like immunoassay, gene transfection, biomolecule separation, cell separation, enzyme immobilization, drug delivery, and biomedical imaging. Nanoparticles also show potential for targeted cancer drug delivery and simultaneous imaging and therapy.
The Molecular Imaging Laboratory at Howard University provides state-of-the-art imaging equipment including high resolution MRI systems for small animal and clinical research. The lab aims to train students and foster multidisciplinary research using imaging to study disease processes and investigate new treatments. Areas of research include in vivo MRI and optical imaging of disease models in small animals, as well as molecular imaging of biological processes and developing new imaging probes and nanoparticles.
Handbook of coherent domain optical methodsSpringer
This chapter discusses light scattering spectroscopy techniques, both elastic and inelastic. Elastic light scattering spectroscopy measures the size, shape, and optical properties of particles or cells based on the spectrum of light they scatter. Inelastic light scattering spectroscopy, also known as Raman spectroscopy, measures the spectrum of light scattered by molecular vibrations or phonons in solids. These techniques are emerging as useful tools in materials science, environmental science, and biomedicine for applications such as measuring the size distribution of cell nuclei, early cancer detection, and disease diagnosis.
It has been almost decades since the “war on cancer” was declared. It is now generally
believed that personalized medicine is the future for cancer patient management.
Possessing unprecedented potential for early detection, accurate diagnosis, and
personalized treatment of cancer, nanoparticles have been extensively studied over the last
decade. In this report, I will try to summarize the current state-of-the-art nanoparticles in
biomedical applications targeting cancer. Multi- functionality nanoparticle-based agents.
Targeting ligands, imaging labels, therapeutic Drugs, and other. And the Role of Chemical
Engineers in this field and the promise that it holds for future.
Impact of detector thickness on imaging characteristics of the Siemens Biogra...Anax Fotopoulos
This document summarizes a Monte Carlo study using GATE simulation of a Siemens Biograph DUO PET/CT scanner. It investigates the impact of detector thickness on imaging characteristics by simulating the scanner with LSO detectors of 2cm and 3cm thickness. The results show that increasing thickness from 2cm to 3cm improves detection efficiency, resulting in a sharper energy peak at 511 keV, higher counts, and better energy resolution and signal-to-noise ratio. However, thicker detectors may also delay signal processing and increase device cost and complexity.
The document discusses the use of quantum dots (QDs) for biomedical applications such as bioimaging and therapy. It provides an overview of the photophysical properties of QDs that make them advantageous over organic dyes for imaging. Various biomedical applications of QDs are described, including in vitro and in vivo imaging, biosensing, photodynamic therapy, drug delivery, and gene delivery. Finally, the document outlines a research proposal to develop MoS2@polyaniline nanohybrids for dual-model imaging and synergistic photothermal/radiation therapy of tumors.
This document summarizes various molecular imaging techniques. It discusses how nuclear medicine involves molecular imaging by combining detectable labels with biologically important molecules to assess cellular functions. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) allow noninvasive assessment and quantification of small differences between patients. New molecular imaging agents currently in clinical trials will help transform imaging and therapy by quantifying targets like gene expression, receptors, and tumor metabolism. The document outlines different imaging modalities including PET, SPECT, magnetic resonance imaging, optical imaging, ultrasound, and identifies various biomarkers that can be used as imaging targets.
The Effect of Gamma Irradiation on the Radiofrequency Dielectric Dispersion P...theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
This document summarizes research conducted by Dr. Shiuan Huei Lin and collaborators on developing photopolymer materials for holographic data storage applications. It lists 16 journal publications, 1 book chapter, and 16 conference presentations from 2003 to 2009 reporting on their work investigating various dopants to improve the holographic recording characteristics of poly(methyl methacrylate) photopolymers. The research involved characterization of materials doped with compounds such as phenanthrenequinone and lanthanide organometallics to optimize their properties for volume holographic recording.
This document discusses various applications of nanotechnology in diagnostic pathology. It begins by defining key terms like nanometer and describing early concepts in nanotechnology. It then explores different nanomaterials like carbon nanotubes, nanorods, cantilevers, and quantum dots; how they are used for cancer detection and DNA analysis; and techniques like microfluidics. The document also covers applications in drug delivery, medical imaging, and surgery. Overall, the document outlines the growing role of nanotechnology across many areas of medical diagnosis and treatment.
Nanomedicine is an interdisciplinary field that uses nanotechnology for medical applications. It aims to diagnose, treat, and prevent disease at the molecular level using nano-scale tools. The document outlines the history of nanomedicine from Richard Feynman's 1959 talk introducing nanotechnology to current applications. Key applications discussed include drug delivery using nanoparticles like liposomes, imaging contrast agents, and miniaturized medical devices. Challenges also remain around potential toxicities of nanomaterials.
This document describes a technique called multispectral angular-resolved dark-field imaging (MARDI) that can automatically identify individual bacteria based on their unique light scattering spectra. An 87-channel microscope system was used to measure the scattering spectra of various bacteria at different angles and wavelengths. A simpler 15-channel system then demonstrated the viability of bacterial identification, accurately identifying four out of six bacterial species in tests of individual bacteria. This technique could provide a simple way to identify bacteria without the need for specialized equipment or skills.
This document describes a study that used optical coherence tomography (OCT) to image microvasculature in human skin in vivo with high spatial resolution. The study analyzed an intensity-based Doppler variance (IBDV) method for quantifying blood flow rates using an OCT system. Results showed that the IBDV method's sensitivity for detecting slow blood flow can be improved by increasing the time interval between adjacent A-lines. The study also proposed a higher sensitivity IBDV method that applies the algorithm along the slower scan direction. In vivo imaging of human skin microvasculature demonstrated the ability to identify vascular networks at depths up to 1.2 mm with exquisite spatial resolution.
The document discusses plasmonic nanoantennas and their use in controlling the radiative properties of nanoemitters. It begins with a brief history of plasmonics and defines key concepts like localized surface plasmon resonances and nanoantennas. It then covers topics like the effects of nanoantenna shape and fabrication techniques. Applications discussed include surface-enhanced spectroscopy, fluorescence, solar cells, and nanomedicine. In conclusion, nanoantennas can modify emission properties and provide field enhancement and confinement, enabling applications while fabrication challenges at the nanoscale remain.
Wei Li has published extensively in international scientific journals and books as well as international conference proceedings. Some of his notable publications include papers on spectral characterization of monolithic mode-locked lasers, superluminescent diodes at 1.55 μm, characterization of 1550 nm Fabry-Perot laser structures, and invited chapters in books on dilute nitride semiconductors and GaInNAs quantum well lasers. He has also published work on extending the emission wavelength of GaInNAs/GaAs quantum well lasers beyond 1300 nm and high performance 1.32 μm GaInNAs/GaAs single-quantum-well lasers.
Study of Mitotic Index and DNA profile when exposure to He-Ne laser and UVC r...IOSR Journals
This study examined the effects of He-Ne laser radiation and UVC radiation on mitotic index and DNA profiles in mice. 100 mice were divided into groups exposed to laser radiation at different time periods, UVC radiation for 1 hour, laser pre-exposure followed by UVC, and UVC pre-exposure followed by laser. Mitotic index, which measures the percentage of cells undergoing cell division, and DNA electrophoresis were analyzed at various time points after exposure. Results showed that UVC radiation decreased mitotic index and increased DNA damage over time, while laser pre-exposure protected against these effects of subsequent UVC exposure by inducing antioxidant defenses and DNA repair mechanisms. Laser exposure alone had minor effects on mitotic index.
NANOPARTICLES IN CANCER DIAGNOSIS AND TREATMENTKeshav Das Sahu
This document discusses the use of nanoparticles in cancer diagnosis and treatment. It introduces several types of nanoparticles that can be used, including nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanowires, nanodiamonds, and nanosponges. Nanoshells and dendrimers are highlighted as promising for targeted drug delivery. The document also discusses magnetic resonance imaging contrast agents, including both paramagnetic gadolinium agents and superparamagnetic iron oxide nanoparticles, which can enhance MRI images and improve cancer diagnosis.
The document discusses small animal imaging techniques. It provides an overview of various imaging modalities including their resolutions and applications. Specific techniques covered include bioluminescence imaging, fluorescence imaging, computed tomography, magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography. The document also discusses fluorescent probes and reagents used for small animal imaging including near-infrared dyes, quantum dots, fluorescent microspheres, and protease-activatable probes. Potential applications highlighted include tumor detection, vascular imaging, and monitoring of enzyme activity.
Use of nanotechnology in medical science (pros and cons)Vikram Kataria
here in this presentation I had shared the basic information regarding use of nanotechnology in medical science and what wonders and improvements that nano technology did in the field of medical science.
This document reviews the use of quantum dots for bioimaging applications. It discusses:
1) The synthesis of quantum dots, particularly CdSe/ZnS core/shell structures, and methods to tune their optical properties.
2) Modification of quantum dot surfaces with ligands to make them water soluble and biocompatible while maintaining fluorescence. Common surface modifications include adding carboxylic acids, silica shells, and encapsulation in micelles.
3) Applications of quantum dots in biology, including labeling of proteins and targeting of specific cell surface receptors due to their photostability and ability to detect multiple signals simultaneously.
Nanoparticles show promise for biomedical imaging and diagnosis due to their large size and multifunctionality compared to small molecules. Magnetic iron oxide nanoparticles are commonly used in MRI because they shorten T2 relaxation times, allowing hydrogen protons to move closer to the magnet and produce clearer images. Various types of functionalized magnetic nanoparticles including amine, carboxyl, epoxy and IDA functionalized nanoparticles are used for applications like immunoassay, gene transfection, biomolecule separation, cell separation, enzyme immobilization, drug delivery, and biomedical imaging. Nanoparticles also show potential for targeted cancer drug delivery and simultaneous imaging and therapy.
The Molecular Imaging Laboratory at Howard University provides state-of-the-art imaging equipment including high resolution MRI systems for small animal and clinical research. The lab aims to train students and foster multidisciplinary research using imaging to study disease processes and investigate new treatments. Areas of research include in vivo MRI and optical imaging of disease models in small animals, as well as molecular imaging of biological processes and developing new imaging probes and nanoparticles.
Handbook of coherent domain optical methodsSpringer
This chapter discusses light scattering spectroscopy techniques, both elastic and inelastic. Elastic light scattering spectroscopy measures the size, shape, and optical properties of particles or cells based on the spectrum of light they scatter. Inelastic light scattering spectroscopy, also known as Raman spectroscopy, measures the spectrum of light scattered by molecular vibrations or phonons in solids. These techniques are emerging as useful tools in materials science, environmental science, and biomedicine for applications such as measuring the size distribution of cell nuclei, early cancer detection, and disease diagnosis.
It has been almost decades since the “war on cancer” was declared. It is now generally
believed that personalized medicine is the future for cancer patient management.
Possessing unprecedented potential for early detection, accurate diagnosis, and
personalized treatment of cancer, nanoparticles have been extensively studied over the last
decade. In this report, I will try to summarize the current state-of-the-art nanoparticles in
biomedical applications targeting cancer. Multi- functionality nanoparticle-based agents.
Targeting ligands, imaging labels, therapeutic Drugs, and other. And the Role of Chemical
Engineers in this field and the promise that it holds for future.
Impact of detector thickness on imaging characteristics of the Siemens Biogra...Anax Fotopoulos
This document summarizes a Monte Carlo study using GATE simulation of a Siemens Biograph DUO PET/CT scanner. It investigates the impact of detector thickness on imaging characteristics by simulating the scanner with LSO detectors of 2cm and 3cm thickness. The results show that increasing thickness from 2cm to 3cm improves detection efficiency, resulting in a sharper energy peak at 511 keV, higher counts, and better energy resolution and signal-to-noise ratio. However, thicker detectors may also delay signal processing and increase device cost and complexity.
The document discusses the use of quantum dots (QDs) for biomedical applications such as bioimaging and therapy. It provides an overview of the photophysical properties of QDs that make them advantageous over organic dyes for imaging. Various biomedical applications of QDs are described, including in vitro and in vivo imaging, biosensing, photodynamic therapy, drug delivery, and gene delivery. Finally, the document outlines a research proposal to develop MoS2@polyaniline nanohybrids for dual-model imaging and synergistic photothermal/radiation therapy of tumors.
This document summarizes various molecular imaging techniques. It discusses how nuclear medicine involves molecular imaging by combining detectable labels with biologically important molecules to assess cellular functions. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) allow noninvasive assessment and quantification of small differences between patients. New molecular imaging agents currently in clinical trials will help transform imaging and therapy by quantifying targets like gene expression, receptors, and tumor metabolism. The document outlines different imaging modalities including PET, SPECT, magnetic resonance imaging, optical imaging, ultrasound, and identifies various biomarkers that can be used as imaging targets.
The Effect of Gamma Irradiation on the Radiofrequency Dielectric Dispersion P...theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
This document summarizes research conducted by Dr. Shiuan Huei Lin and collaborators on developing photopolymer materials for holographic data storage applications. It lists 16 journal publications, 1 book chapter, and 16 conference presentations from 2003 to 2009 reporting on their work investigating various dopants to improve the holographic recording characteristics of poly(methyl methacrylate) photopolymers. The research involved characterization of materials doped with compounds such as phenanthrenequinone and lanthanide organometallics to optimize their properties for volume holographic recording.
This document discusses various applications of nanotechnology in diagnostic pathology. It begins by defining key terms like nanometer and describing early concepts in nanotechnology. It then explores different nanomaterials like carbon nanotubes, nanorods, cantilevers, and quantum dots; how they are used for cancer detection and DNA analysis; and techniques like microfluidics. The document also covers applications in drug delivery, medical imaging, and surgery. Overall, the document outlines the growing role of nanotechnology across many areas of medical diagnosis and treatment.
Nanomedicine is an interdisciplinary field that uses nanotechnology for medical applications. It aims to diagnose, treat, and prevent disease at the molecular level using nano-scale tools. The document outlines the history of nanomedicine from Richard Feynman's 1959 talk introducing nanotechnology to current applications. Key applications discussed include drug delivery using nanoparticles like liposomes, imaging contrast agents, and miniaturized medical devices. Challenges also remain around potential toxicities of nanomaterials.
This document describes a technique called multispectral angular-resolved dark-field imaging (MARDI) that can automatically identify individual bacteria based on their unique light scattering spectra. An 87-channel microscope system was used to measure the scattering spectra of various bacteria at different angles and wavelengths. A simpler 15-channel system then demonstrated the viability of bacterial identification, accurately identifying four out of six bacterial species in tests of individual bacteria. This technique could provide a simple way to identify bacteria without the need for specialized equipment or skills.
This document describes a study that used optical coherence tomography (OCT) to image microvasculature in human skin in vivo with high spatial resolution. The study analyzed an intensity-based Doppler variance (IBDV) method for quantifying blood flow rates using an OCT system. Results showed that the IBDV method's sensitivity for detecting slow blood flow can be improved by increasing the time interval between adjacent A-lines. The study also proposed a higher sensitivity IBDV method that applies the algorithm along the slower scan direction. In vivo imaging of human skin microvasculature demonstrated the ability to identify vascular networks at depths up to 1.2 mm with exquisite spatial resolution.
This document characterizes long gradient index (GRIN) lens endoscope systems for multiphoton imaging of unstained tissues. The authors fabricate a portable, rigid endoscope system using a 1 mm diameter GRIN lens system that is 8 cm in length, capable of imaging a 200 μm field of view at 4 frames/s with 0.85 μm lateral and 7.4 μm axial resolution in water. In vivo images of unstained tissues in live, anesthetized rats are presented using this portable device, showing promise for clinical use of GRIN endoscopy.
Monitoring live cell viability Comparative studyWerden Keeler
This document compares three live cell imaging techniques: fluorescence microscopy, oblique incidence reflection microscopy, and phase contrast microscopy. It finds that oblique incidence reflection microscopy is the simplest, least expensive, and least phototoxic method, causing the least damage to live cells during long-term monitoring of cell viability. The document describes the equipment and cell lines used, including normal and cancerous cell lines tagged with fluorescent proteins or unlabeled, to evaluate the stresses induced by different illumination techniques.
A comparative study of living cell micromechanical properties by oscillatory ...Angela Zaorski
This study used an oscillatory optical tweezer-based technique to measure the frequency-dependent viscoelastic properties of cultured alveolar epithelial cells. Both the storage modulus and complex shear modulus followed a weak power law dependence on frequency. Measurements were taken by oscillating either an intracellular organelle or a bead attached to the cell's exterior through membrane receptors. The exponents of the power law were similar between the two measurement methods, but the modulus magnitudes differed significantly. Comparing intracellular and extracellular probing provided insights into cell mechanical properties.
Single Molecule Sequence Detection Via Microfluidic Planar Extensional FlowUniv of Cincinnati
1) 164 transcription factors (TFs) are responsible for 277 human diseases or syndromes.
2) The document suggests detecting TF binding sites on DNA using microfluidic planar extensional flow at a stagnation point to stretch DNA for single-molecule detection of TFs.
3) Three additional suggestions for stretching DNA for TF detection include using nanochannels, laminar flow, or stretching DNA in a casting film.
This document summarizes research on using metallic nanostructures to enhance fluorescence. Specifically, it proposes using "stair-gratings" - nanostructures with corrugations that have an excavated rectangular section to create a stair-like profile. Experiments show that stair-gratings provide higher fluorescence enhancement and narrower emission directionality compared to conventional gratings, covering both the excitation and emission bands of fluorophores. Finite-difference time-domain simulations agree with experimental results, demonstrating the potential of stair-gratings for applications requiring enhanced and directional single-molecule fluorescence.
Investigation of Chaotic-Type Features in Hyperspectral Satellite Datacsandit
This document analyzes the use of Lyapunov exponents to determine chaotic structure in hyperspectral satellite data. It investigates an EO-1 Hyperion hyperspectral image of a mixed forest site in Turkey. Lyapunov exponents are calculated from reconstructed phase spaces of spectral signals for different object classes. Positive and negative Lyapunov exponents indicate chaotic behavior is present. The results demonstrate Lyapunov exponents can be used as discriminative features to improve hyperspectral image classification accuracy by capturing the chaotic structures in the data.
Hamiltonian design in readout from room-temperature Raman atomic memory XequeMateShannon
We present an experimental demonstration of the Hamiltonian manipulation in light-atom interface in Raman-type warm rubidium-87 vapor atomic memory. By adjusting the detuning of the driving beam we varied the relative contributions of the Stokes and anti-Stokes scattering to the process of four-wave mixing which reads out a spatially multimode state of atomic memory. We measured the temporal evolution of the readout fields and the spatial intensity correlations between write-in and readout as a function of detuning with the use of an intensified camera. The correlation maps enabled us to resolve between the anti-Stokes and the Stokes scattering and to quantify their contributions. Our experimental results agree quantitatively with a simple, plane-wave theoretical model we provide. They allow for a simple interpretation of the coaction of the anti-Stokes and the Stokes scattering at the readout stage. The Stokes contribution yields additional, adjustable gain at the readout stage, albeit with inevitable extra noise. Here we provide a simple and useful framework to trace it and the results can be utilized in the existing atomic memories setups. Furthermore, the shown Hamiltonian manipulation offers a broad range of atom-light interfaces readily applicable in current and future quantum protocols with atomic ensembles.
NIR Three dimensional imaging of breast model using f-DOT Nagendra Babu
NIR three dimensional optical imaging of breast model using f-DOT using f-DOT with target specified contrast agent.
Three dimensional mathematical modeling of DOT,f-DOT.
1. The document summarizes Tijmen G. Euser's research activities and publications. As a PhD student, he studied dynamic changes in light propagation in photonic crystals and demonstrated optical switching of photonic band gap crystals.
2. As a postdoc, his research included developing hollow-core photonic crystal fibers for optofluidic microreactors, waveguide-based micromanipulation techniques, and spatial light modulation applications. This work enabled new experiments in fields like photochemistry, microparticle transport, and fiber-based spectroscopy.
3. His publications include over 40 peer-reviewed papers investigating topics like optofluidic reactors, optical trapping and propulsion in fibers, spatial mode control
This document describes a new microscopy technique called SPRAIPAINT that enables three-dimensional superresolution imaging of intracellular protein structures and the cell surface in living bacteria. SPRAIPAINT uses single molecule localization microscopy to image enhanced yellow fluorescent protein (eYFP) fusions within the cell and a dye binding to the cell surface sequentially using a single laser. This allows colocalization of an intracellular cytoskeletal protein (Crescentin-eYFP) with the cell membrane in living Caulobacter crescentus bacteria with 20-40nm precision over a large field of view. Imaging is done using a double-helix point spread function microscope for three-dimensional localization. SPRAIPAINT provides a
This document discusses microfluidic devices for DNA analysis. It summarizes that:
1) Microfabricated electrophoresis devices allow for faster DNA analysis and sequencing - in under 2 minutes for short-tandem-repeat genotyping assays and under 15 minutes for single-stranded DNA sequencing.
2) The microdevice format is a natural extension of improvements to electrophoresis over 100 years, operating in an almost perfect way limited only by the sieving medium.
3) Procedures for making the microfluidic devices are simple using current semiconductor fabrication technology, requiring one or two lithography steps at low resolution.
This document describes research into using fiber optic sensing in laser catheters to differentiate between tissues in real-time during minimally invasive surgery. Experiments were conducted using a porcine model and tissue phantoms to test a fiber optic sensor's ability to distinguish between blood and different tissue types, as well as measure the distance to a surface. The results demonstrated clear differentiation between blood and tissues, discrimination of different tissue types, and detection of surface contact over 1 mm away. This fiber optic sensing technique shows potential for smart surgical tools to increase safety for procedures where tactile feedback is limited.
This document lists 13 publications authored or co-authored by J. Maria. The publications cover a range of topics including soft embossing of nanostructures in glass, 3D chip stacking using micro-C4 interconnects, nanopost plasmonic crystals, multispectral thin film biosensing using 3D plasmonic crystals, optimization of 3D plasmonic crystal structures for refractive index sensing, and surface plasmon resonance imaging of molecules with high spatial resolution. Many of the publications were in prestigious journals such as ACS Nano, Nanotechnology, Analytical Chemistry, Angewandte Chemie International Edition, and Chemical Reviews.
International Journal of Image Processing (IJIP) Volume (1) Issue (1)CSCJournals
This document discusses a new method for enhancing magnetic resonance images (MRI) using multiscale retinex (MSR) to correct image inhomogeneities and enhance contrast. MSR employs single-scale retinex with different weightings to address non-uniform grayscale, improve contrast, correct inhomogeneity, and clarify deep brain structures in MRIs captured using surface coils. The performance is evaluated using peak signal-to-noise ratio and contrast-to-noise ratio on phantom and animal images, and is shown to outperform other methods like histogram equalization and wavelet-based algorithms. The retinex algorithm is thus a valuable method for MRI image processing.
This document lists 36 publications by A.M. Vossepoel in international journals from 1970 to 2013. The publications cover a range of topics including image and signal processing, medical imaging, and computer vision. Many of the publications describe algorithms and methods for tasks such as image segmentation, object detection, contour extraction, and skeletonization. The list provides titles, authors, publication details, and availability of PDFs for each of the 36 publications.
This document lists 50 peer-reviewed publications authored by A.I. Gusev and colleagues between 1991-2006. The publications cover research using time-of-flight mass spectrometry (TOF-MS) and matrix-assisted laser desorption/ionization (MALDI) for applications including quantitative analysis of peptides, proteins, polymers, and other molecules. Many of the publications describe improvements to MALDI and TOF-MS techniques to enhance signal reproducibility, reduce matrix effects, and enable quantification.
This document provides biographical and career information about David S. Moore. It includes his education history, with PhD and MS degrees in Pharmacology & Toxicology from the University of Kansas. It lists his appointments including director of microscopy laboratories and various research positions. It also provides details of his patents, teaching experience, and publications.
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3. 1. Introduction
Fluorescence microscopy is the most popular technique used in biological imaging
applications [1,2] due to the capabilities to target and modify specific single proteins within
the host organism. However, the resolution of a visible light microscope is limited by the
phenomenon of diffraction [3] to length scales set by the Rayleigh criterion to approximately
half the wavelength of the light [4,5]. Due to this limitation, any object smaller than the
microscope resolution will appear as a diffraction-limited spot with a point spread function
(PSF) given by an Airy function. The desire to observe biological structures and functions at
length scales smaller than the diffraction limit has led to the development of super-resolution
techniques that enable imaging intracellular structures with sub-diffraction-limit accuracy [6–
8].
Single molecule localization microscopy techniques, such as PALM [9] and STORM [10],
use optical control to activate a sparse subset of fluorescently tagged proteins in which the
PSF of each individual activated fluorophore does not overlap with that of its nearest
activated neighbor. This allows for the determination of the position of individual probes to a
much higher accuracy than conventional optical methods. The cycle of activation, imaging,
and photobleaching is repeated until all the fluorophores are exhausted, or a sufficient number
of them have been localized, and the measured molecular positions are then plotted to
generate a composite image [11].
In super-resolution microscopy, ultraprecise localization is usually accomplished by
fitting the diffraction-limited image of individual fluorophores to an ideal PSF using
algorithms such as non-linear least squares [12,13], and maximum likelihood [14]. These
methods can provide precision down to ~10nm, limited primarily by the number of photons
collected from each particular fluorophore [15,16], and have been used to great effect in
single particle tracking as well as in other imaging related applications. In these techniques,
reliable localization of a single fluorescent molecule requires both a sufficient number of
photons (N) in the measured PSF, and that the activated molecules be spatially separated by a
distance greater than several times the width of their PSFs [17]. Overlapping PSFs in standard
localization algorithms are generally discarded; this requires that the activation density remain
low across the field of view, which increases the composite image acquisition time [18]. To
address this problem, several methods have been recently developed with the aim of
localizing overlapping PSFs. One utilizes a maximum likelihood technique with increasing
numbers of point sources within the recorded PSF in the localization algorithm and is built for
graphics processing unit (GPU) analysis, and as such is relatively fast – the analysis time is
on the order of minutes [19–21]. Another paper uses a statistical deconvolution technique that
iterates through the observed PSF with a guess-work of overlapping PSFs. This approach is
very slow and requires ~10 times more computation time/frame than the other methods of
single-emitter fitting [22].
The problem of overlapping PSFs in super-resolution microscopy also affects the ultimate
spatial resolution of the composite image. In particular, the achievable spatial resolution is
determined by the localization density (i.e., the density of successfully localized spots) in
addition to the precision of each localization [17,18,23]. Using conventional localization
algorithms, when the number of photons emitted by the fluorophore is relatively low or the
spots are closely spaced, the localization density decreases since many regions of interest
(ROI: areas on the image that map to actual fluorophore locations on the sample) are
discarded in the analysis. Thus, high localization precision, high labeling density, and near-
unity localization efficiency of those fluorescent labels are all needed to achieve the best
possible spatial resolution. Here, we demonstrate a nonlinear image-factorization algorithm
designed for two distinct but related purposes: to suppress image noise associated with low
signal levels (poor photon statistics) and to differentiate image features associated with
different contrast levels. This approach increases spatial resolution and image acquisition
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4. speed both by requiring fewer photons to achieve the same localization precision and by
allowing for a higher density of activated fluorophores during each acquisition cycle.
The aim of this algorithm is effectively to sharpen the edges of the PSF from a single
point source. This sharpening will simultaneously allow isolated fluorophores to be localized
with higher precision (for a given number of photons), and two overlapping fluorophores to
be effectively isolated such that each can be localized to high precision. Usually, spatial
denoising techniques can be divided into linear and nonlinear approaches [24]. Mean and
Wiener filters [25] are two classical linear solutions which simply mask an image using the
local statistical measurements of pixels. At the same time, they also blur the edge. On the
contrary, nonlinear filtering technologies have the advantages to preserve the edge and fine
details. One such method is a multiscale renormalization algorithm which produces a fused
image by nonlinear recombination of the ratio of low-pass (ROLP) pyramidal decompositions
of the original images [26]. Another method is a cyclic algorithm for shadow removal based
on pulse-coupled neural networks (PCNN), an image processing algorithm derived from
biologically-grounded cortical models that addressed the experimental findings of stimulus-
induced synchronous bursts of pulse activity [27,28]. The PCNN-based factorization is an
efficient image-processing tool for noise smoothing and elementary image segmentation that
operates on local image patches. However, these methods are mathematically intractable and
have a high computational complexity. The adopted method is a non-linear algorithm, which
improves contrast at the PSF edges without increasing spurious noise. In particular, our
method uses the K-factor transformation, which decomposes the image into hierarchical
contrast-ordered factors whose joint product reconstructs the original pattern [29]. The K-
factor decomposition divides the image pattern into factors where noise elements are distinct
from those containing the image structure of interest. In this decomposition, the first few
components, which have the highest contrast depth, contain mainly the desired image
information while the higher orders contain mostly noise components. The K-factor algorithm
is applied to raw image data prior to emitter localization, and does not add much
computational complexity nor processing time, requiring only fractions of a millisecond per
frame. Furthermore, the proposed method reduces the total number of individual frames that
are required for the reconstruction of the final image, thus reducing the overall computation
time.
The paper is organized as follows: First, the theoretical background for imaging single
molecules, the mathematical background for nonlinear filtering algorithms, and the K-factor
transform are presented. Second, numerical simulations for a variety of parameters inherent in
the K-factor algorithm are presented and fully discussed. Third, the proposed algorithm is
applied on experimentally acquired images to validate the approach.
2. Theoretical background
For an aberration-free imaging system, the diffraction-limited point spread function
corresponding to a single point emitter (i.e. fluorescent label) is given by an Airy function
[30]. Near the peak, this function is approximated by a Gaussian, which is mathematically
simpler [31]. The measured intensity of the diffraction limited spot is given by the acquired
data together with photon shot-noise, background noise created most commonly by out-of-
focus fluorescence, charge coupled device (CCD) readout noise, dark current, and extraneous
fluorescence in the microscope. The model for the intensity at (x,y) of a fluorescent particle
located at (x0,y0), is given by [17]:
2 2
0 0
2
x x y y
2σ
B shot2
N
I x,y e η η
2πσ
(1)
where N is the total number of photons collected from the fluorophores' label by the image
acquisition system during the measurement period, σ is the standard deviation of the
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 247
5. Gaussian, that is given by setting the e1
point of the intensity model to be equal to the
Rayleigh radius, ηB is a Poisson distributed random variable with variance Nb that describes
the background noise (assumed constant across the field of view), and ηshot is a Poisson
distributed random variable that describes the shot noise with a mean equal to the square root
of the total intensity in each pixel [32]. In the absence of aberrations, the values will be equal
and given by [33]:
0.6 λ
σ
2N.A.
(2)
where N. A. is the numerical aperture of the objective and λ is the wavelength of the emitted
light.
In existing localization microscopy methods, the assumption is that two fluorophores must
be spatially well separated to achieve minimal penetration of the tails of one PSF into the
other. The detection of each PSF is accomplished by establishing an intensity threshold value
that distinguishes background from signal and determining whether a certain PSF exceeds this
value. If so, the data is fit to a model of a Gaussian profile with a single peak. If the two PSFs
are in close proximity such that the saddle between them is higher than the threshold, they
will be indistinguishable and be considered as one, resulting in an increased localization error.
To mitigate these errors, ROIs exhibiting elongated (asymmetric) intensity profiles are often
discarded from the sample set, effectively reducing the sampling density. Thus, the ability to
reduce the saddle between two overlapping PSFs will allow for fewer discarded point sources
in the localization algorithm, while also allowing for faster data acquisition rates.
3. K-factor algorithm
The K-factor algorithm is an image factorization [24,29] technique, which reduces an image
into a nonlinear finite or infinite set of contrast-ordered pseudoimage factors whose joint
product reassembles the original image.
The K-factor transformation of an image I(x,y) can be described mathematically as:
n
n 1
I x,y f x,y
M
(3)
where M is the number of factors that reconstruct the image, and fn are the pseudo image
factors that when multiplied together reconstruct the image and are given by:
n1 k g x, y
nf x, y
n n1 k
(4)
where the parameter k controls the contrast depth at each level with a value is between 0 and
1, and gn is a binary image computed as:
n 1 n
jj 1
I x, y 1
1
1 k, f x, y
0 .
ng x y
OW
(5)
The factorization algorithm is an iterative process that is based entirely on the depth of
contrast. The choice of k orders the image into different contrast depth factors. A small value,
close to zero, will give large contrast steps and produce a spatially coarse version of the image
with relatively few factors, while a value close to unity produces an image version with fine
geometrical details with a larger amount of factors.
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 248
6. For each n, the function gn that represents each pixel in the image is set to be either 1 or 0
according the threshold set by k. This will set the value for the factor fn. The original image is
then divided by the factor fn and this sets the threshold for the next iteration of gn. As a result,
the first few factors exhibit the largest depth of contrast, and mostly contain the desired image
information along with details related to image distortion. Since the desired image
information and distortion details generally exhibit different contrast levels, they typically
separate into distinct factors, allowing for their differentiation. Random spatial noise usually
falls into the higher-order factors, due to their lower contrast levels.
The K-factor algorithm produces a set of contrast-ordered factors, which when multiplied
together according to Eq. (3) will reproduce the original image with high fidelity, provided
appropriate values of k and M. If, however, the running product is truncated at a lower-order
factor fh, where h<M, then those image components with lower contrast levels (i.e., mostly
noise) will be de-emphasized in the reconstruction. In addition to noise, the truncated high
order factors might also contain some fine spatial information associated with low contrast
levels. Thus, we multiply the truncated product by the original image to restore some of these
fine spatial details:
1
, , ,
h M
h n
n
R x y I x y f x y
(6)
where Rh(x,y) is the truncated reconstruction. The optimal choice of the parameters k and M,
as well as the number of factors h in the truncated reconstruction, depends entirely on the
nature of the image, rather than on a pre-chosen spatial scale, and therefore it is determined de
novo for each application.
Figure 1 illustrates the influence of the K-factor transform on a noiseless image containing
two PSFs separated by a distance of 3σ~600nm. The K-factor parameters are k = 0.9, M = 48,
h = 8. It is clearly seen that the overlap between the two PSFs was reduced.
Fig. 1. Image of two PSFs separated by a distance of 3σ~600nm . Before the K-factor
transform (black) and after the K-factor transform (red).
The proposed technique is an added step during the conventional PALM routine as
illustrated in Fig. 2(b). The suggested super-resolved image acquisition technique begins with
single frame acquisition using repeated cycles of activation and imaging of the activated
fluorophores followed by bleaching to minimize the presence of already visualized particles
in posterior acquired images. The frame is passed on to a computer program like MATLAB
software package (MathWorks, Natick, MA, USA), where it is applied with the K-factor
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 249
7. algorithm. The next step is the localization of the activated fluorophores using methods of
single emitter fitting. Reconstruction and creation of the super-resolved image is done by
summing the molecular images across all frames.
Fig. 2. Super-resolution image acquisition steps for conventional PALM (a) and acquisition
steps for the K-factor algorithm (b). Conventional PALM acquisition is divided into three
independent steps: frame acquisition using repeated cycles of activation and imaging of the
activated fluorophores followed by bleaching to minimize the presence of already visualized
particles in posterior acquired images. Localization of the activated fluorophores using
methods of single emitter fitting. Reconstruction and creation of the super-resolved image by
summing the molecular images across all frames. The K-factor analysis adds a step prior to the
localization and is applied to each frame using computer software.
4. Simulation results
To evaluate the performance of the K-factor transformation routine and its impact on
localization precision, Monte-Carlo simulations were used to generate mock data sets with
two emitters in each set. In these simulations the model was a fluorescence source with λ =
540nm that was imaged through an objective lens onto a CCD camera. The model matches
the parameter of the Zeiss Axiovert 200 microscope using a 63x objective, 1.2N.A. water
immersion objective lens. Each pixel on the CCD sensor array had dimensions of 6.45μm ×
6.45μm, which translates to 102nm × 102nm in the image plane with the 63x objective.
Background noise was introduced by adding a sample from a Poisson distribution with the
parameter Nb. Shot noise was introduced by adding a Poisson distribution with parameter that
is the square root of the total intensity of the light at each pixel. The value for σ in the model
used for the PSF was calculated from Eq. (2) to be 191nm for the given imaging parameters.
Gaussian fitting was performed on both the raw simulated data and K-factor filtered data.
Each image contained a randomly positioned fluorophore, which was fit using the non linear
least-squares minimization routine lsqnonlin in MATLAB to fit a model of the form of:
2 2
0 0
2
x x y y
2σ
psf 2
N
I x, y e
2πσ
(7)
where N,σ, x0 and y0 are all fit parameters. The algorithm initially detected the position of
each particle as the pixel with the highest intensity in its region, and preformed a fit by taking
9 × 9 pixels around this initial location [34]. The fit produces the best estimate of the position
of that particular fluorophore, and the process is repeated many times yielding a set of L
localization positions xi, i) that can be compared with the known positions (xi,yi). The root
mean square (RMS) localization error is computed for both the raw and K-factor filtered
simulated data:
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 250
8.
2 2
1
1 L
rms i i i i
i
yerr x x y
L
(8)
The position of the each fluorophore, the distance between fluorophores, the number of
arriving photons N, the background noise parameter Nb, the K-factor parameter k and the
number of harmonics h, were allowed to vary. All analysis was performed in MATLAB.
A crucial aspect is the choice of the parameter k, and thus it was carefully determined after
numerous simulations and iterations, to result in a reliable decomposition of the image. The
simulated image contained two PSFs separated by 3σ~600nm. The k parameter varied
between 0-1. Additional noise was not introduced in order to reach maximum correlation and
maximize the k parameter. For each k value, the maximum correlation between the
reconstructed image and the original image was calculated using the corr function in
MATLAB (as shown in Fig. 3 (left y axis)), and also the number of factors needed for
achieving maximum correlation (as shown in Fig. 3 (right y axis)).
Fig. 3. Maximum correlation between the reconstructed image and the original image of two
PSFs separated by a distance of 3σ~600nm, as a function of k values (left y axis). Number of
factors needed for maximum correlation as a function of k values (right y axis).
It is clearly seen that for lower k values, the correlation is less than 75%, and therefore the
residual error is relatively large, whereas for higher k values, the correlation can reach 100%,
which means full reconstruction. A value of only k = 0.9 yielded a correlation of 100%; thus
a higher k value would not additionally contribute to image reconstruction. It is also evident
that the higher the k value, the more factors are required for the full reconstruction, as is
predicted by theory, and full image reconstruction results in a longer computation time. Due
to this tradeoff between correlation and the overall number of factors, users with
computational considerations can choose to work with a lower image correlation, for example
a correlation of 95%, with k = 0.5, where the number of factors is only 10. This will result in
a smaller improvement in resolution in the reconstructed image when compared to using a
value of k = 0.9.
Another key merit is the number of harmonics that the reconstructed image is multiplied
by, and its effect on the overlapping of neighboring PSFs. The first factors in the
decomposition contain most of the signal’s information, where the higher-order factors
contain mostly the noise. By multiplying the data with the first harmonics that contain most of
the signal data, the signal intensity increases and can be discriminated from the noise.
Simulations were carried in order to find the number of harmonics that contain only the signal
for different k values. Empirically, the formula that describes the number of harmonics that
contains only the signal:
1
6
h n (9)
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 251
9. where n is the number of factors needed for achieving a correlation of 100% with the original
image prior to the decomposition, for a given k. Figure 4(b) presents the influence of the
number of multiplied harmonics on the RMS error in localization as a function of distances
between centers for k = 0.9, n = 48. The lowest RMS error was obtained for h = 8 calculated
according to Eq. (9). The signal information contained in these h = 8 factors equals 91% of
the entire signal information. A lower value of h results in a reduced impact of the algorithm,
since not all the signal components were enhanced. For the end case of h = 0, the
reconstructed image equals the original image and the error equals the predicted value for the
least squares algorithm [17]. Increasing h also decreases the effect, since both the signal and
the noise were enhanced, and the algorithm's influence becomes negligible. For the end case
of h = n, the error returned is the same as for h = 0.
Figure 4(a) illustrates the relation of the K-factor transform and h on overlapping PSFs
according to Eq. (9) for a relatively low k = 0.4 (red) and for high k = 0.9 (blue). In such a
scenario, the PSF degradation is due to the overlap of the PSFs with each other, and in order
to present the influence of the algorithm on closely spaced PSFs, no additional noise was
added. The saddle was reduced by 56.1% and 54.2% respectively. However, the lower k
distorts the image and reduces the effect of improved localization.
Fig. 4. (a) The K-factor effect on overlapping PSFs for k = 0.4 (red) and k = 0.9 (blue). (b) the
influence of the number of multiplied harmonics on the RMS error in localization as a function
of distances between centers for k = 0.9, n = 48.
The shot noise is a Poisson random process with rate that depends on the total number of
photons detected. It is proportional to N, as can be seen in Fig. 5, e.g. an increased number
of emitted photons results in a more accurate localization precision [35]. Figure 5 compares
the results of the least squares fitting process applied to raw data (black line) and applied to
raw data that underwent processing with the K-factor algorithm (red line). It is clearly seen
that applying the K-factor algorithm on the raw data followed by the least squares fitting
process, resulted in localization error of ~3.5nm for a high photon count and for a distance of
3σ, an error that is lower than the predicted localization accuracy for the least squares
algorithm by itself [17]. The reason for this is due to the influence of the algorithm on the
reduction of the σ of each PSF. It narrows the width of the PSF, while allowing for the
isolation and localization of overlapping PSFs. For example, in Fig. 4(a), applying the K-
factor technique yielded a reduction in σ, by a factor of 1.5, which results in a localization
RMS error that is lower than N.
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 252
10. Fig. 5. Error in localization as a function of detected photons, distance between particles 3σ
~600nm, without background noise. The simulation contained 1000 Monte-Carlo iterations.
The background noise variance for each pixel is a parameter, marked by Nb, that can be
measured experimentally and therefore is considered to be known [36], and varied in our
simulations between 0 to 10 photons. We calculated the improvement (%) in localization
error according to:
100
original K Factor
original
RMS RMS
Improvement
RMS
(10)
The results can be seen in Fig. 6. For a distance of 3σ ~600nm), at low Nb of 0-2 photons, the
improvement was 40-50%, and with the background increased up to Nb = 10 photons, the
improvement reaches up to 85%. For low photon counts, the improvement in localization
accuracy is the most pronounced, and for fluorophores with lower photon yields, such as
fluorescent proteins, this will produce the most dramatic improvements in localization
accuracy. Since our proposed technique is aimed for reducing noises, the higher the
background noise is, the higher the improvement in localization over existing methods. The
improvement is achieved due to the high noise discrimination capabilities of the deshadowing
algorithm.
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 253
11. Fig. 6. The improvement in detection using the K-factor algorithm as a function of detected
photons, for different background noise photons Nb. The distance between particles was 3σ
~600nm.
As can be seen in Fig. 7, without background noise, at close distances of 2.5-4.σ and with
a low photon count, the improvement in detection of each spot can reach up to 85%. At larger
distances, the obtained improvement is reduced to ~5-20%, depending on the photon count.
Fig. 7. The improvement in localization using the K-factor algorithm as a function of the
distance between florescence centers for different number of photons N, without background
noise and with k = 0.9, h = 8.
5. Experimental results
Single molecule imaging experiments were performed in an epi-fluorescence microscope
setup consisting of an inverted microscope (Zeiss Elyra P.1, Carl Zeiss Microscopy Inc.), 1.46
N.A. total internal reflection (TIRF) objective, 642nm diode laser, and an electron multiplying
CCD camera Ixon (897, Andor Technologies PLC.) with EM gain set to 200. The epi-
fluorescence filter setup consisted of a dichroic mirror (650nm, Semrock) and an emission
filter (692/40, Semrock). The sample chamber was mounted in a 3D piezostage (P-737
PIFOC Specimen-Focusing Z Stage, Physik Instrumente). 10,000 images were taken in a
TIRF configuration at 40frames/second. Frames were 512 × 512 pixels with an effective pixel
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 254
12. size of 99.8nm. The K-factor technique was tested experimentally by imaging microtubules
from BSC-1 African green monkey kidney epithelial cells (American Type Culture
Collection-ATCC). These cells were cultured and stained with Alexa 647 phalloidin
fluorescent probes, using Abcam rat antibody to tubulin, ab6160 as the primary antibody, and
Invitrogen Alexa Fluor® 647 Goat Anti-Rat, A-21247 as the secondary antibody. Cell culture
methods were standard cell culture techniques for primary/secondary antibody labeling
methods. The peak emission wavelength of Alexa 647 is 671nm. Image pre-processing of the
raw data that contained noise filtering and application of the K-factor algorithm on each
frame was performed using MATLAB. The localization and the reconstruction of the super-
resolved image was done in ImageJ [37] using QuickPALM plugin [38].
The fluorescence data included individual frames in which pairs or larger groups of
fluorophores were simultaneously activated and produced a data set with overlapping
molecules (Fig. 8(a)). The reconstruction of the final super-resolution PALM image was
achieved using extracted data from 10,000 frames. The K-factor method was performed on
each of the 10,000 individual frames followed by the same localization and reconstruction
procedures to construct a super-resolution image. By applying the K-factor algorithm on each
individual frame (Fig. 8(b)), areas with overlapping molecules became distinguishable (Figs.
8(d), 8(f), 8(h)) compared to the original image (Figs. 8(c), 8(e), 8(g)).
Fig. 8. Individual PALM frame without processing (a) and after K-factor processing(b).
Marked regions is where the difference can be clearly seen. (c),(e) and (g) is the magnification
of the marked areas in (a). (d),(f) and (h) is the magnification of the marked areas in (b).
The proposed method's performance was tested as follows: first a conventional PALM
analysis was performed on the data (Fig. 9(a)), in which frames that contained overlapping
emitters were localized (Fig. 9(b)). Subsequently, the K-factor algorithm with parameters of k
= 0.9, n = 48, was applied to the data (Fig. 9(d)) followed by the same single molecule fitting
technique (Fig. 9(e)). As can be seen from the fitting process using both the conventional
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 255
13. PALM and the K-factor algorithm in (Fig. 9(b) and 9(e)), when neighboring molecules were
in close proximity, like in the upper section of the image, the K-factor routine enabled the
identification of two overlapping molecules, whereas in its absence, only one molecule was
identified. Figure 9(c) and 9(f) show the cross section of the dashed line that passes through
the center of the overlapping two emitters with σ = 195nm in the upper part of the image of
Fig. 9(b) and 9(e) respectively. As can be seen, applying the proposed method prior to the
localization reduces the saddle of overlapping PSFs and enables their localization.
Fig. 9. Single molecule fitting process preformed on individual frames of conventional PALM
(Upper row) and on frames preprocessed by the K-factor algorithm (Lower row). (b) is the
magnification of the marked area in (a). PALM analysis (blue circles) localize 2 emitters. (e) is
the magnification of the marked area in (d). K-factor preprocessing followed by PALM
analysis (red crosses) localize 3 emitters. (c) and (f) show the cross section of the dashed line
that passes through the center of the overlapping two emitters with σ = 195nm in the upper part
of the image in (b) and (e) respectively.
In order to create the fully reconstructed image, all the single emitter estimations of
individual frames were summed, using both the unprocessed images and the K-factor
processed images. The use of the K-factor algorithm enabled the extraction of data that
otherwise would have been discarded from each frame, which resulted in the ability to
reconstruct the full image with fewer frames than the conventional PALM image. To quantify
the reduction in number of frames that require the full reconstruction using the K-factor
process, an iterative approach was used. First, the correlation coefficient between the two
images was calculated for a total number of 10,000 frames used for the reconstruction. Then,
for this given correlation coefficient, the number of frames in the K-factor reconstruction
process was reduced, until the point where the correlation coefficient decreased by 5%. This
was set to be the lowest number of frames needed for the full reconstruction in comparison to
the conventional PALM.
Using the K-factor algorithm, only 5,735 frames were required for the full reconstruction
of the image (Fig. 10(b)) whereas conventional PALM analysis required 10,000 (Fig. 10(c)).
Taking only 5,735 frames using the conventional PALM produced a less detailed image
estimation with gaps in locations where there was a high density of overlapping emitters (Fig.
10(a)). Taking 10,000 frames using the K-factor algorithm resulted in a sharper image
estimate, due to the higher accuracy in localization (Fig. 10(d)).
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 256
14. Fig. 10. Reconstruction of imaging data from Alexa647 labeled microtubules sample without
processing and using the K-factor algorithm prior to the localization. Images in the upper row
represent reconstructed image using 5,735 frames and those in the lower row represent
reconstructed image using 10,000 frames. (a,c) conventional PALM analysis. (b,d) K-factor
algorithm applied to raw data followed by conventional PALM analysis.
The effective resolution of the reconstructed image was measured using the method of
Fourier ring correlation (FRC) [23,39]. FRC evaluates the degree of correlation of two
independent reconstructions of the same object in frequency space and determines the
resolution threshold (the spatial frequency) at which both reconstructions are consistent and
considered to be resolved. Reconstruction using 10,000 individual frames without additional
processing resulted in effective resolution of 55.74nm, whereas taking the same amount of
frames and using the K-factor algorithm additional processing yielded a resolution of
43.21nm. When taking only 5,735 frames, the resolution obtained without additional
processing was 80.16nm, compared to 55.15nm with the K-factor algorithm processing. The
use of the proposed method in comparison to conventional PALM analysis improves the
resolution of the obtained image. In addition, the same effective resolution of a given super-
resolution image can be obtained using the proposed method with a lower number of frames,
and as a result, decreases image acquisition time while increases the sampling density. For the
experimental results presented, acquisition of each frame of an Alexa647 labeled
microtubules image with a field of view: 51.1μm × 51.1μm takes 40ms. For an effective
resolution of ~55nm, the amount of individual frames required for the generation of the super-
resolution image was 10,000 using conventional PALM, in comparison to 5,735 individual
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 257
15. frames using the K-factor algorithm. The use of the K-factor processing enabled the decrease
of 42% of the total super-resolution image acquisition time for the same resolution. The
penalty is the increase in processing time, which is 0.3ms per frame in a simple PC (HP
Compaq Elite 8300 Microtower PC with Windows 7 professional 64 bit operation system,
Intel® Core i5-3470 processor, 3.20 GHz, 12 GB RAM).
6. Conclusions
Implementation of an image decomposition K-factor algorithm on images acquired by
fluorescence microscopy techniques like PALM and STORM, prior to the fitting process,
allows for the analysis of images with a higher density of activated fluorophores per frame,
thereby offering an increase in the image acquisition speed. In addition, it can improve the
precision of localization of individual fluorophores.
The proposed method was compared to localization performed by standard methods of
single molecule fitting, such as least-squares fitting and maximum likelihood. In the
simulation section, data fitting was performed using the method of least-squares, while in the
experimental section, the fit was performed using both methods. The technique showed
maximum impact and effect on closely spaced fluorescent spots, such as two neighboring
PSFs overlaping extensively. This paper examined the effects of changing the parameters of
the K-factor algorithm, such as the depth of contrast k, the number of factors needed for
achieving maximum correlation for a given k and the number of harmonics as a function of
the distance between particles, the number of arriving photons and the background noise
parameter.
The best way to appropriately choose parameters for working with the K-factor algorithm
is first determine the k parameter that yields the best reconstruction of the image in terms of
maximum correlation, and subsequently derive the parameters n and h. Our validation studies
using simulated and experimental data showed that the K-factor algorithm can increase image
collection time of super-resolution data by 42% while maintaining the same image resolution,
or improve the obtained resolution by 37% for the same total super-resolution image
acquisition time (provided the sample still has active fluorophores). Therefore, the method
can be a useful tool for fast single molecule fitting super-resolution microscopy techniques
operating at high density, allowing for an increase in activated fluorophore density (in units of
μm2
) of ~50%.
When comparing the performance of the K-factor algorithm to the two multi-emitter
fitting methods presented in the introduction section, both techniques – utilizing a maximum
likelihood technique for GPU analysis and statistical deconvolution – do not improve the
obtained resolution of the super-resolution image as does the K-factor method, and are aimed
at reducing the number of frames needed for acquisition by factor of ~5 and ~8 respectively.
The multi-fitter method has an analysis times on the order of minutes, similar to the K-factor,
whereas the statistical deconvolution method requires ~10 times more computation
time/frame than standard methods of single-emitter fitting, and in total does not decrease
image acquisition time, as the K-factor technique does.
Acknowledgments
This work was supported by NSF grant CAREER #DBI-0845193 and NIH grant R01
NS034307. We would also like to thank Edward J. Hujber for his help with imaging and data
collection.
#198546 - $15.00 USD Received 30 Sep 2013; revised 20 Nov 2013; accepted 21 Nov 2013; published 16 Dec 2013
(C) 2013 OSA 1 January 2014 | Vol. 5, No. 1 | DOI:10.1364/BOE.5.000244 | BIOMEDICAL OPTICS EXPRESS 258