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The dynamics of a diffusing particle in a potential field is ubiquitous in physics, and it plays a pivotal role in single-molecule studies. We present a formalism for analyzing the dynamics of diffusing particles in harmonic potentials at low Reynolds numbers using the time evolution of the particle probability distribution function. We demonstrate the power of the formalism by simulation and by measuring and analyzing a nanobead tethered to a single DNA molecule. It allows one to simultaneously extract all the parameters that describe the system, namely, the diffusion coefficient and the restoring-force constant., IST/Imaging Science and Technology, Applied Sciences

The dynamics of a diffusing particle in a potential field is ubiquitous in physics, and it plays a pivotal role in single-molecule studies. We present a formalism for analyzing the dynamics of diffusing particles in harmonic potentials at low Reynolds numbers using the time evolution of the particle probability distribution function. We demonstrate the power of the formalism by simulation and by measuring and analyzing a nanobead tethered to a single DNA molecule. It allows one to simultaneously extract all the parameters that describe the system, namely, the diffusion coefficient and the restoring-force constant., IST/Imaging Science and Technology, Applied Sciences

Using an optimized combination of tethered particle motion method, total internal reflection, and a gold nanobead, we measured the three-dimensional distribution of the free end of a tethered DNA molecule. The distribution along the axial z direction (perpendicular to the surface) is found to be Rayleigh-like, in agreement with wormlike chain and freely jointed chain simulations. Using these simulations, we show that the presence of the wall increases the correlations between the orientations of neighboring chain segments compared to free DNA. While the measured and the simulated planar (xy) distributions always agree with that of a Gaussian-random-walk (GRW) model, for short DNA lengths (1 ?m) studied in our experiment, the corresponding axial (z) distributions deviate from those predicted for a GRW confined to half-space., BT/Biotechnology, Applied Sciences

HU is a highly conserved protein that is believed to play an important role in the architecture and dynamic compaction of bacterial DNA. Its ability to control DNA bending is crucial for functions such as transcription and replication. The effects of HU on the DNA structure have been studied so far mainly by single molecule methods that require us to apply stretching forces on the DNA and therefore may perturb the DNA-protein interaction. To overcome this hurdle, we study the effect of HU on the DNA structure without applying external forces by using an improved tethered particle motion method. By combining the results with DNA curvature analysis from atomic force microscopy measurements we find that the DNA consists of two different curvature distributions and the measured persistence length is determined by their interplay. As a result, the effective persistence length adopts a bimodal property that depends primarily on the HU concentration. The results can be explained according to a recently suggested model that distinguishes single protein binding from cooperative protein binding., Imaging Science and Technology, Applied Sciences

Using an optimized combination of tethered particle motion method, total internal reflection, and a gold nanobead, we measured the three-dimensional distribution of the free end of a tethered DNA molecule. The distribution along the axial z direction (perpendicular to the surface) is found to be Rayleigh-like, in agreement with wormlike chain and freely jointed chain simulations. Using these simulations, we show that the presence of the wall increases the correlations between the orientations of neighboring chain segments compared to free DNA. While the measured and the simulated planar (xy) distributions always agree with that of a Gaussian-random-walk (GRW) model, for short DNA lengths (1 ?m) studied in our experiment, the corresponding axial (z) distributions deviate from those predicted for a GRW confined to half-space., BT/Biotechnology, Applied Sciences

HU is a highly conserved protein that is believed to play an important role in the architecture and dynamic compaction of bacterial DNA. Its ability to control DNA bending is crucial for functions such as transcription and replication. The effects of HU on the DNA structure have been studied so far mainly by single molecule methods that require us to apply stretching forces on the DNA and therefore may perturb the DNA-protein interaction. To overcome this hurdle, we study the effect of HU on the DNA structure without applying external forces by using an improved tethered particle motion method. By combining the results with DNA curvature analysis from atomic force microscopy measurements we find that the DNA consists of two different curvature distributions and the measured persistence length is determined by their interplay. As a result, the effective persistence length adopts a bimodal property that depends primarily on the HU concentration. The results can be explained according to a recently suggested model that distinguishes single protein binding from cooperative protein binding., Imaging Science and Technology, Applied Sciences

We measured individual trajectories of fluorescently labeled telomeres in the nucleus of eukaryotic cells in the time range of 10-2–104 sec by combining a few acquisition methods. At short times the motion is subdiffusive with (r2)?t? and it changes to normal diffusion at longer times. The short times diffusion may be explained by the reptation model and the transient diffusion is consistent with a model of telomeres that are subject to a local binding mechanism with a wide but finite distribution of waiting times. These findings have important biological implications with respect to the genome organization in the nucleus., Imaging Science and Technology, Applied Sciences

Department of Imaging Science and Technology, Applied Sciences

We measured individual trajectories of fluorescently labeled telomeres in the nucleus of eukaryotic cells in the time range of 10-2–104 sec by combining a few acquisition methods. At short times the motion is subdiffusive with (r2)?t? and it changes to normal diffusion at longer times. The short times diffusion may be explained by the reptation model and the transient diffusion is consistent with a model of telomeres that are subject to a local binding mechanism with a wide but finite distribution of waiting times. These findings have important biological implications with respect to the genome organization in the nucleus., Imaging Science and Technology, Applied Sciences

Department of Imaging Science and Technology, Applied Sciences

Imaging Science and Technology, Applied Sciences

Single-molecule techniques continue to gain in popularity in research disciplines such as the study of intermolecular interactions. These techniques provide information that otherwise would be lost by using bulk measurements that deal with a large number of molecules. We describe in this report the motion of tethered DNA molecules that have been tagged with gold nanobeads and observed under dark field microscopy to study single molecular interactions (SMI). We further report on the derivation and use of several physical parameters and how these parameters change under differing experimental conditions., Quantitative Imaging Group, Applied Sciences

Imaging Science and Technology, Applied Sciences

Single-molecule techniques continue to gain in popularity in research disciplines such as the study of intermolecular interactions. These techniques provide information that otherwise would be lost by using bulk measurements that deal with a large number of molecules. We describe in this report the motion of tethered DNA molecules that have been tagged with gold nanobeads and observed under dark field microscopy to study single molecular interactions (SMI). We further report on the derivation and use of several physical parameters and how these parameters change under differing experimental conditions., Quantitative Imaging Group, Applied Sciences

The study of DNA-protein interactions is gaining increased attention due to their importance in cellular processes. Only a well-functioning interaction guaranties that such a process can take place without errors. So far, only a small percentage of these interactions have been unraveled, partially due to their complexity but also due to the fact that there are only a few techniques that permit the study of these interactions. In this report we describe the development of a research tool based on tethered bead motion and Resonance Light Scattering (RLS) from gold beads. This method permits the study of DNA-protein interactions and the screening of proteins binding to a specific DNA sequence., Quantitative Imaging Group, Applied Sciences

We present a study on the far-field patterns of light transmitted through sub-wavelength metallic hole-arrays. Spectral imaging measurements are used here on hole arrays for the first time. It provides both spatial and spectral information of the transmission in far-field. The visibility of the images, measured in two illumination modes: Köhler and collimated, is calculated for different planes in and out of focus. The transmission under collimated illumination reveals that 75% of the beam if non-divergent. The results are in agreement with the low divergence measured by Lezec [Science 297, 820 (2002)]., Imaging Science and Technology, Applied Sciences

The knowledge of the near-field of extra-ordinary transmission through hole-arrays is mostly theoretical; there is less experimental validation of the theory. We study the near-field properties by measuring fluorescent molecules that are immersed in a solution and their Brownian motion. The measurements are performed by filling the space above the hole-array with fluorescent solution and exciting these molecules through the hole-array. By measuring both the fluorescence and the direct exciting light, it is possible to learn about the near-field properties., Imaging Science and Technology, Applied Sciences

While fluorescence microscope systems remains an essential tool in modern biology and medical work, no compact instrumentation has been developed for the rapid calibration of such systems. Almost invariably results are presented in terms of the [AU], "arbitrary units". To remedy this situation we have developed a small, portable instrument - the size of a microscope slide - that uses low-power LEDs at different wavelengths to produce calibrated amounts of light. A computer controls the instrument—through a USB connector—so that the current to the selected LED can be swept through an increasing range of values. The amount of light measured by the microscope's total imaging system (lenses, filters, EO sensor, and digitizer) is then recorded to provide a “current in, digital value out” calibration. Further, the current can be translated easily to optical power and thus photons per second at the chosen LED wavelength. We have built and programmed such a system, tested it for accuracy and precision, and used it to calibrate several microscopes and microscope/lens combinations. The results will be presented., Quantitative Imaging Group, Applied Sciences

The invention relates to a titre plate for chemical and/or biochemical analysis based on an illumination measurement comprising a transparent substrate, applied to the substrate there is a metallic layer, which is provided with an array of reaction chambers, wherein the reaction chambers extend from the top side of the metallic layer to, or into, the substrate thereunder, wherein at the side of the metallic layer facing away from the substrate, the same is provided with a transparent cover plate and wherein, viewed in the plane of the metallic layer, the cross-sectional dimension of the reaction chambers is smaller than the illumination wavelength., Electrical Engineering, Mathematics and Computer Science

We present a study on the far-field patterns of light transmitted through sub-wavelength metallic hole-arrays. Spectral imaging measurements are used here on hole arrays for the first time. It provides both spatial and spectral information of the transmission in far-field. The visibility of the images, measured in two illumination modes: Köhler and collimated, is calculated for different planes in and out of focus. The transmission under collimated illumination reveals that 75% of the beam if non-divergent. The results are in agreement with the low divergence measured by Lezec [Science 297, 820 (2002)]., Imaging Science and Technology, Applied Sciences

The study of DNA-protein interactions is gaining increased attention due to their importance in cellular processes. Only a well-functioning interaction guaranties that such a process can take place without errors. So far, only a small percentage of these interactions have been unraveled, partially due to their complexity but also due to the fact that there are only a few techniques that permit the study of these interactions. In this report we describe the development of a research tool based on tethered bead motion and Resonance Light Scattering (RLS) from gold beads. This method permits the study of DNA-protein interactions and the screening of proteins binding to a specific DNA sequence., Quantitative Imaging Group, Applied Sciences

The knowledge of the near-field of extra-ordinary transmission through hole-arrays is mostly theoretical; there is less experimental validation of the theory. We study the near-field properties by measuring fluorescent molecules that are immersed in a solution and their Brownian motion. The measurements are performed by filling the space above the hole-array with fluorescent solution and exciting these molecules through the hole-array. By measuring both the fluorescence and the direct exciting light, it is possible to learn about the near-field properties., Imaging Science and Technology, Applied Sciences

While fluorescence microscope systems remains an essential tool in modern biology and medical work, no compact instrumentation has been developed for the rapid calibration of such systems. Almost invariably results are presented in terms of the [AU], "arbitrary units". To remedy this situation we have developed a small, portable instrument - the size of a microscope slide - that uses low-power LEDs at different wavelengths to produce calibrated amounts of light. A computer controls the instrument—through a USB connector—so that the current to the selected LED can be swept through an increasing range of values. The amount of light measured by the microscope's total imaging system (lenses, filters, EO sensor, and digitizer) is then recorded to provide a “current in, digital value out” calibration. Further, the current can be translated easily to optical power and thus photons per second at the chosen LED wavelength. We have built and programmed such a system, tested it for accuracy and precision, and used it to calibrate several microscopes and microscope/lens combinations. The results will be presented., Quantitative Imaging Group, Applied Sciences

The invention relates to a titre plate for chemical and/or biochemical analysis based on an illumination measurement comprising a transparent substrate, applied to the substrate there is a metallic layer, which is provided with an array of reaction chambers, wherein the reaction chambers extend from the top side of the metallic layer to, or into, the substrate thereunder, wherein at the side of the metallic layer facing away from the substrate, the same is provided with a transparent cover plate and wherein, viewed in the plane of the metallic layer, the cross-sectional dimension of the reaction chambers is smaller than the illumination wavelength., Electrical Engineering, Mathematics and Computer Science

Quantitative analysis in combination with fluorescence microscopy calls for innovative digital image measurement tools. We have developed a three-dimensional tool for segmenting and analyzing FISH stained telomeres in interphase nuclei. After deconvolution of the images, we segment the individual telomeres and measure a distribution parameter we call ?T . This parameter describes if the telomeres are distributed in a sphere-like volume (?T ? 1) or in a disk-like volume (?T » 1). Because of the statistical nature of this parameter, we have to correct for the fact that we do not have an infinite number of telomeres to calculate this parameter. In this study we show a way to do this correction. After sorting mouse lymphocytes and calculating ?T and using the correction introduced in this paper we show a significant difference between nuclei in G2 and nuclei in either G0/G1 or S phase. The mean values of ?T for G0/G1, S and G2 are 1.03, 1.02 and 13 respectively., Quantitative Imaging Group, Applied Sciences

When light passes through a hole smaller than the wavelength of the light, the transmission is very low and the light is diffracted. This however changes if holes are arranged in a periodic array on metal. In that case the light couples to surface plasmons; this results in enhanced transmission, spectral selection and a small angular diffraction. We develop a novel microscopic method based on a periodic hole-array, which will be used as a multiple-apertures near-field source for illuminating a biological sample while the light is collected in far-field. The measurement speed is high, due to the use of an array instead of a single source. The main advantage of this microscope originates from the low diffraction of light through a relatively thick sample with enhanced transmission. It results in the ability to measure the samples interior and 3D reconstruction can be made by semi-confocal techniques. This overcomes the major limitation of near-field methods for which only a shallow layer of the surface (~20 nm) is detectable. For our measurements we use glass coated devices. The holes are processed with a focused ion beam. The photonplasmon coupling process is characterized as a function of the wavelength. Our experiments aim on gaining a better understanding of the transmission process. We tested the dependence of the transmitted spectrum on angle of incidence was tested as well as far-field spectral imaging measurements of the transmission in both Koehler and collimated light illumination. The results as well as the description of the microscope that we are constructing are presented., Imaging Science and Technology, Applied Sciences

We report on the progress of a novel nanofluidic device for detecting and manipulating single molecules in solution. This paper discusses the development of an earlier proposed molecule separation method, where electrokinetic forces separate different molecules based on their masses and charges. Optical imaging using confocal microscopy is applied to perform the detection of the single molecules. Potential applications of this device will be assessed. This research aims for the high spatial and spectral resolutions, both in manipulation and detection, which can lead to molecular identification., Quantitative Imaging Group, Applied Sciences

The invention relates to an optical microscope, comprising, at least a light source, a carrier for an object to be examined, a detector for registering the illuminated object, and a light path that during operation runs substantially from the light source to the object and form the object to the detector, wherein a metallic film having a periodic hole array is placed in the light path between the light source and the object, and wherein the carrier of the object is provided with a drive to allow the same to be adjusted in the plane of the carrier, wherein the holes of the metallic thin film have a diameter that is smaller than approximately 250 nm, in that the drive is designed for adjusting the carrier for the object in an orientation perpendicular to the plane of the carrier, and in that a processing device is provided that is connected with the detector for constructing a three-dimensional image of the object., Applied Sciences

In previous publications we have shown that we can perform enzymatic reactions in nanoarrays by means of a microarray-reader based on a conventional microscope. In this publication we report on a modification of this system in order to monitor the aggregation kinetics of the natively unfolded protein ?-synuclein. We describe the motivation for this development, the problems associated with the miniaturization of the aggregation assay, and the validation of our modifications., Imaging Science and Technology, Applied Sciences

When light passes through a hole smaller than the wavelength of the light, the transmission is very low and the light is diffracted. This however changes if holes are arranged in a periodic array on metal. In that case the light couples to surface plasmons; this results in enhanced transmission, spectral selection and a small angular diffraction. We develop a novel microscopic method based on a periodic hole-array, which will be used as a multiple-apertures near-field source for illuminating a biological sample while the light is collected in far-field. The measurement speed is high, due to the use of an array instead of a single source. The main advantage of this microscope originates from the low diffraction of light through a relatively thick sample with enhanced transmission. It results in the ability to measure the samples interior and 3D reconstruction can be made by semi-confocal techniques. This overcomes the major limitation of near-field methods for which only a shallow layer of the surface (~20 nm) is detectable. For our measurements we use glass coated devices. The holes are processed with a focused ion beam. The photonplasmon coupling process is characterized as a function of the wavelength. Our experiments aim on gaining a better understanding of the transmission process. We tested the dependence of the transmitted spectrum on angle of incidence was tested as well as far-field spectral imaging measurements of the transmission in both Koehler and collimated light illumination. The results as well as the description of the microscope that we are constructing are presented., Imaging Science and Technology, Applied Sciences

We report on the progress of a novel nanofluidic device for detecting and manipulating single molecules in solution. This paper discusses the development of an earlier proposed molecule separation method, where electrokinetic forces separate different molecules based on their masses and charges. Optical imaging using confocal microscopy is applied to perform the detection of the single molecules. Potential applications of this device will be assessed. This research aims for the high spatial and spectral resolutions, both in manipulation and detection, which can lead to molecular identification., Quantitative Imaging Group, Applied Sciences

The intense research in proteomics is demanding for fast, reliable and easy-to-use methods in order to study the proteome. In this proceeding we report the development of such a novel research tool based on spectral imaging and Resonance Light Scattering gold particles. This method will allow the study of DNA-protein interactions. We suggest a broad range of applications: the screening of proteins binding to a specific DNA sequence, the analysis of binding affinities between protein and DNA, and the investigation of the influence of environmental conditions on the binding. We will explain the principle, first experiments and first results based on Brownian motion., Imaging Science and Technology, Applied Sciences

In previous publications we have shown that we can perform enzymatic reactions in nanoarrays by means of a microarray-reader based on a conventional microscope. In this publication we report on a modification of this system in order to monitor the aggregation kinetics of the natively unfolded protein ?-synuclein. We describe the motivation for this development, the problems associated with the miniaturization of the aggregation assay, and the validation of our modifications., Imaging Science and Technology, Applied Sciences

Quantitative analysis in combination with fluorescence microscopy calls for innovative digital image measurement tools. We have developed a three-dimensional tool for segmenting and analyzing FISH stained telomeres in interphase nuclei. After deconvolution of the images, we segment the individual telomeres and measure a distribution parameter we call ?T . This parameter describes if the telomeres are distributed in a sphere-like volume (?T ? 1) or in a disk-like volume (?T » 1). Because of the statistical nature of this parameter, we have to correct for the fact that we do not have an infinite number of telomeres to calculate this parameter. In this study we show a way to do this correction. After sorting mouse lymphocytes and calculating ?T and using the correction introduced in this paper we show a significant difference between nuclei in G2 and nuclei in either G0/G1 or S phase. The mean values of ?T for G0/G1, S and G2 are 1.03, 1.02 and 13 respectively., Quantitative Imaging Group, Applied Sciences

The invention relates to an optical microscope, comprising, at least a light source, a carrier for an object to be examined, a detector for registering the illuminated object, and a light path that during operation runs substantially from the light source to the object and form the object to the detector, wherein a metallic film having a periodic hole array is placed in the light path between the light source and the object, and wherein the carrier of the object is provided with a drive to allow the same to be adjusted in the plane of the carrier, wherein the holes of the metallic thin film have a diameter that is smaller than approximately 250 nm, in that the drive is designed for adjusting the carrier for the object in an orientation perpendicular to the plane of the carrier, and in that a processing device is provided that is connected with the detector for constructing a three-dimensional image of the object., Applied Sciences

Traditional light sources for fluorescence microscopy have been mercury lamps, xenon lamps, and lasers. These sources have been essential in the development of fluorescence microscopy but each can have serious disadvantages: lack of near monochromaticity, heat generation, cost, lifetime of the light source, and possible distortions due to coherence effects. We are examining the possibility of using the new high-power light-emitting diode (LED) sources as alternatives to the above mentioned sources. LED sources are near monochromatic, are inexpensive, produce little heat, have no coherence problems, have extended lifetimes, are small, and can easily be modulated. We describe experiments comparing various LEDs to other light sources. We compare, for example, a 530 nm LED to the 546 nm line from a mercury lamp on a fluorophore whose absorption maximum is broad and in the middle between these two wavelengths., Quantitative Imaging Group, Applied Sciences

Applied Sciences

Recently, an extraordinary transmission of light through small holes (<200 nm) in a thin metallic film has been described. This phenomenon has been shown to be the result of the photon-plasmon interaction in thin films where a periodic structure (such as a set of holes) is embedded in the film. One of the extraordinary results is that the beam that passes through a hole has a very small diffraction in extreme contrast to the wide angle predicted by diffraction theory. Based on this effect, we propose here a new type of microscopy that we term mid-field microscopy. It combines an illumination of the sample through a metallic hole-array with far-field collection optics, a scanning mechanism and a CCD. When compared to other high resolution methods, what we suggest here is relatively simple because it is based on a thin metallic film with an array of nano-sized holes. Such a method can be widely used in high-resolution microscopy and provide a novel simple-to-use tool in many life-sciences laboratories. When compared to near-field scanning optical microscopy (NSOM), the suggested mid-field method provides a significant improvement. This is chiefly for three reasons: 1. The penetration depth of the microscope increases from a few nanometers to a few micrometers, hence the name mid-field microscope. 2. It allows one to measure an image faster because the image is measured through many holes in parallel rather then through a single fiber tip used in conventional near-field microscopy, and 3. It enables one to perform three-dimensional reconstruction of images due to a semi-confocal effect. We describe the physical basics of the photon-plasmon interaction that allows the coupling of light to the surface plasmons and determines the main spectral characteristics of the device. This mechanism can be ascribed due to the super-periodicity of the electron oscillations on the metallic surface engendered by the grating-like structure of the holearray., Imaging Science and Technology, Applied Sciences

We present the latest progress on a novel technology for detecting and manipulating solution of single molecules in nanofluidic channels. This paper explains the design and fabrication of nanofluidic chip and its interface, molecule manipulation technique being used, and the optical detection method employed. Single molecule detections are performed using optical imaging as well as metal microelectrodes. The ultimate goal is to get high spatial and spectral resolutions that can lead to molecular identification., Quantitative Imaging Group, Applied Sciences

Traditional light sources for fluorescence microscopy have been mercury lamps, xenon lamps, and lasers. These sources have been essential in the development of fluorescence microscopy but each can have serious disadvantages: lack of near monochromaticity, heat generation, cost, lifetime of the light source, and possible distortions due to coherence effects. We are examining the possibility of using the new high-power light-emitting diode (LED) sources as alternatives to the above mentioned sources. LED sources are near monochromatic, are inexpensive, produce little heat, have no coherence problems, have extended lifetimes, are small, and can easily be modulated. We describe experiments comparing various LEDs to other light sources. We compare, for example, a 530 nm LED to the 546 nm line from a mercury lamp on a fluorophore whose absorption maximum is broad and in the middle between these two wavelengths., Quantitative Imaging Group, Applied Sciences

Applied Sciences

Recently, an extraordinary transmission of light through small holes (<200 nm) in a thin metallic film has been described. This phenomenon has been shown to be the result of the photon-plasmon interaction in thin films where a periodic structure (such as a set of holes) is embedded in the film. One of the extraordinary results is that the beam that passes through a hole has a very small diffraction in extreme contrast to the wide angle predicted by diffraction theory. Based on this effect, we propose here a new type of microscopy that we term mid-field microscopy. It combines an illumination of the sample through a metallic hole-array with far-field collection optics, a scanning mechanism and a CCD. When compared to other high resolution methods, what we suggest here is relatively simple because it is based on a thin metallic film with an array of nano-sized holes. Such a method can be widely used in high-resolution microscopy and provide a novel simple-to-use tool in many life-sciences laboratories. When compared to near-field scanning optical microscopy (NSOM), the suggested mid-field method provides a significant improvement. This is chiefly for three reasons: 1. The penetration depth of the microscope increases from a few nanometers to a few micrometers, hence the name mid-field microscope. 2. It allows one to measure an image faster because the image is measured through many holes in parallel rather then through a single fiber tip used in conventional near-field microscopy, and 3. It enables one to perform three-dimensional reconstruction of images due to a semi-confocal effect. We describe the physical basics of the photon-plasmon interaction that allows the coupling of light to the surface plasmons and determines the main spectral characteristics of the device. This mechanism can be ascribed due to the super-periodicity of the electron oscillations on the metallic surface engendered by the grating-like structure of the holearray., Imaging Science and Technology, Applied Sciences

We present the latest progress on a novel technology for detecting and manipulating solution of single molecules in nanofluidic channels. This paper explains the design and fabrication of nanofluidic chip and its interface, molecule manipulation technique being used, and the optical detection method employed. Single molecule detections are performed using optical imaging as well as metal microelectrodes. The ultimate goal is to get high spatial and spectral resolutions that can lead to molecular identification., Quantitative Imaging Group, Applied Sciences