Qiaole Zhao, Ben Schelen, Raymond Schouten, Rein van den Oever, Rene Leenen, Harry van Kuijk, Inge Peters, Frank Polderdijk, Jan Bosiers, Marcel Raspe, Kees Jalink, Jan Geert Sander de Jong, Bert van Geest, Karel Stoop, Ian Young
We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.
We have developed a model-based, parallel procedure to estimate fluorescence lifetimes. Multiple frequencies are
present in the excitation signal. Modeling the entire fluorescence and measurement process produces an analytical ratio
of polynomials in the lifetime variable τ. A non-linear
model-fitting procedure is then used to estimate τ. We have
analyzed this model-based approach by simulating a 10 μM fluorescein solution (τ = 4 ns) and all relevant noise sources.
We have used real LED data to drive the simulation. Using 240 μs of data, we estimate τ = 3.99 ns. Preliminary
experiments on real fluorescent images taken from fluorescein solutions (measured τ = 4.1 ns), green plastic test slides
(measured τ = 3.0 ns), and GFP in U2OS (osteosarcoma) cells (measured τ = 2.1 ns) demonstrate that this model-based
measurement technique works.
Qiaole Zhao, Ian Ted Young, Ben Schelen, Raymond Schouten, Rein van den Oever, René Leenen, Harry van Kuijk, Inge Peters, Frank Polderdijk, Jan Bosiers, Kees Jalink, Sander de Jong, Bert van Geest, Karel Stoop
We have built an all-solid-state camera which is directly modulated at the pixel level for frequency
domain fluorescence lifetime imaging microscopy (FLIM) measurement. This novel camera eliminates
the need for an image intensifier through the use of an application-specific CCD design,
which is being used in a frequency domain FLIM system. The first stage of evaluation for the
camera has been carried out. Camera characteristics such as noise distribution, dark current influence,
camera gain, sampling density, sensitivity, linearity of photometric response, and contrast
modulation transfer function have been studied through experiments. We are able to do lifetime
measurement using MEM-FLIM cameras for various objects, e.g. fluorescence plastic test slides,
fluorescein solution, fixed GFP cells, and GFP - Actin stained live cells.
We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon "budget." These measures are relevant to many fluorescence microscope users and the results are not restricted to FLIM but applicable to widefield fluorescence microscopy in general. Limitations in photon numbers, however, are more of an issue with FLIM compared to other less quantitative types of imaging. By modeling a typical experimental configuration, examples are given for fluorophores whose absorption peaks span the visible spectrum from Fura-2 to Cy5. We have performed experiments to validate the assumptions and parameters used in our mathematical model. The influence of fluorophore concentration on the intensity of the fluorescence emission light and the Poisson distribution assumption of the detected fluorescence emission light have been validated. The experimental results agree well with the mathematical model. This photon budget is important in order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems.
We measured light scattered from gold nanoparticles with darkfield microscopy in order to perform single molecule detection based on tethered particle motion (TPM). This combination results in a signal to noise ratio of about 40 dB, which allowed us to use 80 nm diameter gold particles as reporters instead of the typically used polystyrene particles whose sizes are up to 1 µm. The particle size is crucial in TPM experiments as it can induce a volume-exclusion effect, which results in a stretching force acting on the DNA tether. This affects both the biophysical and statistical properties of the anchored DNA and hence the interpretation of the experimental data. We demonstrated that the gold nanoparticles and darkfield microscopy can be used to characterize the confined Brownian motion of dsDNA-tethered gold particles with a spatial precision of 3 nm. Physical parameters such as the spring constant of the tethered DNA fragment and the persistence length can be derived from the two dimensional (2D) (x, y) projected image data. We have applied this method to various MgCl2 and glycerol concentrations as a proof of principle.
Based upon a collection of compact LEDs (light-emitting diodes) and a compact photodiode, we
have developed a calibration tool for fluorescence microscopes that are used as digital imaging
devices. The entire device (excluding a USB connector) measures 25 mm × 80 mm × 12 mm.
Virtually all commonly-used fluorophores can be simulated with one of the six LEDs. An LED is
chosen from the host computer and its current range is selected (digitally) so as to provide a test of
the complete dynamic range of the imaging system. Thus by varying the current through an LED in a
controlled way, a controlled amount of "emission" light can be produced, transmitted through the
chosen optical path of the microscope, and measured by the image sensor. The digitized intensity
can then be determined as a function of the LED current. Any other (fluorescence) intensity
measured through the same electro-optical path can then be characterized (and thus calibrated) by an
equivalent electrical current.
The excitation light is calibrated by a photodiode which has a dynamic range of 10^5:1 and thus is
suitable for a variety of light sources: mercury lamps, lasers, LEDs, etc. The integration time of the
photodiode as well as its gain can be digitally selected from the host computer. Further, using a
Spectralon® reflector, the inherent non-linearity of the LED emission versus current can be measured
by the photodiode and used to provide a look-up table compensation independent of the image
sensor used in the fluorescence microscope system.
A new microscopy method for multi diffraction-limited spot illumination is based on extraordinary light transmission through a periodic metal grid (typical period of 600 nm) of sub-wavelength holes (150 nm). Multiple spots illuminate a fluorescently labeled sample and the emission is collected by far-field optics. Theoretical comparison with a confocal microscope reveals equivalent spot sizes and a scanning method with the advantage of multiple illumination spots. The system is used to measure the actual transmitted field with a fluorescent sample in far-field. The obtained results are consistent with the theoretical prediction and provide a proof of concept of the midfield microscope.
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.
The knowledge of the near-field of extraordinary 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.
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.
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.
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 photon-plasmon 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.
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 α-synuclein. We describe the motivation for this development, the problems associated with the miniaturization of the aggregation assay, and the validation of our modifications.
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 ρT. This parameter describes if the telomeres are distributed in a sphere-like volume (ρT ≈ 1) or in a disk-like volume (ρT >> 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 ρT 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 ρT for G0/G1, S and G2 are 1.03, 1.02 and 13 respectively.
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.
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.
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.
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 LED light 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.
In this presentation we will describe experiments comparing various LEDs to other light sources. We will 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.
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 hole-array.
In previous publications and presentations we have described our construction of a laboratory-on-a-chip based on nanoliter capacity wells etched in silicon. We have described methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the production or consumption of NADH in enzyme-catalyzed reactions such as those found in the glycolytic pathway of yeast. In this paper we describe the use of light sensors (photodiodes) in each well to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence in 100 μM and for ATP/luciferase bioluminescence is 2.4 μM.
We are participating in the international competition to develop robots that can play football (or soccer as it is known in the US and Canada). The competition consists of several leagues each of which examines a different technology but shares the common goal of advancing the skills of autonomous robots, robots that function without a central hierarchical command structure. The Dutch team, Clockwork Orange, involves several universities and the contribution of our group at the TU Deift is in the domain of robot vision and motion. In this paper we will describe the background to the project, the characteristics of the robots in our league, our approach to various vision tasks, their implementation in computer architectures, and the results of our efforts.
Yeast-Saccharomyces cerevisiae - it widely used as a model system for other higher eukaryotes, including man. One of the basic fermentation processes in yeast is the glycolytic pathway, which is the conversion of glucose to ethanol and carbon dioxide. This pathway consists of 12 enzyme-catalyzed reactions. With the approach of microarray technology we want to explore the metabolic regulation of this pathway in yeast. This paper will focus on the design of a conventional microscope based microarray reader, which is used to monitor these enzymatic reactions in microarrays. These microarrays are fabricated in silicon and have sizes of 300 by 300 micrometers 2. The depth varies from 20 to 50 micrometers . Enzyme activity levels can be derived by monitoring the production or consumption rate of NAD(P)H, which is excited at 360nm and emits around 450nm. This fluorophore is involved in all 12 reactions of the pathway. The microarray reader is equipped with a back-illuminated CCD camera in order to obtain a high quantum efficiency for the lower wavelengths. The dynamic range of our microarray reader varies form 5(mu) Molar to 1mMolar NAD(P)H. With this microarray reader enzyme activity levels down to 0.01 unit per milliliter can be monitored. The acquisition time per well is 0.1s. The total scan cycle time for a 5 X 5 microarray is less than half a minute. The number of cycles for a proper estimation of the enzyme activity is inversely proportional to the enzyme activity: long measurement times are needed to determine low enzyme activity levels.
We are developing a method for high-throughput screening using arrays of `nanowells' built into a silicon substrate. These wells can serve as bioreactors for studying a variety of biochemical reactions such as the enzymatic activity that occurs in yeast metabolism. For a variety of studies it is important to know the volume of liquid that has been deposited in a given well and/or to monitor the evaporation of the liquid. Using silicon as our substrate means that we can take advantage of the ability to build microelectronics into the wells in order to develop `smart' wells.
Intelligent Molecular Diagnostic Systems (IMDS)- The objective of this multidisciplinary research program is to design and develop an analytical system that is able to measure and interpret concentrations of various analytes which are dispensed on a micro-array. The analytes are detected by means of fluorescence or (chemi)luminescence measurement. Furthermore, the collected data are combined and interpreted using modern reasoning techniques. Micro-injection- Dispensing picoliters (pl) of reagents (enzymes, antibodies, etc.) and liquid samples on a micro-array requires special techniques. At the moment we are working on a technique which will allow for accurately dispensing liquid volumes less than 100 pl on a micro-array. Detection of (beta) -D-glucose- (beta) -D-glucose standards are dispensed on a micro-array, after which a solution of Amplex Red reagent, horse radish peroxidase (HARP), and glucose oxidase in a mixture of ethylene glycol and water is added. Ethylene glycol is added to prevent evaporation. The (beta) -D-glucose reacts with glucose oxidase to D-gluconolactone and H2O2. The H2O2 reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) with a 1:1 stoichiometry to produce highly fluorescent resorufin. The formation of resorufin with time is followed with a Zeiss Axioskop microscope equipped with a KAF Photometrics CCD camera, in order to determine the sensitivity, concentrations, and volumes associated with the dispensed fluids.
The goal of our TU Delft interfaculty research program is to develop intelligent molecular diagnostic systems (IMDS) that can analyze liquid samples that contain a variety of biochemical compounds such as those associated with fermentation processes. One specific project within the IMDS program focuses on photon sensors. In order to analyze the liquid samples we use dedicated microarrays. At this stage, these are basically miniaturized micro titre plates. Typical dimensions of a vial are 200 X 200 X 20 micrometer3. These dimensions may be varied and the shape of the vials can be modified with a result that the volume of the vials varies from 0.5 to 1.6 nl. For all experiments, we have used vials with the shape of a truncated pyramid. These vials are fabricated in silicon by a wet etching process. For testing purposes the vials are filled with rhodamine solutions of various concentrations. To avoid evaporation glycerol-water (1:1, v/v) with a viscosity of 8.3 times the viscosity of water is used as solvent. We aim at wide field-of-view imaging at the expense of absolute sensitivity: the field-of-view increases quadratically with decreasing magnification. Small magnification, however, implies low Numerical Aperture (NA). The ability of a microscope objective to collect photons is proportional to the square of the NA. To image the entire microarray we have used an epi-illumination fluorescence microscope equipped with a low magnification (2.5 X/0.075) objective and a scientific CCD camera to integrate the photons emitted from the fluorescing particles in the solutions in the vials. From these experiments we found that for this setup the detection limit is on the order of micromolar concentrations of fluorescing particles. This translates to 108 molecules per vial.
We present the results of experiments in which we investigate how the physical gamma maps onto the perceived gamma, and how the subjective quality of a printed image may be influenced by the grey scale mapping that is used to print the image. The grey scale is altered by changing the gamma, the exponent relating the input to the output luminance. Five natural images, widely different in content, have been printed with 16 different values of gamma. First the printer gamma is measured. In general, this gamma differs from 1. In the printer a gamma transform is performed as well. The selected gamma values are corrected using the directly gamma function. The aim of the first experiment is to find out what the relation is between physical gamma and perceived gamma. The stimulus response function derived from our data is found to be quite linear over the range investigated. The aim of the second experiment is to test the subjective preference for a particular gamma. We have found that these gammas are not independent of image content. The mean preference averaged over the subjects for the various images ranges form 1.6 to 2.2.
A well-known distortion of objects in three-dimensional microscopy manifests itself as an elongation in the axial direction. Authors such as Visser and Hell have seemingly contradicted one another on the cause as well as the magnitude of the effect. We have examined these theories and performed simulations and experimental measurements to better understand the nature of the effect. We simulate point spread functions (based on the work of Gibson) taking into account the various refractive indices involved as well as the magnification, the numerical aperture, the working distance of the objective, the depth of the object under the coverslip, and the object's size. We measure the axial and lateral dimensions of digitized images of microspheres that have been 'acquired' using a simulated point spread function that changes as the depth of the object changes. These simulations are done for conventional (optical sectioning) microscopy as well as for confocal microscopy. Further, we have performed experimental measurements on real microspheres on a conventional microscope to relate theory, simulation, and practice. Our measurements and simulations show that (1) the object's size, (2) its depth under the coverslip, (3) the refractive index mismatch between the immersion fluid (nimmersion) and embedding material for the object (nembedded), and (4) the NA of the lens play a pivotal role in the effect.
One of the promising recent developments in fluorescence microscopy is fluorescence lifetime imaging microscopy. This type of microscopy images the lifetime of fluorescence molecules (in the nano second range) rather than the amount of light emitted by these molecules. This physical property is of interest while it gives information about the local environment of the molecule, such as molecular concentration of O2, Ca2+, pH, and conjugation. Our goal is to design a affordable, robust and easy-to-use FLIM workstation which is completely automated and does not need any difficult calibration. Therefore we are developing a workstation which applies a homodyne detection scheme (frequency range: 1 - 100 MHz) with use of an intensity modulated laser-diode (635 nm) and a gain modulated intensified CCD camera to image fluorescence lifetimes in the range of 1 - 100 ns. Using these components it is possible to make a FLIM workstation based on a normal fluorescence microscope by just replacing the light source and image detector. The FLIM image acquisition procedure in software allows automatic optical measurements of fluorescence lifetimes in different ranges and mixtures of lifetimes by adjusting the modulation frequency.
KEYWORDS: Objectives, 3D image processing, Optical spheres, 3D metrology, Microscopes, Optical transfer functions, Point spread functions, Image processing, Luminescence, Microscopy
An important point in the automated evaluation of molecular cytogenetic preparations is focusing the specimen before taking images. The in-focus position can vary for different positions on the slide, thus every new field of view has to be refocused. Another problem is that objects in one field of view can lie in different planes with the effect that one object is out-of-focus while the other one is in-focus. We want to investigate the importance of focus with regard to measurements like object intensity. Diffraction wave theory leads to a formula, which can be used to determine the depth-of-focus (DOF), the tolerance in the in-focus position where the image is still sharp. However practice shows that it is possible to get good measurement results even if the specimen is not imaged perfectly.the relation between the theoretical and practical DOF is analyzed. The objects under investigation are either 2D or 3D and are imaged in absorption and fluorescence mode. It appears that the DOF based on measurements can be 5 to 10 times bigger than a DOF based on wave theory with only little loss of accuracy.
In this paper we present methods for characterizing CCD cameras. Interesting properties are linearity of photometric response, signal-to-noise ratio, sensitivity, dark current, and spatial frequency response. The techniques to characterize CCD cameras are carefully designed to assist one in selecting a camera to solve a certain problem. The methods described were applied to a variety of cameras: an Astromed TE3/A with P86000 chip, a Photometrics CC200 series with Thompson chip TH7882, a Photometrics CC200 series with Kodak chip KAF1400, a Xillix' Micro Imager 1400 with Kodak chip KAF1400, an HCS MXR CCD with a Philips chip and a Sony XC-77RRCE.
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