Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1429—Signal processing
  • G01N15/1433—Signal processing using image recognition
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
  • G01N21/553—Attenuated total reflection and using surface plasmons
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/0008—Microscopes having a simple construction, e.g. portable microscopes
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/008—Details of detection or image processing, including general computer control
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N2015/0038—Investigating nanoparticles
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
  • G01N2015/019—Biological contaminants; Fouling
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
  • G01N21/553—Attenuated total reflection and using surface plasmons
  • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance

Definitions

• the invention relates to a heatsink containing
• the invention further relates to a method for optical detection of individual nanoparticles on two-dimensional measuring surfaces.
• optical arrangements i. the ability to observe small objects in optical microscopy, for example, is limited by the diffraction.
• the smallest particles which can be resolved with a light microscope have a diameter in the range of 0.2 ⁇ m corresponding to 200 nm.
• the arrangements required for this purpose are expensive.
• a label-free method which allows selectively viruses or particles of interest with a diameter in the nanometer range (nanoparticles) in water or
• the change in light intensity is analyzed at certain points in the image. When viewed with the eye, this is done on the retina.
• the detection with a detector takes place for example with a batch
• Coupled Device Since these changes are due to the difference in the optical properties of the object, such as transmission, refractive index or color and the environment, one can characterize the properties of the object.
• DE 40 24 476 C1 describes a Kretschmann arrangement which is used for the determination of analytes in a fluid sample.
• Layer thickness of the molecules bound to the surface can be achieved so that a required signal-to-noise ratio is achieved. Individual particles can not be detected by the method.
• the object of the invention is achieved in that the resolution of the observation optics and the detector is greater than the achievable with the radiation source under classical conditions, diffraction-limited resolution. It has surprisingly been found that the observation of nanoparticles is also possible with wavelengths which are significantly larger than the particle diameter. For example, the observation of particles with a
• a reference signal is formed, with which the signal is normalized.
• the reference signal may be generated by averaging the signals detected at the same location on the sensor surface prior to attachment of a particle to the sensor surface.
• the detection of the plasmon resonance can be done with a so-called Kretschmann arrangement.
• a local change in reflectivity is caused by the interaction of the nanoparticles with the evanescent field.
• particles smaller than 40 nm can be detected.
• areas with several square millimeters can be detected simultaneously. It is important in the arrangement that the particles are very close to or touch the sensor surface at a distance below 200 nm.
• the device is suitable for use in virological research and in the detection of viruses in public areas. The procedure can also be used for highly sensitive clinical diagnostics.
• the change in the reflectivity of the sensor surface is observed, which is caused by a particle.
• This change is in a preferred one
• Embodiment of the invention stepwise and localized in a few microns.
• the change is made at a location unknown in advance and at an unknown time measured.
• the location and time are found individually for each particle during the measurement.
• the signal is measured at pre-defined relatively large (> 100x100 ⁇ m 2 ) locations. At these places have been known
• the signal increases continuously, in proportion to the layer density of the already bound particles or molecules.
• the sensor used is a surface which ensures the greatest possible change in reflectivity by changing the refractive index.
• Such a surface with a strong change in reflectivity can be realized by means of a gold surface in Kretschmann arrangement.
• the reflectivity at an angle of incidence near the resonance angle depends very much on the refractive index of the medium that is in contact with the gold layer.
• the selectivity of the detection is ensured, as in the conventional SPR imaging method, by selectivity of the binding of particles to receptors attached to the sensor surface.
• the reflectivity changes are due to the size and refractive index of the particle, they can be used to characterize the particle.
• the area with high reflectivity change can be realized by periodic structures that allow plasmon excitation. Also anti-glare glass or crystal surfaces are suitable. The anti-reflective coating is broken by the particles and the reflectivity in the environment increases.
• the surface with high reflectivity change is realized by a surface with metallic nanoparticles which allow the excitation of localized plasmon resonance at selected wavelengths. These metallic nanoparticles serve as sensor particles. Near the resonance, the scattering of a particle is dependent on the refractive index. When another non-metallic particle attaches to a sensor particle, the reflectivity of the sensor changes
• the surface with high reflectivity change can be realized by a multi-layered optical structure in which surface plasmon resonance can also be excited.
• Embodiments of the invention are the subject of the dependent claims.
• Fig.l is a schematic representation of a Kretschmann- arrangement for
• FIG. 2 shows the course of a reflection signal in the vicinity of the resonance wavelength with and without particles.
• Fig. 5 shows the image of an observed area after different
• Fig. 6 is a schematic illustration of a regular array
• FIG. 1 shows a Kretschmann arrangement in an imaging configuration, generally designated 10. Such an arrangement 10 is already known. The operation of the surface plasmon spectrometer therefore need not be described in detail here.
• a glass sheet 12 is coated with a 50 nm thick gold layer 14. With the side facing away from the gold layer 14 16, the glass sheet 12 is attached to a prism 18. For fixing and improving the optical contact immersion oil is used.
• the gold layer 14 is illuminated with radiation 20 from a superluminescent diode 22.
• a superluminescent diode 22 is a QSDM-680-9 of
• Illumination takes place through the prism 18 at a fixed angle of incidence 24.
• a fixed angle of incidence 24 is also included in the prism 18.
• a superluminescent diode has no irregularities in the
• the angle of incidence 24 is chosen so that the wavelength of the diode appears on the left side of the resonance minimum, ie at a smaller angle.
• a lens 26 is used to generate a parallel radiation beam. It is understood that a curved mirror can also be provided here.
• a sample space for liquids in the form of a flow cell 28 is attached on the gold layer.
• the flow cell 28 is formed by a 1 mm thick S-shaped PDMS seal.
• the rear part of the flow cell 28 (not shown) is made of Plexiglas.
• an inlet and an outlet in the form of hoses are provided on the flow cell 28.
• the cell volume of the flow cell 28 is about 300 ⁇ L.
• the glass surface of the disk 12 forms a sensor surface.
• the sensor surface is illuminated by a standard Minolta Photo Lens 30 on a Charge Coupled Device
• the aperture of the objective 30 is 1 / 1.7.
• the CCD detector was a Cappa-100 CCD with a pixel size of 6.45x6.45 ⁇ m.
• the saturation capacity of the detector is 40000 electrons per pixel.
• the detector 32 has an area of -1000 x 1000 pixels. At a 7x magnification, one pixel corresponds to a sensor area of about ⁇ l ⁇ m. In the horizontal plane (p-plane), the image is compressed due to the slope of the sensor surface relative to the optical axis. Here a pixel corresponds to a sensor surface of about ⁇ l, 4 ⁇ m. The slope additionally causes a significant limitation of the area on the sensor surface, which can be sharply imaged.
• the images are possible at a rate of up to 100 frames / second.
• the pictures are saved and evaluated.
• Intensity is formed for each pixel and stored for further processing.
• the intensity distribution of the original image is very inhomogeneous. This is due to the high sensitivity of the sensor surface to small irregularities on the surface. There are usually many points to see that have a deviation from a mean value of up to 70%.
• the local inhomogeneity of ⁇ 10% within a 20 ⁇ m spot on the sensor surface is typical for the rest of the image. This intensity distribution is relatively stable over time. Normalization is used to reduce inhomogeneity.
• a reference image is generated by averaging the images over the period 34 ( Figure 3) in which no particle suspension is coming.
• the shot noise is the essentially limiting factor for the minimum detectable intensity level.
• the maximum product of the pixel capacitance C and the read-out frequency F is preferably used. With a pixel capacity of 40,000 photoelectrons, only ⁇ 20,000 can accumulate. Otherwise, saturation of some pixels occurs due to the significant inhomogeneities of the image.
• the present embodiment is integrated with an average over 40 images / second over 10 pixels.
• 2x10 6 electrons are accumulated in the area associated with the bound particle.
• the present method can detect steps of the order of 10 -3 . This means that the shot noise is the limiting factor.
• FIG. 5 The images in FIG. 5 were taken with the CCD camera 32. On successive shots one can directly observe the binding of particles.
• the particles were injected in the 140th second.

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• Physics & Mathematics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Immunology (AREA)
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• Optics & Photonics (AREA)
• Engineering & Computer Science (AREA)
• General Engineering & Computer Science (AREA)
• Computer Vision & Pattern Recognition (AREA)
• Signal Processing (AREA)
• Dispersion Chemistry (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)

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• 2009
  • 2009-02-27 DE DE102009003548A patent/DE102009003548A1/de not_active Ceased
• 2010
  • 2010-02-23 WO PCT/EP2010/052229 patent/WO2010097369A1/de active Application Filing
  • 2010-02-23 EP EP10706196A patent/EP2401602A1/de not_active Withdrawn
  • 2010-02-23 JP JP2011551477A patent/JP2012519271A/ja active Pending
• 2011
  • 2011-08-26 US US13/218,804 patent/US8587786B2/en active Active

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