EP3824272A1 - Diffraktiver biosensor - Google Patents
Diffraktiver biosensorInfo
- Publication number
- EP3824272A1 EP3824272A1 EP19719788.2A EP19719788A EP3824272A1 EP 3824272 A1 EP3824272 A1 EP 3824272A1 EP 19719788 A EP19719788 A EP 19719788A EP 3824272 A1 EP3824272 A1 EP 3824272A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- light
- phase
- detector
- biogrid
- measuring
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- 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
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/4788—Diffraction
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
- G01N21/774—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides the reagent being on a grating or periodic structure
- G01N21/7743—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides the reagent being on a grating or periodic structure the reagent-coated grating coupling light in or out of the waveguide
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
- G02B26/02—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the intensity of light
- G02B26/04—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the intensity of light by periodically varying the intensity of light, e.g. using choppers
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
- G02B6/24—Coupling light guides
- G02B6/42—Coupling light guides with opto-electronic elements
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
- G02B6/24—Coupling light guides
- G02B6/42—Coupling light guides with opto-electronic elements
- G02B6/4201—Packages, e.g. shape, construction, internal or external details
- G02B6/4204—Packages, e.g. shape, construction, internal or external details the coupling comprising intermediate optical elements, e.g. lenses, holograms
- G02B6/4215—Packages, e.g. shape, construction, internal or external details the coupling comprising intermediate optical elements, e.g. lenses, holograms the intermediate optical elements being wavelength selective optical elements, e.g. variable wavelength optical modules or wavelength lockers
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- 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
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/4788—Diffraction
- G01N2021/479—Speckle
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/063—Illuminating optical parts
- G01N2201/0633—Directed, collimated illumination
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/063—Illuminating optical parts
- G01N2201/0635—Structured illumination, e.g. with grating
Definitions
- the present invention relates to a diffractive biosensor.
- Such sensors are based on the adsorption of biomolecules to be detected on a diffractive grating for diffraction of light.
- the signal from a photodetector for the diffracted light serves as a measure of the mass loading of the biosensor with the biomolecules.
- Planar waveguides arranged on a substrate are known from optics and have an optical grating for coupling in or coupling out light.
- Such an optical grating is, for example, structures etched into the substrate or into the waveguide and thus consist of the material of the substrate or of the waveguide.
- the grating period required depends on the wavelength of the light used and on the refractive index of the waveguide. Depending on the coupling angle, the grating period is in the range of the effective wavelength of the light Waveguides. It is typically about half the vacuum wavelength of the light.
- biogrid In the field of biosensors, grids for coupling light in and out are also known which consist of biological matter and act as a receptor for the biomolecules to be examined. If such biomolecules attach themselves to the receptors structured to form a lattice, the biomolecules form an optically effective lattice. Such receptors structured to form a lattice with or without adsorbed biomolecules are referred to below as biogrid. Since the diffraction efficiency of such a biogrid depends on the mass occupancy of the grating with the biomolecules, a quantitative statement about the mass occupancy can be made on the basis of the intensity of the diffracted light measured by means of a detector.
- a diffractive biosensor is known from W02015004264A1, in which divergent light falls through a substrate onto an optical grating for coupling light into a waveguide.
- the light propagating in the waveguide then hits a biogrid that acts as a coupling-out grating.
- the outcoupled light is focused through the substrate onto a detector.
- the light intensity measured in the detector is a measure of the occupancy of the coupling-out grating with the biomolecule to be examined.
- the use of two biogrids means a very low signal due to the double weak coupling. Since the desired measurement light is also overlaid by undesired scattered light, which is also in a fixed phase relationship to the measurement light and can interfere with it, optimal measurement signals are not obtained.
- a diffractive biosensor is also described in US7008794B2. Here it is proposed to subtract a background diffraction pattern from the measurement signal in order to emphasize the actually desired signal. However, the fixed phase relationship of the scattered light to the measurement light is also neglected here.
- Other biosensors are also described in EP2618130A1, EP2757374A1 and EP2929326B1.
- a diffractive biosensor for the selective detection of biomolecules having a substrate and an optical biogrid arranged on the substrate, the biogrid having periodically arranged receptors for the biomolecules, and the efficiency of a diffraction of incident light and thus the intensity of an in a measuring light beam arriving at a detector depends on a mass occupancy of the biogrid with the biomolecules to be detected.
- the biosensor has a device for generating a reference light beam directed at the detector, with which the phase position of scattered light arriving in the detector can be determined relative to the measurement light beam.
- speckle occurs when illuminating scattering surfaces with coherent light. It is scattered light that interferes with itself with a random phase position and thus generates a random phase and amplitude distribution. This phenomenon also occurs with diffractive biosensors (e.g. as in the prior art cited above), and affects the measuring accuracy.
- the diffractometric measuring field is coherently overlaid by a stray field and falsified by this.
- E M or Es are the electrical field strengths of the measuring field or stray field
- y M or cps the respective phases
- IM + S the associated overall intensity in the detection plane.
- the present invention therefore provides an apparatus and an associated method for measuring this phase difference in order to be able to draw off the stray field correctly, and thus to be able to infer the undisturbed measuring field E M or its intensity I M.
- Unwanted scattering centers ie disturbances of all kinds such as surface roughness, contamination of the surface, grain boundaries in the waveguide, non-specifically attached particles / biomolecules, etc.
- the unwanted scattering centers are not structured but arranged randomly, the scattered light is emitted in a wide solid angle and not specifically in the direction of the detection location. This is the reason why diffractive biogrids are extremely robust against non-specific deposits.
- the unwanted scattering centers - especially in the case of scattering due to substrate roughness - are unstructured, but are nevertheless arranged in a stationary manner.
- the phase position of the resulting stray field Es is thus random, but is constant over time. Only a very small, but not negligible part of the stray field is emitted in the direction of the detection location.
- An optimal design of the detection optics allows only the light mode (hereinafter referred to as measurement mode) to reach the detector that generates the diffractive biogrid of the biomolecules to be detected and blocks all other modes through suitable apertures and apertures. The stray light is thus suppressed in these other modes and does not reach the detector.
- the scattered light that is emitted into the measuring mode cannot, however, be suppressed due to the principle. With a field strength Es and phase cps, it contributes to the detected total intensity IM + S.
- Scattered light in the measurement mode which is generated by non-stationary, ie fluctuating, scattering centers, can be suppressed by averaging the detected intensity over time, since the expected value of the interference term is zero.
- Unwanted stray light that is generated in a process that differs in some parameter (e.g. location, wavelength, polarization, etc.) from the generation of the measurement light and is therefore not emitted into the measurement mode can be suppressed using this parameter.
- the proportion of the scattered light that is emitted into the measuring mode arises at the same locations on the substrate as the measuring light and is generated in the same direction and with the same polarization.
- the measuring light and the scattered light component in the measuring mode are thus inseparably mixed in an optical mode and can no longer be separated.
- stray field So far only speckle has been considered, which occurs when illuminating scattering surfaces with coherent light.
- Another concrete example (and a special case) of a stray field is the optical bias of a biogrid.
- bias a constant zero signal appears as a background, which is referred to as bias, even without connecting biomolecules to be detected.
- This bias can either overlap constructively or destructively with the measuring field and falsify it. It is therefore advantageous to fill the bias by filling it out (so-called "backfilling") To minimize or even eliminate grid gaps with a suitable material.
- phase difference ACP MS between measuring field E M and stray field It cannot be distinguished whether the electrical field Es has its origin in speckle or bias.
- the proposed method for measuring the phase difference ACP MS between the measuring field E M and the stray field Es and the subsequent coherent subtraction of the stray field thus also represent a possibility for measuring or eliminating the bias.
- the associated increase in measuring accuracy of the biosensor is particularly advantageous since the measuring signal is no longer falsified by the bias or by speckle.
- Another advantage is the fact that the bias does not have to be completely eliminated by backfilling, which allows greater tolerances in the production of such biogrids.
- the present invention provides to measure the unknown phase difference ACP MS between measuring field E M and stray field Es in order to then be able to completely subtract the stray field by coherent difference formation.
- the flow chart is as follows, the individual steps are explained in more detail below:
- the accessible intensity distributions are measured in step (i).
- the unknown phase of a light wave can be determined by interference with a known reference wave. Therefore, in addition to the already defined field strengths of the measuring field EM and stray field Es, the field strength of the reference field ER is defined here.
- IM EM 2
- the total intensity of different combinations (ie coherent overlays) of the measuring field, stray field and reference field is called IM + S + R, IM + S USW. designated.
- the intensity distribution is understood to mean in particular the spatial intensity distribution on a 2-dimensional detector (for example a camera).
- the evaluation of these intensity distributions can take place both per camera pixel and in areas, ie in order to save computing power, certain areas of the camera image can be summarized at the expense of accuracy and then the various evaluations can be carried out for these areas.
- the reference field IR can be switched on and off by simply stopping down.
- the whole Are emitted from the region of the measuring field and leakage field Biogitter be dimmed to only receive IR. Basically, the following five combinations of measuring field, stray field and reference field are experimentally accessible as measured variables:
- the unknown phase of a light wave can be determined by interference with a known reference wave.
- Either carrier wave methods in which the reference phase is impressed as carrier frequency are suitable for this (see D. Malacara, Interferogram analysis for optical testing, Chapter 8 "Spatial Linear and Circular Carrier analysis"), or phase-shifting methods in which the reference phase is in at least three Steps are varied (cf. D. Malacara, Interferogram analysis for optical testing, Chapter 7 "Phase shifting interferometry").
- both methods are the same: The unknown phase of the output wave to be analyzed is determined. Both methods are briefly explained below.
- the reference phase CP R is modulated by impressing a carrier frequency fo, for example by obliquely irradiating the reference wave.
- the reference phase CP R is given depending on the geometry by:
- the resulting intensity distribution on a detector is then characterized by the appearance of a stripe pattern, so-called “fringes”.
- Output shaft is the same at all points.
- the maxima of the stripe pattern shift for any output wave due to the phase distribution of the output wave: the stripe pattern shifts transversely to the stripe direction.
- the sought phase information about the output wave to be analyzed is encoded, which can be extracted, for example, by Fitten, Hilbert or Fourier transformation.
- Corresponding algorithms are described in multiple versions in the literature. The algorithms according to Takeda, J. Opt. Soc. At the. 72 (1) (1982) (short: Fourier transformation of the intensity distribution, deletion of unnecessary frequency components, shifting the carrier frequency peak to the origin and reverse transformation) or S.
- the reference phase While the reference phase, defined by the geometry dependence, varies spatially by itself in the carrier wave method, the reference phase must be actively varied in the phase-shifting method, i.e. slide.
- phase delay of the reference wave can be achieved by many different methods.
- Known in the literature is e.g. the introduction of a plane-parallel (delay) glass / plastic plate in the reference beam path, the introduction of an electro-optical phase delay element, e.g. a liquid crystal element, the displacement of a mirror in the reference beam path by means of a linear actuator, or the displacement of a diffractive grating perpendicular to the beam in the reference beam path.
- the relative phase of the reference wave is set to several (at least 3) fixed values, and the resulting intensity distributions of the coherent superimpositions of the reference wave and the output wave are recorded.
- a large number of algorithms three, four and five, etc. step methods, continuous methods) are known for the subsequent calculation of the unknown phase (cf. D. Malacara, Interferogram analysis for optical testing, Chapter 6 “Phase Detection Algorithms ").
- the three-step algorithm with 120 ° phase difference between the steps is described here as an example:
- phase-shifting method is the acquisition of at least three images per intensity distribution, which means a certain additional effort.
- the advantage of this method is that, in contrast to the carrier wave method, the reference wave does not necessarily have to be irradiated at an angle in order to separate it in k-space.
- step (iii) Using the accessible intensity distributions described in step (i) and the methods described in step (ii), measure the phase of the measuring field and stray field relative to an irradiated reference field can, in step (iii), one can infer the sought undisturbed measurement intensity IM.
- a first evaluation method (iii a) images of the intensity distributions of IS + R and IM + S + R are taken and the phase difference of the output wave (of the stray field Es or the addition of stray field and measuring field ES + EM) to the reference wave is calculated accordingly one of the above procedures. Then you take a picture of the intensity distribution IR and calculate the amounts of the electric fields Es and ES + EM with knowledge of the respective phase difference from the above formulas for IS + R and I M + S + R. Since the electrical fields Es or the addition of stray field and measuring field ES + EM according to amount and phase are now known, these can be subtracted from each other in vector form and obtain the EM sought.
- phase difference (CPR - cps) is calculated therefrom by using one of the methods for phase calculation described in step (ii).
- phase-shifting methods several equations are obtained with the cos term containing the phase difference, from which the phase can be reconstructed.
- the phase information sought is obtained from the deviations of the stripe pattern from the carrier frequency.
- the scattering intensity Is can then be calculated as follows:
- phase difference (CP R - CP M ) is calculated from this.
- phase-shifting processes see above, several equations with cos term are obtained from which the phase can be reconstructed, and only one with the carrier wave process.
- the measurement intensity IM is then calculated as follows:
- ls the intensity of the reference wave IR SO should be set to correspond approximately to that of the stray light background ls.
- phase stability and intensity of the reference wave are incorporated into the following embodiments of the invention, which primarily involve the generation of a reference wave. There are also other advantages and details of the present invention from the following description of various embodiments with reference to the figures.
- Figures 1 - 4 a first embodiment according to the
- Carrier wave method with a flat reference wave and a focusing biogrid
- FIGS. 5-9 show a second embodiment according to a phase-shifting method, with a spherical
- FIGS. 10-13 show a third embodiment according to a phase-shifting method, with an external reference wave and a focusing biogrid
- FIGS. 14-16 show a fourth embodiment with a Bragg
- Figure 17 shows a fifth embodiment with an external
- Reference wave and a focusing biogrid which is imaged on the detector with optics.
- Figures 1 to 4 show a first embodiment in the two side views XZ (Fig. 1) and YZ (Fig. 4) and in plan views for the components biochip and aperture plate (Fig. 2) and shutter (Fig. 3).
- Light L of a coherent laser light source is coupled via a coupling grating EKG into a planar waveguide W of a biochip BC arranged on a substrate SUB.
- Biochip BC here denotes the substrate SUB with the elements arranged on the front and back of the substrate SUB. Together with the other elements such as light source and detector as well as the movable screens and other elements, this results in a biosensor.
- the wavelength of the coherent laser light source is preferably in the range from 400 nm to 1000 nm.
- the coupling-in grating EKG is located on the underside of the planar waveguide W.
- the light L coupled into the planar waveguide W propagates in the X direction (outside the waveguide W, this falls Light mode exponentially) and strikes a first reference grating RG.
- This first reference grating RG is designed as a linear grating on the underside of the planar waveguide W and is preferably produced by the same process steps that also generate the coupling-in grating EKG.
- the decoupled first reference light bundle RL is collimated by the linear grating shape of the reference grating RG. It arrives at a detector D with several individual detectors, which is preferably designed as a CMOS or CCD image sensor. Only a small part of the light L propagating in the planar waveguide W is coupled out through the first reference grating RG. The majority continue to propagate to a first BG biogrid.
- the first biogrid BG consists of the first capture molecules that are lattice-shaped, i.e. how the bars of a grid are connected to the surface of the BC biochip. These first capture molecules specifically bind first analyte molecules, which are thus also lattice-like and whose mass occupancy is to be measured.
- the grating shape of the biogrid BG is selected - as described in WO 2015004264 A1 cited above - in such a way that the decoupled first measuring light bundle ML is focused on the detector D on a small focal area.
- the grating shape thus represents a diffractive lens with the focal length f.
- the first reference grating RG and the first biogrid BG are selected such that the first reference light bundle RL and the first measuring light bundle ML are superimposed at the location of the detector D.
- This coherent superimposition results in a first intensity stripe system, which is detected by the detector D and evaluated in an evaluation unit (not shown).
- the superposition of the two light beams RL, ML at the location of the detector D can be achieved, for example, by choosing the coupling-out angle of the first reference grating RG, which is given by the grating orientation and by the grating constant of the first reference grating RG.
- the first biogrid BG also only couples out a very small proportion of the light L propagating in the planar waveguide W. The majority continue to propagate to a second reference grid RG and a subsequent second biogrid BG.
- the second reference grating RG is identical to the first reference grating RG, likewise in the form of a linear grating, and decouples a second reference light bundle RL, which arrives at the detector D offset from the first reference light bundle.
- the second biogrid BG consists of second capture molecules, which in turn are attached in a lattice shape. The lattice shape is identical to the lattice shape of the first biogrid BG and thus also represents a diffractive lens.
- the second catcher molecules differ from the first catcher molecules of the first biogrid BG and thus bind other specific analyte molecules whose mass assignment is also to be measured.
- the second reference and the second measurement light bundle RL, ML are again superimposed at the location of the detector D, where they strike the first reference and measurement light bundle so that they can be detected independently.
- a second intensity stripe system is created, that is detected by the detector D and evaluated in the evaluation unit, not shown.
- biochip BC in addition to the first and second reference and biogrid RG, BG, further reference and biogrid RG, BG are arranged in order to be able to detect further analyte molecules. With a single biochip BC from this exemplary embodiment, four different analyte molecules can therefore be examined.
- a diaphragm plate BP is introduced in the beam path between the biochip BC and the detector D. It has openings OR, OM for the multiple reference and measurement light bundles RL, ML and blocks stray light which arises outside of these light bundles RL, ML.
- the openings OR, OM are therefore chosen to be as small as possible in order to achieve high suppression of stray light, but sufficiently large so that the reference and measurement light bundles RL, ML are not appreciably impaired.
- the aperture plate BP can be designed as a thin metal plate with openings OR, OM.
- an absorbent layer can also be applied to a glass plate and provided with openings OR, OM accordingly. This second alternative has the advantage that this glass plate can at the same time be a cover plate of an optical module, which protects the detector D and other optical components from contamination which can arise when the biochip is inserted or removed.
- the evaluation unit evaluates the intensity stripe systems on the focus areas of the measuring light bundles ML.
- Intermediate individual detectors or pixels of the image sensor are not used for the evaluation, since they only detect scattered light that arises outside the measurement mode and is not relevant for the evaluation.
- This selection of pixels only in the area of the focus areas corresponds to a virtual diaphragm structure at the location of the detector D.
- OM of the diaphragm plate BP a diaphragm system is created which only allows light to pass through, with respect to location and Direction of the measuring mode corresponds. All other modes that differ from the measuring mode in terms of the location and / or direction of light are blocked. This results in the desired mode filter.
- the determination of the scattered measurement intensity I M of the biogrid BG requires a series of measurements in which either only the reference light bundle RL or the measurement light bundle ML or both are detected together. It is therefore necessary to insert a shutter S in the beam path from the biochip BC to the detector D.
- This shutter S has openings or transparent areas SO through which the reference and / or measuring light bundles RL, ML are transmitted.
- beam-blocking regions SB can be pushed into the beam path of the reference light bundle RL or into the beam path of the measurement light bundle RB, so that a measurement of the intensity values IM + S + R, IM + S, and IR is enabled.
- the intensity values IS + R and ls are measured before the analyte molecules are attached.
- a partition T thus separates the area of the beam coupling from the area of the beam detection.
- a beam trap F also absorbs the light transmitted through the coupling-in grating EKG. Both reduce the stray light.
- Figures 5 to 9 show a second embodiment of the invention in the two side views XZ (Fig. 5) and YZ (Fig. 9) as well as in plan views for the components biochip (Fig. 6), aperture plate (Fig. 7) and the combined Shutter / delay plate carrier (Fig. 8).
- the reference grids RG are now (in the z direction) below the associated biogrid BG and each couple a small proportion of the light in the planar waveguide W as a reference RL light bundle in the form of spherical waves.
- the reference grids RG are designed as chirped gratings with curved grating lines and act as diffractive diverging lenses.
- the Biogitter BG are linear grids with a constant grating period and couple collimated measuring light bundles ML out of the waveguide W.
- the coupling takes place at an angle a + 90 °.
- the reference grids RG are delimited in a circle and are each surrounded by circular rings with biogrids BG.
- the emerging reference light bundles RL are thus each enclosed by associated measuring light bundles ML.
- the decoupled measuring and reference light bundles ML, RL pass through a fixed diaphragm plate BP (FIG. 7) and subsequently meet a combined diaphragm and phase delay plate BPV that can be displaced in the x and y directions (FIG. 8).
- aperture elements B1, B2 are provided on the combined aperture and phase delay plate BPV, which can block either the reference or the measurement light bundles RL, ML.
- phase delay elements V1, V2, V3 which can be inserted into the beam path of the reference light bundle RL and which delay the phases of the reference light bundle RL by 60 °, 180 ° or 300 °.
- All aperture and phase delay elements B1, B2, V1, V2, V3 are arranged in the grid of the measurement and reference light bundles RL, ML, so that the optical effect is always the same for all reference light bundles RL and also for all measurement light bundles ML.
- the aperture structures B1, B2 and a corresponding x and y shift of the combined aperture and delay plate make it possible to determine the intensity values IR and IM + S + R, IM + S (after adding the analyte) or IS + R and ls (before adding the analyte).
- phase delay elements V1, V2, V3 When inserting the phase delay elements V1, V2, V3 into the reference Light bundles RL can also delay the phase of the reference light bundles and thus determine the phases of the corresponding interference terms in accordance with the phase shifting method.
- the phase delay elements V1, V2, V3 consist of a transparent, optically denser material such as the surrounding medium air, for example glass or a transparent polymer of suitable thickness, in order to obtain the desired phase delay.
- the reference and the measurement light bundles RL, ML hit a lens array plate.
- Collective lenses SL are arranged on this in the grid of the measuring light bundles RL, ML. They each focus the measuring light bundles ML on the detector D located underneath.
- the distance between converging lenses SL or lens array plate and detector D is therefore chosen to be equal to the focal length of the converging lenses SL.
- the reference light bundles RL are also concentrated on the detector D by the converging lenses SL of the lens array plate. However, the detector D is not in relation to the reference light bundle RL
- Biogitter BG as a linear lattice structure is much easier than for a diffractive lens structure.
- a diffractive lens structure shows a continuous variation of the local one
- the measurement light bundles ML have disturbing fluctuations in intensity across their cross sections, which lead to a larger focus area on the detector D and thus to greater measurement noise. These disadvantages do not occur when producing biogrids BG with linear lattice structures.
- the lithography can be optimized for this one grating constant, for example by choosing an optimal exposure divergence, an optimal exposure distance or the choice of an optimal exposure wavelength. If there are unavoidable fluctuations in the exposure intensity, there are corresponding fluctuations in the web-gap ratio of the biogrid BG, but these are constant over their transverse extent. The diffraction efficiency or the intensity of the measurement light bundle ML thus also remains constant over the transverse extent, so that the measurement light bundle ML can be focused on the detector D with limited diffraction. This in turn results in low measurement noise.
- the fixed lattice constant of BG biogrids with a linear lattice structure enables the optimization of the bridge-gap ratio for this lattice constant. This leads to increased decoupling efficiency and thus increased intensity of the measuring light bundle ML.
- the decoupling in this embodiment should be slightly oblique to the normal of the biochip BC in order to suppress multiple reflections and back reflections on optical elements and in the planar waveguide W.
- this angle can again be aligned perpendicular to the detector D if necessary.
- the zone without grating lines described in the prior art EP 2618130 A1
- EP 2618130 A1 which are provided within the focusing grating to avoid back reflection in the waveguide
- biogrid BG as a linear grating structure
- the constant polarization of the collimated measuring light bundle ML in contrast to a polarization which varies over the transverse extent in the case of a diffractive grating structure.
- biogrids BG with linear grating structures requires - as described above - subsequent focusing of the measurement light bundles and is therefore associated with the use of at least one converging lens SL or a lens array plate (for several biogrids BG on a biochip BC).
- the positional tolerances of the converging lenses SL with respect to one another are sufficiently small only in the case of a lens array plate.
- the adjustment of individual lenses that have to be arranged in a very narrow grid is far too complex.
- the converging lenses SL of the lens array plate can be designed both refractive and diffractive.
- the diffractive variant can be produced as a single-stage, binary structure or advantageously in several stages as a blazed structure. Since the position between detector D and converging lenses SL or lens array plate is fixed, the position of the focal areas on detector D does not change when the biochip BC is displaced relative to the scanning optics, which simplifies the evaluation. Such shifts in all three spatial directions can occur when the biochip is inserted into one Readout unit or occur due to thermal drift processes. Only rotation of the biochip BC around the x or y axis would shift the focus areas. The Biochip BC must therefore be aligned with stops close to the edges of the Biochip BC. A rotation around the z-axis is only of minor influence due to the slight deviation of the coupling angle a from 90 ° and can also be easily controlled by stops.
- the reference light bundles RL in the form of spherical waves used in this embodiment are advantageous since the transverse extent of the reference light bundles RL on the detector D can be selected by the focal length of the reference grids RG in the form of diffractive diverging lenses. It can thus be designed in such a way that a uniform intensity of the reference light bundle RL is produced on the detector D over the focus areas of the measuring light bundle ML.
- the phase-shifting method used here only requires light beams with low beam inclinations, ie with a low numerical aperture.
- the measuring accuracy is increased accordingly.
- the measuring and the associated reference beams ML, RL are coupled out at very adjacent locations from the biochip BC.
- the temperature influence on the phase shift between the measurement and reference light bundles ML, RL, which arises from changes in the refractive index in the planar waveguide W, is accordingly correspondingly low.
- the computational outlay in the evaluation unit is less than in the carrier wave method, since no stripe patterns have to be evaluated for the phase determination, but only simple arithmetic calculations and arctangent formation are necessary.
- Figures 10 to 13 show a third embodiment in the side views XZ (Fig. 10) and in plan views for the components biochip waveguide (Fig. 1 1), top of the diaphragm plate with reference grating waveguide (Fig. 12) and bottom of the diaphragm plate (Fig. 13). Only the differences from the first embodiment are described below.
- the biogrids BG are again designed as diffractive lenses and focus the measuring light bundles ML again on the detector D.
- the reference light bundles R pass through an aperture plate BP.
- the aperture plate BP has a substrate SUB ', a coupling grating EKG and a separate planar waveguide W'.
- Part of the light L of the coherent laser light source (not shown) is phase-shifted via an electro-optical phase delay element PVE in the form of a liquid crystal element or an electro-optical modulator and coupled into the planar waveguide W 'of the aperture plate BP via the coupling-in grating EKG.
- the light propagates there in the + x direction to reference grids RG, which couple out reference light bundles RL.
- the reference grids RG are designed as linear grids, so that the reference light beams are collimated.
- the grating constant of the reference grating is selected such that the reference light bundles RL couple out slightly obliquely (a F 90) to the normal direction of the biochip BC in order to avoid back reflections in the waveguide W '. This takes place in analogy to the Bragg zones described in EP 2618130 A1, in which grating lines are left out in the respective biogrids in order to avoid reflections in the planar waveguide according to the Bragg condition.
- the reference grids RG are positioned relative to the biogrids BG in such a way that the reference light bundles RL each overlap with the associated measurement light bundles ML at the location of the detector D and interfere therewith.
- phase shift of the reference light bundle RL by the electro-optical phase delay element PVE allows the relative phase to be shifted between the measuring and reference light bundles ML, MR and thus the determination of the relative phase in an evaluation unit, not shown, according to the phase shifting method described above.
- a shutter S movable in the x direction allows the measurement or reference light bundles ML, RL to be blocked before the light L hits the respective coupling grating EKG.
- the biochip BC also carries a referencing grid, also referred to as a phase drift reference grid PDBG.
- a referencing grid also referred to as a phase drift reference grid PDBG.
- a small part of the light L propagating in the planar waveguide W of the biochip is coupled out through this first referencing grating PDBG and generates a first referencing light bundle RZL.
- the referencing grating PDBG is designed as a linear grating, so that the first referencing light bundle RZL1 emerges collimated. It is subsequently detected by detector D.
- the aperture plate BP carries a further reference grating RG, which is arranged under the first referencing grating PDBG on the biochip BC and is likewise designed as a linear grating.
- a small part of the light L propagating in the planar waveguide W of the diaphragm plate BP is coupled out, so that a collimated, second referencing light bundle RZL2 is produced.
- the first and the second referencing light bundles RZL1, RZL2 overlap and interfere at the location of the detector D.
- the second referencing light bundle RZL2 can be phase shifted by the electro-optical phase delay element PVE and also allows the determination of the relative phase of the first and second referencing light bundles RZL1, RZL2.
- This relative phase depends on the relative position of the biochip BC and the aperture plate BP.
- the relative position also influences the relative phases between the measuring and the associated reference light bundles ML, RL.
- the part of the relative phases of measurement and associated reference light bundles ML, RL can be determined and subtracted which depends on the relative position of the biochip BC and the aperture plate BP.
- a drift of the relative position of the biochip BC relative to the aperture plate BP during the measurement period can thus be compensated for.
- there is a sensitive direction for the relative position which determines the relative phases of measurement and associated reference light bundles ML, RL.
- the condition for an identical coupling direction for the measuring light bundles ML, the reference light bundles and the first and second referencing light bundles RZL1, RZL2 results. This is possible through a suitable grating constant and grating orientation of the reference grating RG and referencing grating PDBG.
- the particular advantage of this embodiment is that the reference grating RG only has to be structured in a waveguide W permanently installed in the detection apparatus and does not have to be present in every biochip waveguide W. Calibration is also simplified. Also, no moving parts are required for the phase shift of the reference light bundle RL.
- the shutter S is arranged in front of the coupling grating EKG, so that less stray light is produced when one of the two light beams is blocked.
- FIGS. 14 to 16 show a fourth embodiment in the side view XZ (FIG. 14) and in plan views for the components biochip (FIG. 15), as well as the aperture plate and the combined shutter / delay plate carrier (FIG. 16).
- This embodiment is based on an arrangement described in EP2929326B1.
- the biogrids BG now only deflect the light L in the planar waveguide W, but do not couple it out of the planar waveguide W.
- the BG biogrids are designed as linear grids.
- the coupling out of the waveguide W takes place by means of additional coupling-out gratings AG.
- the light L first passes through electro-optical phase delay elements PVE (for the portion of the light that is later directed onto the reference grating RG) and a shutter S in order to be able to separate the light portions on the bio and reference grids BG, RG separately ,
- the light is coupled in analogously to the first embodiment via an EKG coupling grid.
- the light L propagating in the x direction then strikes a first linear, two-part biogrid BG, in the middle of which a first reference grid RG is executed.
- the grating lines of both grids BG, RG are equidistant and oblique to the direction of propagation of the light L, and meet the Bragg condition for deflecting the light in the waveguide in the direction of the coupling-out grating AG.
- the distance between the grating lines d is thus linked via the angle Q of the light L to the grating lines with the wavelength of the light in the waveguide l.
- the diffraction order n is usually 1 in order not to generate any additional, disturbing diffraction orders and to increase the diffraction efficiency.
- the small portion of the total light L deflected by the biogrid BG and the reference grating RG strikes a focusing decoupling grating AG on the underside of the waveguide W, which directs both portions onto the detector D and superimposes them there.
- the reference grating RG can be made slightly curved in order to ensure a small divergence of this reference light bundle RL.
- a continuous variation of the coupling angle on the coupling grid EKG in the y direction via a suitable actuator is also useful in order to meet the Bragg condition of the biogrid BG and thus optimize the measurement intensity.
- the light fraction remaining in the waveguide W is redirected and coupled out at another location in the next bio and reference grating BG, RG.
- a particle spacer M with a suitable pore size is provided. This measure can also be useful in connection with all other embodiments.
- the aim is to keep unwanted scattering particles SP (eg cells) away from the waveguide W by filtration.
- the particle spacer M is provided outside the evanescent field of the waveguide W, and the pore size is selected so that it can be passed by the biomolecules or analytes A to be analyzed, while undesired larger particles SP are retained in the liquid supernatant.
- the particle spacer M can be in the form of a membrane, or as a molecular layer or more porous Cover layer in the vicinity of the waveguide W or brought to the waveguide W.
- the particle spacer M prevents larger particles such as tumor cells (typical diameter 10-30 pm) from changing the scattered light background when they reach the evanescent field on or near the waveguide W.
- the advantage of this embodiment with particle spacer M is that it is ensured that the scattered light background can be measured with and without a measuring wave under identical conditions, without this being changed by larger particles or cells introduced with the analyte medium.
- Figure 17 shows the fifth embodiment in the XZ side view. Above all, the differences from the first embodiment are explained.
- Light L of a coherent laser light source LQ is split into two parts with the aid of a first beam splitter ST1, which are used below to generate the measuring light bundles ML (first part) and the external reference light bundle RL (second part) separately.
- a first portion of the light split at the beam splitter ST1 is coupled via a coupling grating EKG into a planar waveguide W of a biochip BC arranged on a substrate SUB.
- the biogrids BG are again designed as diffractive lenses with focal length f and focus the measuring light bundles ML on a focal plane BE which is at a distance f from the waveguide W.
- the shutter S1 which can be moved in the x direction, allows the measuring light bundles ML to be blocked even before light hits the coupling grid EKG.
- the focal plane BE is imaged with the aid of two objectives 01, 02 with focal lengths fo bj .i and fo bj, 2 on a detector D, which is located at a distance 2fob j, i + 2fobj , 2 from the focal plane BE.
- a detector D which is located at a distance 2fob j, i + 2fobj , 2 from the focal plane BE.
- this results in a 4f image with magnification M -1.
- the optical imaging of the focal plane BE used in this embodiment on the detector D is particularly advantageous, since at a distance 2fobj , i from the focal plane BE or at a distance 2fo bj, 2 V from the detector D there is a Fourier plane into which a Fourier diaphragm FB is introduced with a suitable opening OF, so that k-space filtering (ie angular filtering) is realized.
- the numerical aperture of the detection optics can be adapted in such a way that undesired scattered light, which is emitted in modes other than the measurement mode, is blocked. This thus results in a desired mode filter.
- the Fourier diaphragm FB is designed to be displaceable in the X and Y directions, so that tilting of the measuring light bundle ML coupled out of the biochip BC can be compensated for about the R x and Ry axes.
- a second portion of the light L split at the beam splitter ST1 is collimated with the aid of suitable second beam shaping optics SF02 and used as an external reference light bundle RL.
- a second shutter S2 movable in the z direction allows the reference light bundle RL to be blocked.
- the reference light bundle RL is directed onto the detector D with the aid of a second beam splitter ST2 (or another deflection element which serves to deflect and combine the beams) and overlapped with the measuring light bundle ML, so that both interfere at the location of the detector D.
- the angle R y of the second beam splitter ST2 must be selected such that the incident reference light RL is irradiated at an angle greater than the numerical aperture of the measuring light and scattered light component in the measuring mode.
- the second beam splitter ST2 can be designed to be adjustable by R y , so that when the angle of the measuring light bundle ML and scattered light drifts, the angle of the To be able to track the reference light beam RL accordingly. If no mechanical tracking is provided, the period of the intensity stripe system, which is caused by the interference of measurement light bundle ML and reference light bundle RL, should be estimated and the measured phases should be corrected for the associated slope error.
- the phase measurement can also be carried out using the phase-shifting method.
- the angle R y of the second beam splitter ST2 is again freely selectable and does not necessarily have to be adjustable.
- a phase delay element In order to delay the phase of the reference light bundle RL by 60 °, 180 ° or 300 °, a phase delay element must then be introduced into the beam path of the reference light bundle RL at a suitable point.
- the second portion of the light split at the first beam splitter ST1 can also be used to illuminate a small opening which, in addition to the first opening OF, is offset in the Xier direction from the optical axis in the Fourier diaphragm FB located.
- This small illuminated opening acts like a point light source in the Fourier plane from the second objective 02, so that a plane reference light beam directed towards the detector D is created, which is overlapped with the measuring light beam ML and thus brought into interference at the location of the detector D.
- the distance between this small second opening and the optical axis determines the angle R y by which the reference light bundle RL is irradiated against the measuring light bundle ML and the scattered light is irradiated onto the detector D, and can in turn be selected to be adjustable at one Drifting the angle of measuring light bundle ML and scattered light to be able to track the angle of the reference light bundle RL accordingly.
- the light path from the first beam splitter ST1 to the Fourier diaphragm FB can also be bridged by guiding the light in an optical fiber.
- the reference light bundle RL is not coupled out by a plurality of reference gratings RG, but by a first beam splitter ST1.
- Only one reference light bundle RL is generated and used to measure all measuring light bundles ML.
- This embodiment is particularly advantageous since no space for reference grids RG has to be provided on the surface of the biochip BC, so that the biogrid BG - in contrast to the first and fourth embodiments - is arranged more densely or - in contrast to the second and third embodiments - is used over the entire surface can be. It is also advantageous that the complex structuring of reference gratings RG is dispensed with, and only beam splitters ST1, ST2 are required which are permanently installed in the detection apparatus.
- a disadvantage of this embodiment is first of all that the optical paths of the reference light bundle RL and measurement light bundle ML do not match.
- a so-called “common path” geometry is usually chosen, in which the optical paths of the reference light bundle RL and measurement light bundle ML largely correspond, i.e. the same optical elements pass (as in the first, second and fourth embodiment, with restrictions also in the third embodiment).
- mechanical or thermal drift processes have the same effect on both light beams RL, ML, so that the relative phase remains constant.
- the solution described in this fifth embodiment represents a so-called “double-path” geometry, which is inherently susceptible to such drift processes.
- the phase distribution cps of the resulting speckle background is constant in time and space, and can be used as an intrinsic phase standard to drift to measure and compensate for the relative phase between the reference light bundle RL and the measuring light bundle ML.
- a common phase offset of the speckle background which can occur, for example, due to a drift of the biochip relative to the light source and / or relative to the detector, is here assigned to a phase shift of the reference wave, which would have the same effect.
- the intensity distributions IS + R, IS and IR are recorded at a first point in time ti - the relative phase position between the stray field and the irradiated reference field is cps-cpR.
- the intensity distribution IS + R ' is measured again - the relative phase position between the stray field and the irradiated reference field is now (PS- (PR'.
- PS- PR'.
- the pixel drift ⁇ cp R between the reference light bundle RL and scattered light can then be estimated using a wavefront model that contains the corresponding degrees of freedom for different drift processes (phase shift, phase tilt etc.), so that an overall phase drift ⁇ cp R is obtained and compensated accordingly can - usually by subtraction.
- the time t 2 at which the phase drift between the reference light bundle RL and the measurement light bundle ML is to be determined is after the time of adding the analyte.
- the intensity distribution IS + R ' is no longer accessible.
- M + S + R I S + R '.
- the unchanged speckle background outside the focus area can thus continue to be used as an intrinsic phase standard and the corresponding evaluation of the phase drift ⁇ cp R is carried out analogously according to the above formula.
- a lateral shift of the biochip between the measurements can also be determined by correlating the speckle background.
- the intensity distribution of the speckle background is used for this correlation.
- the lateral shift can easily be corrected by a software shift of the pixel assignments.
- the advantage of this method is that the speckle background can be used as an intrinsic phase standard over the entire detector area, and therefore no additional reference grids are required.
- coupling-in gratings EKG and / or reference gratings RG can be designed not only on the underside of the waveguide W but also on the top of the waveguide W.
- the excitation can also be carried out by a light beam totally reflected at the interface of the biochip BC.
- the biogrid BG and reference grid RG are located at this interface.
- the evanescent electrical field of the totally reflected light then interacts completely analogously to the variant with excitation via the waveguide with the respective gratings.
- a flat single detector per detection location and an aperture with the diameter of the measuring field, ie the diameter of the measuring mode, can be used as detector D.
- a diffraction grating that can be displaced perpendicular to the beam is also known as a phase shifter, since in its diffraction orders the phase varies depending on the position of the webs and gaps relative to the beam.
- a mirror moved, for example, by a piezo actuator can vary the beam path of the reference beam in order to vary the phase.
- the reference wave is polarized, for example, by means of a suitably oriented 1 ⁇ 2 plate inserted into the reference beam perpendicular to the Measuring wave, you can use a rotatable polarization filter in front of the detector to select which beam components you want to observe. Only the respective wave is then measured parallel to the polarization of the reference or measuring wave; with a setting between 0 ° and 90 ° relative to the polarization of the reference wave, the relative strength of the two partial waves can be set so that optimal interference contrast (see above) ), and bring them to interference on the detector.
- separators can be inserted between the individual detection locations in all variants to prevent crosstalk.
- the detector resolution and the focus diameter of the biogrid BG should be chosen so that the focus diameter is in the range of 5-50 pixels.
- the detector resolution and the stripe spacing in the carrier wave method should be selected so that the stripe spacing is in the range of 5-50 pixels.
- the reference grids RG can be applied offset in the x direction both before (as shown in the first exemplary embodiment) and after the biogrid BG in the carrier wave method.
- An arrangement with an offset in the y direction or combinations thereof are also possible.
- the advantage of an offset only in the y direction to the biogrid BG is that the optical path length in the waveguide W for the reference and biogrid RG, BG is identical, which minimizes drift in the phase between the reference and the biogrid.
- the phase drift can also be compensated for by calculating the phase difference with one of the two reference grids Biogrid is calculated. The phase drift of the two reference gratings behaves in opposite directions and can thus be corrected.
- a reference grid RG (e.g. by superimposing two grid structures that are rotated relative to one another or generating a strongly divergent wave) can also generate reference waves for several surrounding biogrid BG.
- the aperture structure below selects the appropriate partial wave for the respective biogrid BG from the generated reference waves.
- movable shutter can also electronically switchable elements such as LCDs to block or release
- Beams of light can be used.
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Abstract
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DE102018212013 | 2018-07-18 | ||
PCT/EP2019/059853 WO2020015872A1 (de) | 2018-07-18 | 2019-04-16 | Diffraktiver biosensor |
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US (1) | US20210270736A1 (de) |
EP (1) | EP3824272A1 (de) |
JP (1) | JP7278363B2 (de) |
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US11946930B2 (en) | 2018-03-01 | 2024-04-02 | Hoffmann-La Roche Inc. | Device for use in the detection of binding affinities |
DE102020212031A1 (de) * | 2020-09-24 | 2022-03-24 | Dr. Johannes Heidenhain Gmbh | Vorrichtung und Verfahren zur Bestimmung der Intensität des in einem planaren Wellenleiter geführten Lichts IWG(x, y) |
DE102020212029A1 (de) * | 2020-09-24 | 2022-03-24 | Dr. Johannes Heidenhain Gmbh | Vorrichtung und Verfahren zur simultanen Abbildung zweier Objektebenen |
WO2023187070A1 (en) * | 2022-03-30 | 2023-10-05 | Miltenyi Biotec B.V. & Co. KG | Space filtering in optical biomolecule interaction analysis |
EP4306940A1 (de) | 2022-07-15 | 2024-01-17 | lino Biotech AG | Vorrichtung zur verwendung bei der detektion von bindungsaffinitäten |
ES2958785A1 (es) * | 2023-07-17 | 2024-02-14 | Univ Valencia Politecnica | Dispositivo de medida para el analisis de la respuesta optica de materiales biosensibles difractivos |
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US4537861A (en) * | 1983-02-03 | 1985-08-27 | Elings Virgil B | Apparatus and method for homogeneous immunoassay |
DE69208413T2 (de) * | 1991-08-22 | 1996-11-14 | Kla Instr Corp | Gerät zur automatischen Prüfung von Photomaske |
EP1266207B1 (de) | 2000-03-22 | 2010-10-27 | Axela Inc. | Verfahren und vorrichtung zur bestimmung mehrerer analyten |
US7023544B2 (en) * | 2000-10-30 | 2006-04-04 | Sru Biosystems, Inc. | Method and instrument for detecting biomolecular interactions |
DE10162180A1 (de) * | 2001-12-18 | 2003-07-03 | Bosch Gmbh Robert | Interferometrische Messvorrichtung |
US7123363B2 (en) * | 2003-01-03 | 2006-10-17 | Rose-Hulman Institute Of Technology | Speckle pattern analysis method and system |
EP2618130A1 (de) | 2012-01-17 | 2013-07-24 | F. Hoffmann-La Roche AG | Vorrichtung zur Verwendung bei der Bindeaffinitätserkennung |
EP2741074A1 (de) | 2012-12-04 | 2014-06-11 | F. Hoffmann-La Roche AG | Vorrichtung zur Verwendung bei der Bindeaffinitätserkennung |
EP2757374A1 (de) | 2013-01-17 | 2014-07-23 | F. Hoffmann-La Roche AG | Verfahren zur Herstellung einer äußeren Oberfläche eines planaren Wellenleiters zur Bindung von Zielproben entlang einer Vielzahl festgelegter Linien sowie planarer Wellenleiter |
EP2824446A1 (de) | 2013-07-12 | 2015-01-14 | F. Hoffmann-La Roche AG | Vorrichtung zur Verwendung bei der Bindeaffinitätserkennung |
EP3862747A1 (de) * | 2015-07-07 | 2021-08-11 | Furuno Electric Co., Ltd. | Messchip, messvorrichtung und messverfahren |
WO2018020331A1 (en) * | 2016-07-29 | 2018-02-01 | Semiconductor Energy Laboratory Co., Ltd. | Display device, input/output device, and semiconductor device |
US10274807B2 (en) * | 2016-12-08 | 2019-04-30 | Northwestern University | Optical quantification of interfacial charge states |
CN107219590B (zh) * | 2017-06-05 | 2018-12-25 | 峻立科技股份有限公司 | 具有监控分光路径的光学元件 |
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US20210270736A1 (en) | 2021-09-02 |
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