DIFFRACTIVE BIOSENSOR
20210270736 · 2021-09-02
Inventors
Cpc classification
G02B6/4215
PHYSICS
G02B26/04
PHYSICS
International classification
Abstract
A diffractive biosensor for the selective detection of biomolecules includes a substrate and an optical biograting situated on the substrate, the biograting having periodically arranged receptors for the biomolecules and the efficiency of a diffraction of incident light, and thus the intensity of a measuring light beam arriving in a detector, is a function of a mass coverage of the biograting with the biomolecules to be detected. The biosensor has a device for generating a reference light beam directed to the detector, by which the phase position of stray light arriving in the detector is able to be determined.
Claims
1-10. (canceled)
11. A diffractive biosensor for selective detection of biomolecules, comprising: a substrate; an optical biograting arranged on the substrate and including periodically arranged receptors for the biomolecules, an efficiency of a diffraction of incident light and an intensity of a measuring light beam arriving in a detector being a function of a mass coverage of the biograting with the biomolecules to be detected; and a reference light beam generation device adapted to generate a reference light beam directed to the detector by which a phase position of stray light arriving in the detector relative to a measuring light beam is determined.
12. The diffractive biosensor according to claim 11, wherein the reference light beam generation device includes a reference grating and/or a beam splitter adapted to deflect a portion of incident light as the reference light beam to the detector.
13. The diffractive biosensor according to claim 11, further comprising a movable element having transparent regions and light blocking regions adapted to selectively block the reference light beam or the measuring light beam.
14. The diffractive biosensor according to claim 11, further comprising an electronically switchable diaphragm adapted to selectively block the reference light beam or the measuring light beam.
15. The diffractive biosensor according to claim 11, wherein the biograting is provided on a waveguide arranged on the substrate in a planar manner.
16. The diffractive biosensor according to claim 15, wherein a reference grating of the reference light beam generation device is provided in the waveguide and arranged to a side of the biograting.
17. The diffractive biosensor according to claim 11, wherein the reference light beam generation device includes a first beam splitter adapted to deflect a portion of incident light as the reference light beam to a second beam splitter adapted to superpose the reference light beam with the measuring light beam and deflect the superposed light beams to the detector.
18. The diffractive biosensor according to claim 11, wherein the reference light beam generation device includes a first beam splitter adapted to deflect a portion of incident light as the reference light beam to a deflection element adapted to superpose the reference light beam with the measuring light beam and deflect the superposed light beams to the detector.
19. The diffractive biosensor according to claim 17, wherein a first objective and a second objective are arranged in a beam path of the measuring light beam downstream from the biograting and upstream from the second beam splitter, and a Fourier diaphragm is arranged in a Fourier plane between the first objective and the second objective.
20. The diffractive biosensor according to claim 18, wherein a first objective and a second objective are arranged in a beam path of the measuring light beam downstream from the biograting and upstream from the deflection element, and a Fourier diaphragm is arranged in a Fourier plane between the first objective and the second objective.
21. The diffractive biosensor according to claim 11, wherein the diffractive biosensor includes a plurality of reference light beam generation devices and a plurality of biogratings, the diffractive biosensor further comprising a lens array including a plurality of collimation lenses arranged in a raster of the biogratings, the lens array adapted to direct light of the reference light beam generation devices and of the biogratings onto the detector.
22. A method for determining a phase drift between a reference light beam and a measuring light beam in a device as recited in claim 11, wherein a speckle background, which is stable in space and time, is used as an intrinsic phase standard.
23. The method according to claim 22, wherein a phase offset between the speckle background and the reference light beam and/or the measuring light beam is ascertained at different points in time, and a compensation value for the phase drift is ascertained from the phase offset, to establish interferometric stability between the reference light beam and the measuring light beam.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
First Example Embodiment
[0062]
[0063] Via a coupling grating EKG, light L from a coherent laser light source is coupled into a planar waveguide W, situated on a substrate SUB, of a biochip BC. In this instance, biochip BC denotes substrate SUB with the elements situated on the front and rear side of substrate SUB. Together with the further elements such as a light source and detector as well as the movable diaphragms and additional elements, these results in a biosensor.
[0064] The wavelength of the coherent laser light source preferably lies in the range of 400 nm to 1000 nm. Coupling grating EKG is situated on the underside of planar waveguide W. Light L coupled into planar waveguide W propagates in the X-direction (this light mode drops exponentially outside waveguide W) and impinges upon a first reference grating RG. This first reference grating RG is arranged as a linear grating on the underside of planar waveguide W and is preferably produced by the same process steps that also produce coupling grating EKG.
[0065] Because of the linear grating form of reference grating RG, decoupled first reference light beam RK is collimated. It impinges upon a detector D having a plurality of individual detectors, which is preferably arranged as a CMOS or a CCD image sensor. Only a small portion of light L propagating in planar waveguide W is decoupled by first reference grating RG. The predominant portion propagates further to a first biograting BG. First biograting BG includes first capture molecules, which are adsorbed in the form of a grating, i.e. like the webs of a grating on the surface of biochip BC. These first capture molecules specifically adsorb first analyte molecules, which are therefore likewise adsorbed in the form of a grating and whose mass coverage is to be measured.
[0066] Because of the grating-type adsorption of the first analyte molecules at the capture molecules, a small portion of light L propagating in planar waveguide W is decoupled as a first measuring-light beam ML and also reaches detector D. The grating form of biograting BG, as described in the above-cited PCT Patent Document No. WO 2015/004264, is selected so that the decoupled first measuring-light beam ML is focused on a small focus area on detector D. The grating form thus represents a diffractive lens having the focal length f. First reference grating RG and first biograting BG are selected such that first reference light beam RL and first measuring light beam ML superposition at the location of detector D. This coherent superposition results in a first intensity stripe system, which is detected by detector D and evaluated in an evaluation unit. The superposition of both light beams RL, ML at the location of detector D is able to be achieved by the choice of the decoupling angle of first reference grating RG, for instance, which is given by the grating orientation and by the grating constant of first reference grating RG.
[0067] First biograting BG also decouples only a very small portion of light L propagating in planar waveguide W. The predominant part propagates further to a second reference grating RG and a following second biograting BG. Second reference grating RG, identical to the first reference grating RG, is likewise arranged as a linear grating and decouples a second reference light beam RL, which reaches detector D at an offset from the first reference light beam. Second biograting BG includes second capture molecules, which once again are adsorbed in grating form. The grating form is identical to the grating form of first biograting BG and thus also represents a diffractive lens. The second capture molecules differ from the first capture molecules of first biograting BG and thus adsorb different specific analyte molecules whose mass coverage is to be measured as well. Second reference light beam and second measuring light beam RL, ML once again superposition at the location of detector D, impinging at an offset from the first reference and measuring light beam so that they are able to be detected independently. A second intensity stripe system is produced, which is detected by detector D and evaluated in the evaluation unit.
[0068] As illustrated in the top view of biochip BC, still further reference and biogratings RG, BG are situated next to first and second reference gratings and biogratings RG, BG in order to allow for the detection of further analyte molecules. As a result, it is possible to examine four different analyte molecules with the aid of a single biochip BC from this exemplary embodiment.
[0069] A diaphragm plate BP is introduced into the beam path between biochip BC and detector D. It has openings OR, OM for the plurality of reference and measuring light beams RL, ML and blocks stray light that is produced outside these light beams RL, ML. Openings OR, OM are therefore selected to be as small as possible in order to achieve a high stray light suppression but are sufficiently large so that reference and measuring light beams RL, ML will not be adversely affected to any significant extent. Diaphragm plate BP may be arranged as a thin metal plate having openings OR, OM. As an alternative, it is also possible to apply an absorbent layer on a glass plate and to appropriately provide it with openings OR, OM. This second alternative offers the advantage that this glass plate may simultaneously be a cover plate of an optics module which protects detector D and further optical components from contamination that may be produced when the biochip is introduced or removed.
[0070] The evaluation unit evaluates the intensity stripe systems on the focus areas of the measuring light beams ML. Interposed individual detectors or pixels of the image sensor are not used for the evaluation because they detect only stray light that is produced outside the measuring mode and is not relevant in the context of the evaluation. This selection of pixels only in the region of the focus areas corresponds to a virtual diaphragm structure at the location of detector D. Together with diaphragm openings OR, OM of diaphragm plate BP, a diaphragm system is created which allows only light to pass that corresponds to the measuring mode with regard to the location and direction. All other modes, which after all differ from the measuring mode in the light location and/or in the light direction, are blocked. This therefore results in the desired mode filter.
[0071] As described above, a series of measurements is required in order to determine scattered measuring intensity I.sub.M of biogratings BG, in which either only reference light beams RL or measuring light beams ML or both together are detected. It is therefore necessary to insert a shutter S into the beam path from biochip BC to detector D. This shutter S has openings or transparent regions SO through which the reference and/or measuring light beams RL, ML are transmitted. By shifting shutter S in the x-direction, beam-blocking regions SB are able to be slipped into the beam path of reference light beams RL or into the beam path of measuring light beams RB so that a measurement of intensity values I.sub.M+S+R, I.sub.M+S, and I.sub.R is possible. The measurements of intensity values I.sub.S+R and I.sub.S are carried out prior to the adsorption of the analyte molecules.
[0072]
Second Example Embodiment
[0073]
[0074] Only the differences from the first example embodiment will be discussed below. Reference gratings RG are situated (in the z-direction) underneath associated biogratings BG where they decouple a small portion of the light in planar waveguide W as a reference light beam RL in the form of spherical waves. To this end, reference gratings RG are arranged as chirped gratings including curved grating lines and act as diffractive dispersion lenses. Biogratings BG, one the other hand, are arranged as linear gratings with a constant grating period and decouple collimated measuring light beams ML from waveguide W. In order to avoid back-reflections of the linear gratings into planar waveguide W by a second order diffraction according to the Bragg condition, the decoupling is carried out under an angle α≠90°. As illustrated in the top view in
[0075] The emerging reference light beams RL are thus enclosed by associated measuring light beams ML.
[0076] Decoupled measuring and reference light beams ML, RL pass through a stationary diaphragm plate BP (
[0077] Due to diaphragm structures B1, B2 and a corresponding x- and y-shift in the combined diaphragm and delay plate, it is possible to acquire intensity values I.sub.R and I.sub.M+S+R, I.sub.M+S (after the addition of the analyte) or I.sub.S+R and I.sub.S (prior to the addition of the analyte). When inserting the phase-delay elements V1, V2, V3 into reference light beams RL, the phase of the reference light beams is able to be delayed as well and the phases of the corresponding interference terms can thus be determined according to the phase-shift method. Phase-delay elements V1, V2, V3 are made of a transparent, optically denser material than the surrounding medium air, e.g., of glass or a transparent polymer of suitable thickness, in order to obtain the desired phase delay.
[0078] In the further beam course, reference and measuring light beams RL, ML impinge upon a lens array plate. Situated thereon in the raster of measuring light beams ML are collimating lenses SL. They focus measuring light beams ML on detector D situated underneath. The distance between collimating lenses SL or the lens array plate and detector D is therefore selected to be equal to the focal length of collimating lenses SL. Reference light beams RL, too, are concentrated on detector D by collimating lenses SL of the lens array plate. However, detector D is not situated in the focal plane in relation to reference light beams RL because reference light beams RL are irradiated in the form of spherical waves.
[0079] The use of linear grating structures of biogratings BG and thus of collimated measuring light beams ML in combination with a lens array plate having collimating lenses SL that focus measuring light beams ML on a detector has considerable advantages, which will be described below.
[0080] The production of biogratings BG as a linear grating structure is much simpler than that of a diffractive lens structure. A diffractive lens structure features a continual variation of the local grating constants. In the contactless lithography, which is required for the production of biogratings BG, Talbot effects of the mask gratings have an adverse effect on the light imaging from the mask toward substrate SUB of biochip BC to be produced, to the effect that disturbing interferences of different diffraction orders of the mask grating arise and lead to undesired light modulations. In the production of diffractive lens structures, not all local grating constants are imaged to the same satisfactory degree and additional modulations occur. The corresponding disturbances of biogratings BG cause the light of planar waveguide W to be decoupled from planar waveguide W at a lower diffraction efficiency and additional light beams are produced, which interfere with the detection in the form of stray light. Moreover, measuring light beams ML have disturbing intensity fluctuations across their cross-sections, which lead to a larger focus area on detector D and thus to greater measuring noise. These disadvantages do not occur in the production of biogratings BG having linear grating structures. In addition, the lithography for this one grating constant is able to be optimized, for instance by the selection of an optimal exposure divergence, an optimal exposure distance or the selection of an optimal exposure wavelength. With unavoidable fluctuations of the exposure intensity, corresponding fluctuations of the web-gap ratio of biogratings BG do actually occur, but these are constant across their transverse extension. The diffraction efficiency or the intensity of measuring light beams ML thus also remains constant across the transverse extension, so that measuring light beam ML is able to be focused on detector D in a diffraction-restricted manner. This in turn results in less measuring noise.
[0081] The fixed grating constant of biogratings BG having a linear grating structure allows the web-gap ratio for this grating constant to be optimized. This leads to a greater decoupling efficiency and thus to a greater intensity of measuring light beams ML.
[0082] The decoupling in this example embodiment should be performed at a slight incline to the normal of biochip BC in order to suppress multiple reflections and back-reflections at optical elements and in planar waveguide W. If required, this angle may be realigned again perpendicular to detector D using a suitable lens form of collimation lenses SL of the lens-array plate.
[0083] Moreover, in biogratings BG having a linear grating structure, the zone without grating lines described, for example, in European Patent Document No. 2 618 130, is also omitted, the grating lines within the focusing gratings being provided in order to avoid a back-reflection into the waveguide. This reduces the production outlay and increases the intensities of measuring light beams ML due to the larger surfaces of biograting BG.
[0084] Another advantage of a biograting BG as a linear grating structure is the constant polarization of collimated measuring light beam ML in contrast to a polarization that varies across the transverse extension in a diffractive grating structure. Also, because of the omission of the curvature, the polarization of the propagating waves is always arranged in parallel with the grating lines, which increases the decoupling efficiency.
[0085] As previously described, the use of biogratings having linear grating structures requires subsequent focusing of the measuring light beams and therefore entails the use of at least one collimation lens SL or a lens-array plate (for multiple biogratings BG on a biochip BC). The position tolerances of collimation lenses SL relative to each other are sufficiently small only in a lens-array plate. The adjustment of individual lenses which have to be arranged in a very tight raster is much too complex.
[0086] The collimation lenses SL of the lens-array plate may have a refractive as well as a diffractive configuration. The diffractive variant may be produced as a one-stage, binary structure or advantageously in multi-stage form as a blazed structure.
[0087] Since the position between detector D and collimation lenses SL or the lens-array plate is fixed, the position of the focus areas on detector D does not change when biochip BC is displaced relative to the scanning optics, which simplifies the evaluation. Such displacements in all three directions in space may occur when the biochip is inserted into an evaluation unit or as a result of thermal drift processes. Only a rotation of biochip BC about the x- or y-axis would displace the focus areas. Biochip BC must therefore be aligned with the aid of stops close to the edges of biochip BC. Because of the small deviation of decoupling angle α from 90°, a rotation about the z-axis is of only minor importance and can also easily be controlled by stops.
[0088] Reference light beams RL in the form of spherical waves used in this example embodiment are advantageous because the transverse extension of reference light beams RL on detector D is able to be selected by the focal length of reference gratings RG in the form of diffractive dispersion lenses. It may therefore be configured so that a uniform intensity of reference light beams RL across the focus areas of measuring light beams ML is produced on detector D.
[0089] In comparison with the carrier wave method, the phase-shift method used in this instance requires only light beams having low beam inclinations, i.e. a low numerical aperture. Due to the low beam inclinations, the unavoidable, and also multiple, reflections at optical components such as at diaphragm plate BP do not lead to crosstalk from one measuring light beam ML to an adjacent measuring light beam ML. The measuring accuracy is increased accordingly. In addition, measuring beam bundles and associated reference beam bundles ML, RL are decoupled from biochip BC at closely adjacent points. As a result, the temperature influence on the phase shift between measuring and reference light beams ML, RL, which is produced by changes in the refraction index in planar waveguide W, is correspondingly low. Moreover, the computational effort in the evaluation unit is less than in the carrier wave method because no stripe patterns have to be evaluated for the phase determination, but only arithmetic calculations and an arc tangent formation are required.
Third Example Embodiment
[0090]
[0091] In this example embodiment, biogratings BG are once again arranged as diffractive lenses and focus measuring light beams ML on detector D again. Reference light beams R pass through a diaphragm plate BP. To this end, diaphragm plate BP has a substrate SUB′, a coupling grating EGK, and a separate planar waveguide W. A portion of light L from the coherent laser light source is phase-shifted via an electro-optical phase delay element PVE in the form of a liquid crystal element or an electro-optic modulator and coupled into planar waveguide W of diaphragm plate BP via coupling grating EKG. In waveguide W′, the light propagates in the +x-direction to reference gratings RG, which decouple reference light beams RL. Reference gratings RG are arranged as linear gratings so that the reference light beams are collimated. The grating constant of the reference gratings is selected such that reference light beams RL decouple at a slight angle (α≠90) to the normal direction of biochip BC in order to avoid back-reflections into waveguide W′. This is done analogously to the Bragg zones described in European Patent Document No. 2 618 130, in which grating lines are left out in the respective biogratings in order to avoid reflections into the planar waveguide according to the Bragg condition. Reference gratings RG are positioned relative to biogratings BG such that reference light beams RL come to overlap with the associated measuring light beams ML at the location of detector D and thus interfere. The phase shift of reference light beams RL by the electro-optic phase-delay element PVE allows for a shift of the relative phase between measuring and reference light beams ML, RL, and thus for the determination of the relative phase in an evaluation unit according to the phase-shift method discussed above.
[0092] A shutter S, which is movable in the x-direction, makes it possible to block measuring or reference light beams ML, RL even before light L impinges upon respective coupling gratings EKG.
[0093] In addition to biogratings BG, biochip BC furthermore carries a referencing grating, which is also referred to as a phase-drift reference grating PDBG. A small portion of light L propagating in planar waveguide W of the biochip is decoupled by this first referencing grating PDBG and generates a first referencing light beam RZL. Referencing grating PDBG is arranged as a linear grating so that first referencing light beam RZL1 emerges in collimated form. It is then detected by detector D. Diaphragm plate BP carries a further reference grating RG, which is situated underneath first referencing grating PDBG on biochip BC and also arranged as a linear grating. Here, too, a small portion of light L propagating in planar waveguide W of diaphragm plate BP is decoupled so that a collimated, second referencing light beam RZL2 is produced. First and second referencing light beams RZL1, RZL2 overlap and interfere at the location of detector D.
[0094] With the aid of electro-optic phase-delay element PVE, second referencing light beam RZL2 is able to be shifted in phase and in this case as well, allows for the determination of the relative phase of the first and second referencing light beams RZL1, RZL2. This relative phase depends on the relative position of biochip BC and diaphragm plate BP. However, the relative position also influences the relative phases between measuring light beams and associated reference light beams ML, RL. By determining the relative phase between first and second referencing light beams RZL1, RZL2, the portion of the relative phases of measuring light beams and associated reference light beams ML, RL that is a function of the relative position of biochip BC and diaphragm plate BP is able to be determined and subtracted. Drift of the relative position of biochip BC relative to diaphragm plate BP during the measuring period is thereby able to be compensated. In this context it should be noted that there is a sensitive direction for the relative position that determine the relative phases of measuring and associated reference light beams ML, RL. In the same manner there is a sensitive direction for the relative phase of first and second referencing light beam RZL1, RZL2. Both sensitive directions are given by the direction of light beam L upstream from coupling grating EKG and by the direction of the decoupled light beams. The two sensitive directions should be identical, if possible. At the same coupling angle for biochip BC and diaphragm plate BP, the condition for an identical decoupling direction for measuring light beams ML, the reference light beams and the first and second referencing light beams RZL1, RZL2 comes about. This can be achieved by a suitable grating constant and grating orientation of reference gratings RG and referencing gratings PDBG.
[0095] If needed, additional referencing gratings PDBG are able to be provided on biochip BC and associated reference gratings RG on diaphragm plate BP. Apart from the compensation of linear displacements between biochip BC and diaphragm plate BP, the corresponding further measurements of the relative phases also allow for the compensation of rotations. This results in a particularly accurate variant.
[0096] An advantage of this example embodiment is that reference grating RG simply has to be structured in a waveguide W fixedly installed in the detection apparatus, instead of providing waveguide W in every biochip. In addition, the calibration is simplified as well. Also, no movable parts are required for the phase shift of reference light beams RL.
[0097] Because of the spatial separation of light beams L that impinge upon coupling gratings EKC of the biochip or the diaphragm plate BP, a separate adjustment of their intensity is possible with the aid of corresponding components in the beam path. In this manner, the intensity ratio of measuring and reference light beams ML, RL is able to be adjusted and optimized for an optimal detection. In this case, shutter S is situated upstream from coupling gratings EKG so that less stray light is created when one of the two light beams is blocked.
Fourth Example Embodiment
[0098]
[0099] Only the differences from the first example embodiment will be described below.
[0100] This example embodiment is based on a system which is described in European Patent Document No. 2 929 326. Biogratings BG only deflect light L in planar waveguide W but do not decouple it from planar waveguide W. Biogratings BG are arranged as linear gratings. The decoupling from waveguide W is obtained with the aid of additional decoupling gratings AG.
[0101] Similar to the afore-described example embodiments, light L first passes through electro-optic phase-delay elements PVE (for the portion of the light that will later be directed onto reference grating RG) and a shutter S so that the light components on biograting and reference grating BG, RG are able to be dimmed independently of each other. The coupling of the light is carried out similar to the first example embodiment via a coupling grating EKG.
[0102] Light L propagating in the x-direction then impinges upon a first linear, bipartite biograting BG in whose center a first reference grating RG is provided. The grating lines of both gratings BG, RG are arranged in an equidistant fashion, at an incline to the propagation direction of light L, and satisfy the Bragg condition for the deflection of the light in the waveguide in the direction of decoupling grating AG. Via angle θ of light L to the grating lines, the spacing of grating lines d is therefore linked with the wavelength of the light in waveguide A. The diffraction order n is usually 1 in order not to generate any additional, disturbing diffraction orders and to increase the diffraction efficiency.
[0103] The small portion of total light L deflected by biograting BG and reference grating RG impinges upon a focusing decoupling grating AG on the underside of waveguide W, which directs both components to detector D and brings them to superposition there. In order to ensure a reliable superposition of both components, reference grating RG may be provided with a slight curvature in order to ensure a small divergence of this reference light beam RL. A continual variation of the coupling angle at coupling grating EGK in the y-direction via a suitable actuator is also provided in order to satisfy the Bragg condition of biograting BG and to optimize the measuring intensity in this manner.
[0104] In the next biograting and reference grating BG, RG the light component remaining in waveguide W is deflected at another location and decoupled.
[0105] In addition, a particle spacer M having a suitable pore size is provided. This measure may also be provided in connection with all other example embodiments. The goal is to keep undesired scattering particles SP (e.g., cells) away from waveguide W through filtration. For this purpose, particle spacer M is provided outside the evanescent field of waveguide W and the pore size is selected so that the biomolecules or analytes A to be analyzed are allowed to pass through, whereas undesired larger particles SP are kept behind in the fluid supernatant. Particle spacer M is able to be brought close to waveguide W or onto waveguide W in the form of a diaphragm or also as a molecule layer or porous cover layer. Particle spacer M prevents larger particles such as tumor cells (typical diameter 10-30 pm) from changing the stray light background when they reach the evanescent field or come close to waveguide W.
[0106] One advantage of this example embodiment including particle spacer M is that it is thereby ensured that the stray light background is able to be measured with and without measuring wave under identical conditions without being changed by larger particles or cells introduced with the analyte medium.
Fifth Example Embodiment
[0107]
[0108] With the aid of a first beam splitter ST1, light L from a coherent laser light source LQ is split up into two components, which are subsequently used to generate measuring light beams ML (first component) and external reference light beam RL (second component) separately from one another.
[0109] After passing through a suitable first beam-forming optics SF01 and a first shutter S1, a first component of the light split up at beam splitter ST1 is coupled via a coupling grating EKG into a planar waveguide W of a biochip BC situated on a substrate SUB. In this embodiment, biogratings BG are once again arranged as diffractive lenses having focal length f and focus measuring light beams ML on a focal plane BE, which is located at a distance f from waveguide W. Shutter S1, which is movable in the x-direction, makes it possible to block measuring light beams ML even before light impinges upon coupling grating EKG.
[0110] Focal plane BE is imaged with the aid of two objectives O1, O2 having focal lengths f.sub.Obj,1 and f.sub.Obj,2 onto a detector D, which is situated at a distance 2f.sub.Obj,1+2f.sub.Obj,2 from focal plane BE. 4f-imaging with an enlargement M=−1 thus results in the case of two objectives O1, O2 having identical focal widths f.sub.Obj,1=f.sub.Obj,2=f.sub.Obj. The optical imaging of focal plane BE onto detector D used in this example embodiment is particularly advantageous because a Fourier plane results at distance 2f.sub.obj,1 from focal plane BE or at a distance 2f.sub.Obj,2 from detector D, into which a Fourier diaphragm FB having a suitable opening OF is introduced so that k-space filtering (i.e. angle filtering) is obtained. In this manner, the numerical aperture of the detection optics is able to be adapted such that undesired stray light, which is irradiated into modes other than the measuring mode, is blocked. This therefore results in a desired mode filter. Fourier diaphragm FB is arranged to be displaceable in the X- and Y-directions so that tilting of measuring light beam ML, decoupled from biochip BC, about the R.sub.x- and R.sub.y-axis is able to be compensated.
[0111] A second component of light L split up at beam splitter ST1 is collimated with the aid of a suitable second beam-forming optics SFO2 and used as an external reference light beam RL. A second shutter S2, which is movable in the z-direction, makes it possible to block reference light beam RL. Next, reference light beam RL is directed to detector D with the aid of a second beam splitter ST2 (or some other deflection element used for the deflection and beam convergence) and brought to overlap with measuring light beam ML, so that both interfere at the location of detector D. In order to be able to perform a phase measurement according to the carrier wave method, angle R.sub.y of second beam splitter ST2 is to be selected such that irradiated reference light RL is irradiated under an angle greater than the numerical aperture of the measuring light and stray light component in the measuring mode. In addition, second beam splitter ST2 may be arranged to be adjustable by R.sub.y in order to appropriately correct the angle of reference light beam RL in the event of drift of the angle of measuring light beam ML and stray light. If no mechanical adjustment is provided, then the period of the intensity stripe system that is created by the interference of measuring light beam ML and reference light beam RL should be estimated and the measured phases be corrected by the associated gradient error.
[0112] In a deviation from the illustrated example embodiment, the phase measurement can also be carried out according to the phase-shift method. In this case, angle R.sub.y of second beam splitter ST2 is once again freely selectable and need not necessarily be adjustable. In order to delay the phase of reference light beam RL by 60°, 180° or 300°, a phase-delay element must then be introduced into the beam path of reference light beam RL at a suitable location.
[0113] In a deviation from the illustrated example embodiment, the second portion of the light split up at first beam splitter ST1 may also be used to illuminate a small opening which, in addition to first opening OF, is located in Fourier diaphragm FB in the x-direction at an offset from the optical axis. This small illuminated opening acts like a point light source in the Fourier plane of second objective 02 so that a planar reference light beam is produced, which is directed to detector D and brought to overlap with measuring light beam ML, i.e. is brought to interference therewith at the location of detector D. The distance between this small second opening and the optical axis determines angle R.sub.y at which reference light beam RL is irradiated onto detector D in a tilted manner with respect to measuring light beam ML and the stray light; it may once again be selected to be adjustable in order to allow for a corresponding adjustment of the angle of reference light beam RL in the event of drift of the angle of measuring light beam ML and stray light. The light path from first beam splitter ST1 to Fourier diaphragm FB may also be bridged by conducting the light in an optical fiber.
[0114] In contrast to the example embodiments described up to this point, reference light beam RL is decoupled not by a multitude of reference gratings RG but by a first beam splitter ST1. Only one reference light beam RL is generated and used for measuring all measuring light beams ML. This example embodiment is particularly advantageous because no space has to be reserved on the surface of biochip BC for reference gratings RG so that biogratings BG—in contrast to the first and fourth example embodiments—may be arranged more tightly or—in contrast to the second and third example embodiments—may be arranged across the full surface. It is also advantageous that the complex structuring of reference gratings RG is omitted and only beam splitters ST1, ST2 are required, which are fixedly installed in the detection apparatus.
[0115] A disadvantage of this example embodiment first of all is that the optical paths from reference light beam RL and measuring light beam ML do not agree. In order to achieve interferometric stability, a so-called “common path” geometry is usually selected, in which the optical paths of reference light beam RL and measuring light beam ML largely match, i.e. pass through the same optical elements (as in the first, second and fourth example embodiments, and, with restrictions, also in the third example embodiment). This ensures that mechanical or thermal drift processes affect both light beams RL, ML to the same extent, so that the relative phase remains constant. However, the solution described in this fifth example embodiment represents what is referred to as a “double path” geometry due to the different optical paths for light beams RL, ML, which are susceptible to such drift processes.
[0116] However, this disadvantage can be remedied in a particularly simple manner. Since unavoidable stray field E.sub.S is mainly produced by scattering at the stationary roughness of substrate SUB, phase distribution φ.sub.S of the resulting speckle background is constant over time and space and may be used as an intrinsic phase standard in order to measure and compensate drift of the relative phase between reference light beam RL and measuring light beam ML. A common phase offset of the speckle background, which may occur through drift of the biochip relative to the light source and/or relative to the detector, is allocated to a phase shift of the reference wave in this instance, which would have the same effect.
[0117] For this purpose, intensity distributions I.sub.S+R, I.sub.S and I.sub.R are recorded at a first instant the relative phase position between the stray field and the irradiated reference field being assumed to be φ.sub.S−φ.sub.R. At a later instant t.sub.2, intensity distribution I.sub.S+R′ is measured once again, the relative phase position between the stray field and the irradiated reference field now being assumed to be φ.sub.S−φ.sub.R′. Under the above condition that the phase of the stray light is constant (φ.sub.S=const.), it is then possible to calculate the difference of the reference phase between the instants t.sub.1 and t.sub.2 at every location of two-dimensional detector D (i.e. per pixel), based on the following relationship:
[0118] Phase drift Δφ.sub.R, measured per pixel, between reference light beam RL and stray light can then be estimated with the aid of a wave-front model, which includes corresponding degrees of freedom for different drift processes (phase lift, phase tilt, etc.), so that a total phase drift Δφ.sub.R is obtained and able to be compensated, usually by means of subtraction.
[0119] Because stray light and measuring bundle ML are produced at the same location on waveguide W and are imaged onto detector D along the same optical path, their relative phase positions Δφ.sub.MS=φ.sub.M−φ.sub.S are constant over time. Thus, if the phase of reference light beam RL drifts by Δφ.sub.R relative to phase φ.sub.S of the stray light, it also drifts by Δφ.sub.R relative to phase (pm of measuring light beam ML. Thus, the phase drift between reference light beam RL and measuring light beam ML is determined, so that interferometric stability is able to be established between the two beam bundles.
[0120] As a rule, instant t.sub.2 for which the phase drift between reference light beam RL and measuring light beam ML is to be determined lies after the instant of the analyte addition. In this case, intensity distribution I.sub.S+R′ is no longer accessible. However, since the signal intensity changes only within a small focus area of measuring light beam ML when the analyte is added, I.sub.M+S+R≈I.sub.S+R′ applies outside the focus area. As a result, the unchanged speckle background outside the focus area may continue to be used as the intrinsic phase standard, and the corresponding evaluation of phase drift Δφ.sub.R takes place analogously according to the above formula.
[0121] Lateral shifting of the biochip between the measurements is also able to be determined by a correlation of the speckle background. In a measurement without reference light beams, the intensity distribution of the speckle background is used for this correlation. With a reference light beam, it is more advantageous to utilize the location-dependent phase distribution for the correlation. The lateral shifting is able to be corrected by shifting the pixel allocations with the aid of software.
[0122] One advantage of this method is that the speckle background is able to be used as an intrinsic phase standard across the entire detector area, which means that no additional reference gratings are required.
[0123] In connection with the exemplary embodiments described herein, the generalizations addressed below may be possible either in isolation or in combination. It should be understood that the example embodiments described herein should not be considered as restricting the generality and are able to be modified accordingly without affecting the basic functional principles of the different example embodiments. [0124] For example, coupling gratings EKG and/or reference gratings may be obtained not only on the underside of waveguide W but in the same manner also on the topside of waveguide W. [0125] Instead of the excitation of biograting BG and/or reference grating RG via light L propagating in waveguide W, the excitation may also be obtained by a light beam totally reflected at the boundary surface of biochip BC. Biogratings BG and reference gratings RG are situated at this boundary surface. The evanescent electrical field of the totally reflected light then interacts with the respective gratings in entirely the same manner as in the variant featuring an excitation via the waveguide. [0126] Instead of a camera (i.e. a 2D array of photodetectors), it is also possible to use as a detector D a planar single detector per detection location and a diaphragm having the diameter of the measuring field, i.e. the diameter of the measuring mode. [0127] From the literature, a diffraction grating displaceable perpendicular to the beam is also referred to as a phase shifter because the phase varies in its diffraction orders as a function of the position of the webs and gaps relative to the beam. As an alternative, a mirror, moved via a piezo actuator, for example, may also vary the beam path of the reference beam in order to vary the phase. [0128] If the reference wave is polarized perpendicular to the measuring wave, for instance by a suitably oriented λ/2 lamella introduced into the reference beam, it is possible to select, via a rotatable polarization filter upstream from the detector, which beam components one would like to monitor. Parallel to the polarization of the reference and/or measuring wave, only the particular wave is measured, at a setting between 0° and 90° relative to the polarization of the reference wave. The relative strength of the two partial waves can then be adjusted so that an optimal interference contrast (see above) is obtained and an interference on the detector is thereby able to be brought about. [0129] In addition, separating webs are able to be introduced between the individual detection locations in all variants in order to prevent crosstalk. [0130] The detector resolution and the focus diameter of biograting BG should be selected so that the focus diameter lies in the range of 5 to 50 pixels. [0131] The detector resolution and the stripe spacing in the carrier wave method should be selected so that the stripe spacing lies within the range of 5 to 50 pixels. [0132] Reference gratings RG may be applied in the carrier wave method both upstream (as illustrated in the first exemplary embodiment) and downstream from biograting BG at an offset in the x-direction. A placement with an offset in the y-direction or combinations thereof is possible as well. An advantage of an offset from biograting BG only in the y-direction is that the optical wavelength in waveguide W has an identical length for reference gratings and biogratings RG, BG, which minimizes drift in the phase between the reference grating and biograting. If a reference grating is applied upstream and downstream from the biograting in the x-direction in each case, a compensation of the phase drift is also able to be performed computationally in that the phase difference with respect to the biograting is calculated once in each case using one of the two reference gratings. Phase drift of the two reference gratings acts in reverse and can be corrected in this manner. [0133] In addition, a reference grating RG (e.g., by superposition of two grating structures rotated relative to each other, or the generation of a strongly divergent wave) is also able to generate reference waves for a plurality of surrounding biogratings BG. The diaphragm structure underneath selects from the generated reference waves the matching partial wave for the respective biograting BG. [0134] Instead of the movable shutters (S), it is also possible to use electronically switchable elements such as LCDs for the blocking or the release of light beams.