Devices and methods for determining spatiotemporal refractive index changes

Abstract

A measurement assembly for determining refractive index changes in a sample comprises a light source (2), a tunable wavelength selector (3), an image sensor (4), a processing unit (6), and a Fabry-Perot unit (5) comprising a first plane surface (51) and a second plane surface (52), the Fabry-Perot unit (5) having a sample reception space (53) arranged between the first plane surface (51) and the second plane surface (52) to receive a sample. The Fabry-Perot unit (5) is arranged to act as a wavelength-dependent light intensity filter. The image sensor (4) is configured to record a two-dimensional image of the sample reception space for different spectral subranges and the processing unit (6) is configured to reconstruct the intensity filter function of the Fabry-Perot unit (5) based on the recorded two-dimensional images, and to determine a refractive index map based on the reconstructed intensity filter function.

Description

[0001] TITLE

[0002] DEVICES AND METHODS FOR DETERMINING SPATIOTEMPORAL REFRACTIVE INDEX CHANGES

[0003] TECHNICAL FIELD

[0004] The present invention relates to a measurement assembly for determining spatiotemporal refractive index changes in a sample, in particular in a fluid sample, as well as to an optical microscope setup comprising said measurement assembly. The present invention further relates to a method for determining spatiotemporal refractive index changes using said measurement assembly.

[0005] PRIOR ART

[0006] Chemical gradients on the microscale occur in a variety of situations in nature and in many industrial processes. Electrochemical potentials for ions that move across a membrane are for instance known in biology. For example, in the human gastrointestinal tract within the small gut, the establishment of an electrochemical gradient of sodium enables the transport of nutrients. Such electrochemical gradients also play an important role in many industrial processes, e.g. in batteries, where the electrochemical gradient determines the voltage a battery can produce.

[0007] A different example from material science is the active control of microswimmers that is done by having particles use their surroundings as fuel, leading to a chemical gradient in the trail of the particles. Another example from chemistry is the process of interfacial polymerization, which is often used to create fibers, capsules or ultrathin films. To achieve this, monomers react at an interface between two solvents, leading to a spatial change in concentrations and thus a chemical gradient.

[0008] In terms of reliably experimentally setting up chemical gradients, advancements of microfluidic technology have allowed many of these gradients to easily be set up and controlled. However, whether generated for specific applications or occurring in natural processes, they remain difficult to visualize due to a lack of optical contrast.

[0009] Thus, many of these processes can currently only be measured indirectly. Being able to directly visualize these processes would provide valuable insights about the evolution of microscale chemical gradients. Such measurements and the understanding gained therefrom may in turn be crucial to adapt and optimize existing processes.

[0010] Fabry-Perot interferometry has been developed since the end of the 19th century. In its simplest form, a Fabry-Perot interferometer is an optical cavity formed by two partially reflective surfaces. Electromagnetic waves that are being reflected back and forth between the two partially reflective surfaces interfere either constructively or destructively, leading to a wavelength-dependent intensity filter function, often also termed Fabry-Perot transmission function. Since the wavelength position of the maxima of the intensity filter function depends on the refractive index experienced by the electromagnetic radiation between the two partially reflective surfaces, changes in refractive index occurring in a medium that is placed between the two partially reflective surfaces can be observed by measuring a wavelength shift of these transmission maxima.

[0011] A technique for measuring concentration profiles in a sample based on tracking the transmission maxima of a Fabry-Perot interferometer is disclosed by D.R. Vogus et al., "Measuring concentration fields in microfluidic channels in situ with a Fabry-Perot interferometer", Lab Chip, 2025, 15, 1689, DOI: 10.1039 / c5lc00095e. In the setup disclosed in this publication, an inverted microscope is used to focus white light onto a Fabry-Perot chip that comprises the sample. Light transmitted by the Fabry-Perot chip is focused onto a spectrometer slit with a microscope objective. A CCD camera is used to gather images from the spectrometer. In the images obtained using this setup, the horizontal axis in each image corresponds to the physical dimension in which light is obtained in the slit, while the vertical axis corresponds to the wavelength of diffracted light. In this setup, the spectrum slit position defines a one-dimensional line along which the sample is monitored.

[0012] However, a one-dimensional image only delivers limited information about time-resolved spatial variations occurring in the sample. The setup described by D.R. Vogus et al. does not address the problem of providing an image of the sample along more than one dimension. SUMMARY OF THE INVENTION

[0013] In a first aspect, it is thus an object of the present invention to provide a measurement assembly that enables monitoring of spatiotemporal refractive index changes in a sample along more than one dimension.

[0014] This object is achieved by a measurement assembly according to claim 1. Further embodiments of the invention are laid down in the dependent claims.

[0015] A measurement assembly for determining spatiotemporal refractive index changes in a sample, in particular in a fluid sample, is disclosed. The measurement assembly comprises: a light source having a spectral emission bandwidth; a tunable wavelength selector, wherein the wavelength selector is configured to select a spectral subrange of the spectral emission bandwidth in a tunable manner; an image sensor, a processing unit, and a Fabry-Perot unit, the Fabry-Perot unit comprising a first plane surface with a first non-zero reflectivity Ri and a second plane surface with a second non-zero reflectivity R2, the second plane surface being arranged opposite the first plane surface, wherein the Fabry-Perot unit has a sample reception space arranged between the first plane surface and the second plane surface to receive a sample, in particular a fluid sample, wherein the Fabry-Perot unit is arranged on a light propagation path that extends from the light source to the image sensor, the Fabry-Perot unit thereby acting as a wavelength-dependent light intensity filter with an intensity filter function that depends on a refractive index experienced by light propagating through the Fabry-Perot unit, wherein the image sensor is configured to record a two-dimensional image of at least a portion of the sample reception space for each of the spectral subranges selected by the tunable wavelength selector, and wherein the processing unit is configured to reconstruct the intensity filter function of the Fabry-Perot unit based on the recorded two-dimensional images, and to determine a refractive index map of the imaged portion of the sample reception space based on the reconstructed intensity filter function.

[0016] Such a measurement assembly enables a two-dimensional visualization of refractive index changes in a sample, in particular of spatial gradients and / or temporal variations of the refractive index, without having to spatially scan across the sample, thereby enabling time- resolved visualization. Thus, dynamic processes may be visualized, such as e.g. diffusion processes in a colaminar flow, or temperature-dependent phase transitions of liquid crystals.

[0017] The image sensor preferably comprises a pixel matrix, wherein an intensity value may be read out, in particular by the processing unit, for each pixel. In some embodiments, the image sensor may be configured to provide 2x2 and / or 4x4 pixel binning to increase the intensity values and thus decrease exposure time for faster image generation.

[0018] The processing unit may comprise a display on which one or more refractive index maps may be displayed to a user. If a plurality of refractive index maps is acquired, the processing unit may be configured to display the refractive index maps as frames of a video.

[0019] Preferably, the second plane surface is arranged in parallel to the first plane surface at a geometrical distance d of the first plane surface. The geometrical distance d is ideally constant in such a case, however, even though the first plane surface and the second plane surface may be considered to be arranged "in parallel", the geometrical distance d may vary within a tolerance range due to alignment tolerances and / or manufacturing tolerances of a surface quality of the first plane surface and / or the second plane surface.

[0020] The light source and the tunable wavelength selector may be separate components that are individually controllable, which provides flexibility and facilitates the exchange of damaged components.

[0021] Preferably, the spectral emission bandwidth of the light source ranges from 250 nm to 3000 nm, preferably between 350 nm and 780 nm, in particular from 508 nm to 533 nm

[0022] In particular, the light source may by a white light source, i.e. a source which is configured to emit light simultaneously over its entire spectral emission bandwidth.

[0023] The spectral subrange of the spectral emission bandwidth selectable by the wavelength selector preferably has a spectral width (i.e. full width at half-maximum, FWHM) which is smaller than 0.25 nm, preferably between 0.1 nm and 0.2 nm, in particular 0.17 nm. The spectral subrange selectable by the wavelength selector may be tunable across a tuning bandwidth which corresponds to the entire spectral emission bandwidth of the light source.

[0024] Alternatively, the tuning bandwidth may be smaller than the spectral emission bandwidth of the light source. Especially in cases where the measurement assembly is used to measure relative changes in refractive index of an unknown sample relative to a calibration sample, the tuning bandwidth may generally not need to cover the entire spectral emission bandwidth of the light source. Instead, it may be sufficient to tune the wavelength selector in a small tuning bandwidth around a known wavelength position that corresponds to a maximum of the known intensity filter function of the Fabry-Perot unit when the latter contains the calibration sample. The term "small tuning bandwidth" in this context refers to a bandwidth that is smaller than a wavelength separation between two adjacent transmission maxima of the intensity filter transmission function.

[0025] Preferably, the spectral subrange has a center wavelength that is tunable in increments of less than 0.1 nm, preferably less than 0.06 nm. Ideally, the increments are chosen such that the transmission peaks of the intensity filter function are resolved.

[0026] The tunable wavelength selector may be arranged after Fabry-Perot unit on the light propagation path. This may be advantageous if the measurement assembly is integrated into an existing inverted optical microscope setup, in which space is limited between the light source and a location where the Fabry-Perot unit can be placed.

[0027] Alternatively, the tunable wavelength selector may be arranged before the Fabry-Perot unit on the light propagation path. Having the tunable wavelength arranged before the Fabry- Perot unit, i.e. between the light source and the Fabry-Perot unit, provides the advantage of reducing an amount of light propagating through the Fabry-Perot unit and thus also through a sample placed therein, which may prevent or reduce unwanted radiative heat transfer within the sample.

[0028] In some embodiments, the tunable wavelength selector and the light source may be integrated in a single component to form a tunable light source. In particular, the tunable light source may be a tunable laser. In such a case, the light exiting the light source does not cover the entire spectral emission bandwidth of the light source simultaneously, but only the selected spectral subrange, which may then be shifted in a tunable manner, e.g. via a laser controller.

[0029] The tunable wavelength selector may comprise a single bandpass filter, in particular a dichroic filter, which has a pivot axis about which it is pivotable, the pivot axis being perpendicular to the light propagation path. The selection of the spectral subrange being transmitted through the single bandpass filter may be carried out by pivoting the bandpass filter about its pivot axis. The measurement assembly may comprise a stepper motor to pivot the bandpass filter about its pivot axis.

[0030] When pivoting the bandpass filter about its pivot axis, a spatial offset may be introduced in the beam propagation path perpendicularly to a light incidence direction, wherein the spatial offset varies as the bandpass filter is pivoted. This variable spatial offset may complicate the alignment of the measurement assembly.

[0031] Thus, in preferred embodiments, the tunable wavelength selector may comprise a first bandpass filter and a second bandpass filter that compensates the spatial offset that is introduced by the first bandpass filter, wherein the first bandpass filter and the second bandpass filter each have a bandpass transmission function and a pivot axis about which they are pivotable to spectrally tune the bandpass transmission function, and wherein the pivot axes are preferably parallel to each other and perpendicular to the light propagation path.

[0032] In order to enable full compensation of the spatial offset in the beam propagation path, the first bandpass filter and the second bandpass filter preferably have an equal thickness and are oriented at a filter incidence angle of equal magnitude with respect to the light incidence direction, but with opposite sign, i.e. the first bandpass filter is tilted by a filter incidence angle 0 with respect to the light incidence direction, while the second bandpass filter is tilted by the filter incidence angle -0 with respect to the light incidence direction.

[0033] The selection of the spectral subrange being transmitted through the tunable wavelength selector may be carried out by pivoting the first bandpass filter and the second bandpass filter about their respective pivot axis by an equal angle value, but in opposite directions.

[0034] T o this end, the measurement assembly may comprise a first stepper motor to pivot the first bandpass filter about its pivot axis, and a second stepper motor to pivot the second bandpass filter about its pivot axis. In particular, the stepper motors may be integrated into optical mounts in which the bandpass filters may be mounted.

[0035] The measurement assembly may comprise a control unit for controlling the one of more stepper motors and / or for triggering an image acquisition of the image sensor, in particular to coordinate movements of the stepper motors and the image acquisition such that there is no movement of the stepper motors during image acquisition.

[0036] The first bandpass filter and / or the second bandpass filter may be a dichroic filter. Preferably, the transmission function of the first bandpass filter has a spectral transmission bandwidth that is larger than a spectral transmission bandwidth of the transmission function of second bandpass filter, and the second bandpass filter has a peak wavelength corresponding to maximum light transmission at normal incidence which lies within the spectral transmission bandwidth of the first bandpass filter at normal incidence. This ensures that light is still transmitted through the wavelength selector even if the second bandpass filter is not perfectly angularly aligned with respect to the first bandpass filter.

[0037] Such a tunable wavelength selector enables tuning of the center wavelength of the selected spectral subrange in increments that are sufficiently small to enable a precision exceeding 10'4refractive index units (Rill) at an acquisition rate of at least 1 Hz.

[0038] The tunable wavelength selector may in particular be configured to select at least 150 spectral subranges and control unit may be configured to trigger the image acquisition such that all the selected spectral subranges are recorded within 1 second or less.

[0039] The Fabry-Perot unit may be a microfluidic chip comprising a top plate with a first coating providing the first plane surface, and a bottom plate with a second coating providing the second plane surface, wherein the top plate and the bottom plate are fixed to a spacer layer arranged between the top plate and the bottom plate, and wherein the sample reception space is formed within said spacer layer, in particular wherein the sample reception space is formed as a fluid channel within said spacer layer.

[0040] The spacer layer may comprise or consist of photoresist, in particular Sll-8. Alternatively, double-sided tape, dry film photoresist or a polymer material may be used to form the spacer layer. One or more fluid channels may be patterned into the spacer layer, e.g. using known photolithography processes.

[0041] The top plate and or bottom plate may comprise or consist of glass, in particular borosilicate glass. To provide fluidic access to the sample reception space, in particular to the one of more fluid channels, the top plate and / or bottom plate may comprise one or more holes serving as one or more fluidic connection points. The one or more fluidic connection points may be connectable to tubing, which may be attachable to a syringe pump to allow precise sample introduction into the Fabry-Perot unit.

[0042] The reflectivity of the first plane surface Ri and the reflectivity of the second plane surface R2 for the incident light may be provided by a coating on an inner surface of the top plate T and the bottom plate B, respectively, the inner surfaces facing the sample reception space. The reflectivity R1 and R2 may be the same, i.e. R1 = R2 = R. The coating may comprise several layers. In particular, the coating may comprise four layers.

[0043] In particular, the coating on both the top plate and the bottom plate may be identical and may comprise: a first layer comprising or consisting of titanium (Ti), chromium (Cr) or gold (Au), preferably with a layer thickness in the range of 0.1 nm to 100 nm, the first layer L1 being arranged directly on top the top plate or the bottom plate, respectively, a second layer comprising or consisting of silver (Ag), titanium (Ti), gold (Au) or platinum (Pt), preferably with a layer thickness in the range 0.1 nm to 100 nm, preferably 6 nm, the second layer being arranged on the first layer, a third layer comprising or consisting of titanium (Ti), chromium (Cr) or gold (Au), preferably with a layer thickness in the range of 0.1 nm to 100 nm, the third layer being arranged on the second layer, and a fourth layer comprising or consisting of silicon dioxide (silica, SiC>2), silicon nitride (Si N) , hafnium oxide (HfO), or titanium dioxide (TiCh), preferably with a layer thickness in the range of 0.1 nm to 1000 nm, the fourth layer being arranged on the second layer and facing the sampling reception space.

[0044] The first layer may primarily serve to ensure adhesion between the second layer, which primarily contributes to the desired reflectivity, and the top plate or the bottom plate, respectively. The third layer may primarily serve to ensure adhesion between the second layer and the fourth layer.

[0045] The fourth layer may primarily serve to ensure adhesion between the third layer and the spacer layer.

[0046] In particular, the reflectivity Ri and R2 may be chosen such that the intensity filter function of the Fabry-Perot exhibits a minimum transmission value that is between 10% and 60%, preferably between 35% and 45 %, in particular at 40%.

[0047] A minimum transmission value in the aforementioned range enables sufficient light transmission for simultaneous use of other optical inspection methods, e.g. epifluorescence microscopy or laser scanning confocal microscopy.

[0048] Increasing the minimum transmission enables a reduction of both illumination power density and exposure time leading to an increase in time resolution without compromising a precision of the determined refractive index map.

[0049] In embodiments in which at least one of the first plane surface and second plane surface is not fixed to a spacer layer, said at least one of the first plane surface and second plane surface of the Fabry-Perot unit may be mounted in a moveable manner, for instance on a mounting stage comprising a differential screw, such that the geometrical distance d between the first plane surface and the second plane surface is adaptable.

[0050] Additionally or alternatively, at least one of the first plane surface and second plane surface of the Fabry-Perot unit may be mounted in a removable manner, so as to form a lid that may be removed to place a sample in the sample reception space and may be closed again prior to taking measurements.

[0051] The measurement assembly may further comprise a collimator arranged before the Fabry- Perot unit, wherein the collimator is configured such that light propagating on the light propagation path is collimated when entering the Fabry-Perot unit to avoid or at least reduce additional intensity fringes arising from unwanted spatial interference. The collimator may comprise an optical lens with an appropriately chosen focal length. The measurement assembly may further comprise an adjustable polarizer, wherein the adjustable polarizer is configured to linearly polarize light propagating on the light propagation path in an adjustable polarization direction. Using an adjustable polarizer enhances the measurement quality of samples for which the refractive index is polarization- depend, e.g. birefringent samples such as liquid crystals.

[0052] The measurement assembly may further comprise an angular alignment element to enable an incidence angle between the light propagation path and the Fabry-Perot unit to be adjusted.

[0053] In some embodiments, the Fabry-Perot unit may be fixed in space and the angular alignment element may be a component, such as a mirror, configured for adjustably steering the light.

[0054] Additionally or alternatively, the Fabry-Perot may be mounted, for instance on a rotatable and / or pivotable stage, so as to enable the adjustment of the incidence angle between the light propagation path and the Fabry-Perot unit.

[0055] Preferably, the Fabry-Perot unit is arranged on the light propagation path such that light enters the Fabry-Perot unit at normal incidence, i.e. such that the first plane surface and the plane surface are perpendicular to the light propagation path.

[0056] Preferably, the measurement assembly is configured to be integratable into an optical microscope setup, preferably into an inverted optical microscope.

[0057] In a second aspect, it is an object of the present invention to provide an optical microscope unit for determining spatiotemporal refractive index changes in a sample, in particular in a fluid sample.

[0058] The optical microscope setup comprises: a measurement assembly according to the first aspect of the present invention, and a microscope objective, wherein the measurement assembly is arranged such that the microscope objective is located along the light propagation path between the Fabry-Perot unit and the image sensor. The optical microscope setup may comprise a camera port, and the measurement assembly may comprise an image sensor housing in which the image sensor is arranged, wherein the image sensor housing is configured to be plugged, e.g. screwed or snapped, into the camera port to avoid pollution or dirt entering the image sensor housing and potentially deteriorating the images acquired by the image sensor.

[0059] In a third aspect, it is an object of the present invention to provide a method for determining spatiotemporal refractive index changes in a sample, in particular in a fluid sample, using a measurement assembly according to the first aspect of the present invention. The method comprises: a) introducing a sample in the sample reception space of the Fabry-Perot unit; b) selecting a spectral subrange of the spectral emission bandwidth of the light source using the wavelength selector, c) recording a two-dimensional image of at least a portion of the sample reception space containing the sample for said spectral subranges selected by the tunable wavelength selector, d) repeating steps b) and c) for a plurality of different selected spectral subranges; e) reconstructing the intensity filter function of the Fabry-Perot unit based on the recorded two-dimensional images, and f) determining a refractive index map of the imaged portion of the sample reception space containing the sample based on the reconstructed intensity filter function.

[0060] In particular, between 100 and 200 different selected spectral subranges may be used which are spaced by increments of less than 0.02 nm.

[0061] Preferably, repeating steps b) and c) for the plurality of different selected spectral subranges takes place in 1 second or less.

[0062] The method may further comprise the following steps: g) repeating steps b)-f) to obtain a plurality of refractive index maps, and h) determining temporal changes in the sample based on the plurality of refractive index maps.

[0063] The method may further comprise a calibration step, wherein the calibration step comprises: introducing a calibration sample with a known refractive index into the Fabry-Perot unit, performing steps b)-e), and determining a geometrical distance between the first plane surface and the second plane surface of the Fabry-Perot unit based on the reconstructed intensity filter function.

[0064] BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

[0066] Fig. 1 shows schematical view of an optical microscope setup comprising a first embodiment of a measurement assembly according to the present invention;

[0067] Fig. 2 shows schematical view of an optical microscope setup comprising a second embodiment of a measurement assembly according to the present invention;

[0068] Fig. 3 shows schematical view of an optical microscope setup comprising a third embodiment of a measurement assembly according to the present invention;

[0069] Fig. 4 shows schematical view of an optical microscope setup comprising a fourth embodiment of a measurement assembly according to the present invention;

[0070] Fig. 5 shows a first embodiment of the Fabry-Perot unit in a separate schematical sectional view;

[0071] Fig. 6 shows a second embodiment of the Fabry-Perot unit in a separate schematical view;

[0072] Fig. 7 shows a flow diagram of an embodiment of a method for determining spatiotemporal refractive index changes in a sample according to the present invention;

[0073] Fig. 8A shows two normalized intensity filter functions;

[0074] Fig. 8B symbolizes a two-dimensional image sensor with pixel rows and pixel columns;

[0075] Fig. 9A shows an experimentally measured intensity filter function for one pixel unit with 16 pixels binned in 4x4 binning mode, and

[0076] Fig. 9B shows an experimentally obtained refractive index map showing a diffusive layer of NaCI diffusing into water in a coflowing laminar streams in a microfluidic channel embedded in a Fabry-Perot chip.

[0077] DESCRIPTION OF PREFERRED EMBODIMENTS Fig. 1 shows a schematical view of an optical microscope setup 1 comprising a first embodiment of a measurement assembly according to the present invention and a microscope objective 10. The measurement assembly comprises a light source 2 having a spectral emission bandwidth and a tunable wavelength selector 3. A collimator 7, for instance a lens, is arranged between the light source 2 and the tunable wavelength selector 3, such that light entering the wavelength selector 3 is collimated. As shown in the enlarged excerpt of the wavelength selector 3 in Fig. 1 , the tunable wavelength selector 3 comprises a first bandpass filter 31 that is mounted so as to be tiltable about a pivot axis A1 in order to tune a filter incidence angle 0 under which the light is hitting the first bandpass filter 31 , and thereby tune the bandpass transmission function of the bandpass filter 31. The measurement assembly further comprises a Fabry-Perot unit 5, which is described in further detail below, and an image sensor 4 arranged within an image sensor housing 41.

[0078] The Fabry-Perot unit comprises a sample reception space 53 and is arranged on a light propagation path that extends from the light source 2 to the image sensor 4. The light propagation path is illustrated in Figs. 1-4 by straight or wavy arrows symbolizing light rays. The Fabry-Perot unit 5 acts as a wavelength-dependent light intensity filter with an intensity filter function that depends on the optical path and thus the refractive index experienced by light propagating through the Fabry-Perot unit 5.

[0079] In this first embodiment, the Fabry-Perot unit 5 is arranged between the wavelength selector 3 and the microscope objective 10. The optical microscope setup 1 has a camera port 11 , which is arranged at an output of the microscope objective 10. The image sensor housing 41 is plugged directly into the camera port 11 to prevent any pollution or dirt from entering the sensor housing 41. The image sensor is connected to a processing unit 6, which is configured to reconstruct the intensity filter function of the Fabry-Perot unit 5 based on the recorded two-dimensional images, and to determine a refractive index map of the imaged portion of the sample reception space 53 based on the reconstructed intensity filter function.

[0080] The processing unit preferably comprises a display 61 on which the refractive index map may be displayed to a user. The measurement assembly further comprises a control unit 12 to coordinate the wavelength selection performed by the wavelength selector 3 and the image acquisition performed by the image sensor 4.

[0081] Descriptions of features given in the context of the first embodiment, which have the same reference signs in Figs. 2-6 as in Fig. 1 apply analogously to the embodiments shown in Figs. 2-6, unless specified otherwise.

[0082] Fig. 2 shows a schematical view of an optical microscope setup 1 comprising a measurement assembly according to a second embodiment of the present invention and a microscope objective 10. This second embodiment differs from the first embodiment shown in Fig. 1 in that the tunable wavelength selector 3 is arranged between the microscope objective 10 and the image sensor 4. In addition, the tunable wavelength selector 3 shown in Fig. 2 comprises a second bandpass filter 32. The first bandpass filter 31 and the second bandpass filter 32 each have a pivot axis A1 , A2 about which they are pivotable as indicated by the arrows in Fig. 2. The pivot axes A1 , A2 are parallel to each other and perpendicular to the light propagation path. In order to compensate a spatial offset in the light propagation path that occurs perpendicularly to a light incidence direction after the first bandpass filter 31 , the second bandpass filter 32 is tilted by a filter incidence angle -0 with respect to the light incidence direction, i.e. by the same amount as the first bandpass filter 31 , but in opposite direction. The wavelength selector 3 shown in Fig. 2 may of course also be arranged between the light source 2 and the Fabry-Perot unit 5, i.e. to replace the wavelength selector 3 shown in Fig. 1.

[0083] In a specific embodiment, the first bandpass filter has a spectral transmission bandwidth of 50 nm (FWHM) at a peak wavelength at normal incidence of 353 nm. The second filter has a spectral transmission bandwidth of 0.17nm (FWHM) at a peak wavelength at normal incidence of 532.08 nm to ensure selective binning of the wavelengths of the light. The FWHM of 0.17 nm allows for resolving the transmission peaks of the intensity filter function of a Fabry-Perot unit with a geometrical distance d, i.e. thickness, of up to 200 pm. By tuning the filter incidence angle over a range of + / - 36°, a tuning bandwidth of 23.4 nm, i.e. from 508.64 nm - 532.08 nm is obtained. Two stepper motors (Nanotec, PD2-C4118L1804-E- 01) are used to turn the bandpass filters about their respective pivot axis. These stepper motors provide a total of 4096 steps per revolution with a stopping accuracy of 0.09°or approximately one step. With the angular range of the filters being between ±36° and a range of 23.4 nm, this results in an average wavelength increment per stepper step of 0.0585nm. Close to the edge, i.e. near ±36°, the wavelength increment may be slightly larger, e.g. at about 0.1 nm per stepper step, while it is significantly smaller towards normal incidence, e.g. the very first stepper step away from normal incidence may only results in a 0.00016 nm increment in transmitted wavelength. A control unit in the form of an Arduino Uno can be used to control the measurement assembly. To ensure that the images sensor acquires images when the stepper motors are not moving and vice versa, the image sensor is first triggered to acquire an image.

[0084] Afterwards, the Arduino code waits as long as a set exposure time of the image sensor before sending a movement signal to the stepper motors. Thus, the acquisition time is on the one hand limited by the exposure time needed for the image sensor and on the other hand the time required to move the stepper motors from one position to the next.

[0085] Fig. 3 shows a schematical view of an optical microscope setup 1 comprising a third embodiment of a measurement assembly according to the present invention and a microscope objective 10. This third embodiment differs from the first embodiment shown in Fig. 1 and the second embodiment shown in Fig. 2 in that the tunable wavelength selector 3 and the light source 2 are integrated into a single component forming a tunable light source, i.e. a light source configured to emit light at a tunable wavelength j, e.g. a tunable laser.

[0086] Fig. 4 shows a schematical view of an optical microscope setup 1 comprising a fourth embodiment of a measurement assembly according to the present invention and a microscope objective 10. This fourth embodiment differs from the first embodiment shown in Fig. 1 in that a polarizer 8 is arranged between the light source 2 and the tunable wavelength selector s, more precisely between the collimator ? and the wavelength selector 3. The wavelength selector 3 may be configured as shown in Fig. 1 or as shown in Fig. 2.

[0087] In another embodiment (not shown), it would of course also be conceivable to have a polarizer 8 being arranged between a tunable light source as shown in Fig. 3 and the Fabry- Perot unit 5.

[0088] In the embodiments shown in Fig. 1 , Fig. 2 and Fig. 4, the light source is preferably a white light source, e.g. a "SOLA Light Engine" as provided by Lumencor.

[0089] In all the embodiments discussed above, the image sensor may be a digital camera. In a specific embodiment, the image sensor is a Hamamatsu Orca-Fusion C14440-20UP with a resolution of 2304 x 2304 pixels and a frame rate of between 89.1 frames per second (when capturing 2304 columns) and 41000 frames per second (when capturing 4 columns). Fig. 5 shows a first embodiment of the Fabry-Perot unit in a separate schematical sectional view. The Fabry-Perot unit 5 shown in Fig. 5 is a microfluidic chip which comprises a top plate T, which provides a first plane surface 51 and a bottom plate B, which provides a second plane surface 52. The bottom plate B and the top plate T are arranged parallel to each other and are fixed to a spacer layer 54. In a preferred embodiment, the bottom plate B and the top plate T comprise or consist of glass, e.g. borosilicate glass. The reflectivity Ri and R2, respectively, for the incident light, is provided by a coating on an inner surface of both the top plate T and the bottom plate B, the inner surfaces facing the sample reception space 53. The coating may comprise several layers.

[0090] In a specific embodiment, the coating on both the top plate T and the bottom plate B is identical and comprises: a first layer L1 made of titanium (Ti) with a layer thickness of 2 nm, the first layer L1 being arranged directly on top of a borosilicate glass plate forming the top plate T or the bottom plate B, a second layer L2 made of silver (Ag) with a layer thickness of 50 nm, the second layer L2 being arranged on the first layer L1 , a third layer L3 made of titanium (Ti) with a layer thickness of 2 nm, the third layer L3 being arranged on the second layer L2, and a fourth layer L4 made of silicon dioxide (silica, SiCh) with a layer thickness of 10 nm, the fourth layer L4 being arranged on the second layer L3 and facing the sampling reception space 53.

[0091] In a specific method, the coated top plate G and bottom plate B may be fabricated as follows:

[0092] Two glass wafers are initially cleaned with an oxygen plasma cleaner at 200 W for 1 min (e.g. using TVA Tepla, Gigabatch). The first layer L1 , second layer L2 and third layer L3 are deposited using e-beam vapor (Evatec, BAK 501 LL). The second layer L2 is primarily contributing to the reflectivity R1, R2 necessary to form the Fabry-Perot cavity, whereas the first layer L1 and the third layer L3 are used to ensure adhesion to the glass layers, i.e. the top plate T or bottom plate B, or the fourth layer L4, respectively. The fourth layer L4 is deposited using Plasma Enhanced Chemical Vapor Deposition (PECVD, Plasmalab System 100) to ensure proper adhesion to the spacer layer 54, which may for example comprise or consist of photoresist, in particular Sll-8 photoresist. To create the spacer layer 54, SU8 3025 may be spin-coated with 4000 RPM for a total of 40s. One of more microfluidic channels may then be created via photolithography using a chrome mask and exposing the wafer to UV light for 10 s.

[0093] A further embodiment differs from the embodiment described above in that the second layer L2 made of silver (Ag) has a thickness of 6 nm instead of 50 nm. This reduces the reflectivity Ri, R2 and causes the intensity filter function of the Fabry-Perot unit to have a maximum transmission of 40% instead of 1%.

[0094] Fig. 6 shows a second embodiment of the Fabry-Perot unit 5 in a separate schematical view. Instead of having a fixed geometrical distance d between the first plane surface 51 and the second plane surface 52, the first plane surface and second plane surface of the Fabry-Perot unit are mounted in a moveable manner such that the geometrical distance d between the first plane surface 51 and the second plane surface 52 is adaptable. As indicated by the arrows in Fig. 6, alternatively or additionally the first plane surface 51 and the second plane 52 may be mounted so as to be pivotable with respect to each other, in particular about two pivot axes B1 , B2 that are perpendicular to each other.

[0095] Fig. 7 shows a flow diagram of an embodiment of a method for determining spatiotemporal refractive index changes in a sample according to the present invention, which comprises inserting a sample in the Fabry-Perot 101 , generating a plurality of refractive index maps 102, and determining a temporal in the sample 103 change based on the plurality of refractive index maps.

[0096] In order to obtain a refractive index map, an image recording step 1021 is carried out, which comprises selecting a spectral subrange 10211 with the wavelength selector 3, e.g. by tuning the stepper motors of the first and second bandpass filters, and recording an image 10212 for each spectral subrange, i.e. for each setting of the stepper motors.

[0097] An intensity filter function is then reconstructed for each pixel based on the recorded images in a filter function reconstruction step 1022. Subsequently, in a refractive index map generation step 1023, a refractive index map is generated based on the intensity filter function obtained for each pixel.

[0098] To determine temporal changes in the sample, the step 102 of obtaining a refractive index map is repeated a plurality of times. The refractive index maps then be displayed as frames in a video, for instance on the display 61 of the processing unit 6 shown in Figs. 1-4.

[0099] Fig. 8A shows two theoretical curves of the normalized intensity filter function for a single pixel pij of the image sensor 4 shown schematically in Fig. 8B. The first curve h(t) is obtained at time ti , while the second curve l2(t) was obtained at time t2.

[0100] In a case where both the first plane surface 51 and the second plane surface 52 have the same reflectivity R=RI=R2, the normalized intensity filter function that depends on the geometrical distance d, the refractive index n and the wavelength A can be expressed as follows:

[0101] The larger the reflectivity R, the larger the minimum transmission value becomes, i.e. the less contrast there is between the transmission maxima and the transmission minima of the intensity filter function.

[0102] As shown in Fig. 8A, the maxima of the two curves h(t) and l2(t) are shifted with respect to each other.

[0103] The position Amof the maximum of chromatic order m on the wavelength-axis is defined as follows: mm= 2nd

[0104] Assuming a constant geometrical distance d for both the first and the second measurement, it is thus possible to determine a relative change of refractive index, i.e. the ratio between the refractive index m experienced by the light at time h and the refractive index n2 at time t2 by calculating the ratio of the wavelength positions A1mand A2m:

[0105] If the exact geometrical distance d is initially unknown, it may be measured in a calibration step by filling the sample reception space with a medium that has a known refractive index n, and the geometrical distance d may then be obtained by using the position Amof the maximum of chromatic order m and the position Am+i of the maximum of chromatic order m+1 :

[0106] Fig. 9A and Fig. 9B show an example of an experimental measurement carried out with an optical microscope setup as shown in Fig. 2. Fig. 9A shows the normalized intensity filter function for one pixel unit of the image sensor as a function of the steps of the stepper motors of the wavelength selector 3, and Fig. 9B shows a 2D-map of refractive index showing a diffusive layer of NaCI diffusing into water in a coflowing laminar streams in a microfluidic channel embedded in the Fabry-Perot chip. In a top half of Fig. 9B water flowing from right to left is visible, while in a bottom half of Fig. 9B, water mixed with NaCI flowing from right to left is visible, while the diffusive layer (darkest shading) is visible in the middle of Fig. 8B. The images recorded to generate the 2D-map were captured in 4x4-binning mode, i.e. a pixel unit thus consisting of 16 pixels, resulting in a resolution of 576x576 pixels, with an exposure time of 0.001 s per image, and using a 4x microscope objective.

[0107] LIST OF REFERENCE SIGNS

[0108] 1 optical microscope setup

[0109] 2 light source

[0110] 3 tunable wavelength selector

[0111] 31 first bandpass filter

[0112] 32 second bandpass filter

[0113] 4 image sensor

[0114] 41 image sensor housing

[0115] 5 Fabry-Perot unit

[0116] 51 first plane surface (51)

[0117] 52 second plane surface

[0118] 53 sample reception space

[0119] 54 spacer layer

[0120] 6 processing unit

[0121] 61 display

[0122] 7 collimator

[0123] 8 polarizer

[0124] 9 angular alignment element

[0125] 10 microscope objective

[0126] 11 camera port

[0127] 12 control unit

[0128] T top plate

[0129] B bottom plate

[0130] A1, A2 pivot axes

[0131] B1, B2 pivot axes

Claims

CLAIMS1. A measurement assembly for determining spatiotemporal refractive index changes in a sample, in particular in a fluid sample, the measurement assembly comprising: a light source (2) having a spectral emission bandwidth; a tunable wavelength selector (3), wherein the wavelength selector (3) is configured to select a spectral subrange of the spectral emission bandwidth in a tunable manner; an image sensor (4), a processing unit (6), and a Fabry-Perot unit (5), the Fabry-Perot unit (5) comprising a first plane surface (51) with a first non-zero reflectivity Ri and a second plane surface with a second non-zero reflectivity R2, the second plane surface (52) being arranged opposite the first plane surface (51), wherein the Fabry-Perot unit (5) has a sample reception space (53) arranged between the first plane surface (51) and the second plane surface (52) to receive a sample, in particular a fluid sample, wherein the Fabry-Perot unit (5) is arranged on a light propagation path that extends from the light source (2) to the image sensor (4), the Fabry-Perot unit (5) thereby acting as a wavelength-dependent light intensity filter with an intensity filter function that depends on a refractive index experienced by light propagating through the Fabry-Perot unit (5), wherein the image sensor (4) is configured to record a two-dimensional image of at least a portion of the sample reception space (53) for each of the spectral subranges selected by the tunable wavelength selector (3), and wherein the processing unit (6) is configured to reconstruct the intensity filter function of the Fabry-Perot unit (5) based on the recorded two-dimensional images, and to determine a refractive index map of the imaged portion of the sample reception space (53) based on the reconstructed intensity filter function.

2. The measurement assembly of claim 1 , wherein the light source (2) and the tunable wavelength selector (3) are separate components that are individually controllable.

3. The measurement assembly of claim 1 or 2, wherein the tunable wavelength selector (3) is arranged after Fabry-Perot unit (5) on the light propagation path.

4. The measurement assembly of claim 1 or 2, wherein the tunable wavelength selector (3) is arranged before the Fabry-Perot unit (5) on the light propagation path.

5. The measurement assembly of claim 4, wherein the tunable wavelength selector (3) and the light source (2) are integrated in a single component to form a tunable light source (2), in particular wherein the tunable light source (2) is a tunable laser.

6. The measurement assembly of any one of claims 1-4, wherein the tunable wavelength selector (3) comprises a first bandpass filter (31) and a second bandpass filter (32), wherein the first bandpass filter (31) and the second bandpass filter (32) each have a bandpass transmission function and a pivot axis (A1 ,A2) about which they are pivotable to spectrally tune the bandpass transmission function, and wherein the pivot axes (A1 , A2) are preferably parallel to each other and perpendicular to the light propagation path.

7. The measurement assembly of any one of the preceding claims, wherein the Fabry-Perot unit (5) is a microfluidic chip comprising a top plate (T) with a first coating providing the first plane surface (51), and a bottom plate (B) with a second coating providing the second plane surface (52), wherein the top plate (T) and the bottom plate (B) are fixed to a spacer layer (54) arranged between the top plate (T) and the bottom plate (B), and wherein the sample reception space (53) is formed within said spacer layer (54), in particular wherein the sample reception space (53) is formed as a fluid channel within said spacer layer (54).

8. The measurement assembly of any one of claims 1-6, wherein at least one of the first plane surface (51) and second plane surface (52) of the Fabry-Perot unit (5) is mounted in a moveable manner such that a geometrical distance between the first plane surface (51) and the second plane surface (52) is adaptable.

9. The measurement assembly of any one of the preceding claims, further comprising a collimator (7) arranged before the Fabry-Perot unit (5), wherein the collimator (7) is configured such that light propagating on the light propagation path is collimated when entering the Fabry-Perot unit (5).

10. The measurement assembly of any one of the preceding claims, further comprising an adjustable polarizer (8), wherein the adjustable polarizer (8) is configured to linearly polarize light propagating on the light propagation path in an adjustable polarization direction.

11. The measurement assembly of any one of the preceding claims, further comprising an angular alignment element (9) to enable an incidence angle between the light propagation path and the Fabry-Perot unit (5) to be adjusted.

12. An optical microscope setup comprising: a measurement assembly according to any one of claims 1-11 , and a microscope objective (10), wherein the measurement assembly is arranged such that the microscope objective (10) is located along the light propagation path between the Fabry-Perot unit (5) and the image sensor (4).

13. The optical microscope setup of claim 12, wherein the optical microscope setup comprises a camera port (11), and wherein the measurement assembly comprises an image sensor housing (41) in which the image sensor (4) is arranged, wherein the image sensor housing (41) is configured to be plugged into the camera port (11).

14. A method for determining spatiotemporal refractive index changes in a sample, in particular in a fluid sample, using a measurement assembly according to any one of claims 1-11 , the method comprising: a) introducing a sample in the sample reception space (53) of the Fabry-Perot unit (5); b) selecting a spectral subrange of the spectral emission bandwidth of the light source (2) using the wavelength selector (3), c) recording a two-dimensional image of at least a portion of the sample reception space (53) containing the sample for said spectral subranges selected by the tunable wavelength selector (3), d) repeating steps b) and c) for a plurality of different selected spectral subranges; e) reconstructing the intensity filter function of the Fabry-Perot unit (5) based on the recorded two-dimensional images, andf) determining a refractive index map of the imaged portion of the sample reception space (53) containing the sample based on the reconstructed intensity filter function.

15. The method of claim 14, further comprising: g) repeating steps b)-f) to obtain a plurality of refractive index maps, and h) determining temporal changes in the sample based on the plurality of refractive index maps.

Images