FIELD OF THE INVENTION

[1] The present invention is in the field of optics. In particular, the invention relates to a device and a method for multicolor optical imaging of a sample with wavelength-dependent optical path length enhancement.

BACKGROUND

[2] Optical imaging is employed for a wide range of applications, e.g. for optical micros- copy of mesoscopic objects like thin-film solid state structures or biological cells as well as for optical spectroscopy of chemical or biological samples. Correspondingly, a large variety of imaging techniques has been developed, which are adapted for different purposes. These in- clude for example absorption imaging for samples with spatially vaiying absorption proper- ties and phase contrast imaging for samples with a spatially varying refractive index as well as fluorescence spectroscopy of fluorophore-labeled objects and Raman spectroscopy for probing of vibrational spectra.

[3] While spectacular progress has been made in recent years, e.g. in terms of spatial res- olution with the advent of super-resolution microscopy, many imaging techniques are limited to optically dense samples, which interact strongly with incident light. This holds in particu- lar for samples with a low damage threshold, for which the light intensity e.g. for illumination or resonant excitation is limited to low intensities in order to avoid damage to the sample. This is frequently the case when studying biological samples. For this reason, imaging of samples with a low optical density, e.g. thin samples like adherent single-cell layers as well as cells or cell aggregates in flow, can be challenging, in particular if the thickness of the sample cannot be increased, for example if properties of individual cells are to be studied, e.g. meta- bolic conditions or chemical/biochemical changes due to interactions with the cellular envi- ronment.

[4] One approach to overcome this limitation is to employ optical resonators, in which light coupled into the resonator is reflected back and forth multiple times before leaving the resonator. Thereby, the effective optical path length through a sample placed in the resonator can be increased as the light passes through the sample repeatedly, leading to an enhanced interaction between the light and the sample, e.g. a larger absorption, scattering and/or phase shift of the light. This concept is for example used in scanning cavity microscopy, which relies on an optical microcavity formed between the tip of an optical fiber and a sample holder to enhance a detection signal, see e.g. M. Mader et al., Nat. Commun. 6:7249 (2015). As the light field within the microcavity is localized to a small volume, only a single point of the sample can be probed with this technique and the fiber tip has to be scanned over the sample to obtain a complete image such that the use of this technique is typically restricted to static samples.

[5] To directly image extended samples inside an optical resonator, resonators with fo- cusing elements like lenses or curved mirrors have been employed, wherein the focusing ele- ments are configured to image the sample onto itself, allowing for sharp images independent of the number of round trips that the imaging light performs in the resonator, see e.g. Y. Arai et al, PLoS ONE 10(5): 60125 33 (2015), T. Juffmann et ah, Nat. Commun. 7:12858 (2016) and US 2017/0261739 Al. This, however, requires complicated resonator configurations, which can be difficult to align. Moreover, one particular drawback of these methods is the fact that the image quality strongly depends on the imaging technique that is used, which can prevent simultaneous use of different imaging techniques.

WO 2013/164642 Al describes a device for chemical sensing using an optical microcavity. The device comprises a cavity connected to channels for providing a sample medium. Light from a radiation source is coupled into the cavity and light transmitted through the cavity is detected by a detector. The optical path length of the cavity is tunable and on the micrometer scale such that the free spectral range is sufficiently large so that only a single longitudinal cavity mode is resonant with the light emitted by the radiation source.

Devices for cavity ring down spectroscopy are known from WO 2018/106321 Al and US 5528040 A.

SUMMARY OF THE INVENTION

[6] The object of the invention is thus to provide a device and a method for imaging of a sample with optical path length enhancement that facilitates simultaneous imaging with dif- ferent imaging techniques. This object is met by a device and a method according to claim 1 and 21, respectively. Embodiments of the present invention are detailed in the dependent claims.

[7] The device for multicolor optical imaging of a sample with wavelength-dependent optical path length enhancement comprises: (a) an optical resonator for enhancing an optical path length, wherein the optical resonator has a first finesse at a first wavelength and a sec- ond finesse at a second wavelength; (b) a sample holder for mounting the sample in the opti- cal resonator, wherein the sample holder is configured to hold the sample such that an optical axis of the optical resonator intersects with the sample; (c) a first imaging system for imaging the sample at the first wavelength with a first imaging technique, wherein the imaging is per- formed along an outgoing optical axis of the optical resonator; and (d) a second imaging sys- tem for imaging the sample at the second wavelength with a second imaging technique, wherein the imaging is performed along an outgoing optical axis of the optical resonator and wherein the second wavelength is different from the first wavelength. The first finesse and the second finesse are chosen such that the optical resonator enhances a first image quality indicator of the imaging with the first imaging system and a second image quality indicator of the imaging with the second imaging system.

[8] The optical resonator can be formed by two or more reflective surfaces, e.g. mirrors. Light propagating along an optical axis of the optical resonator is reflected by the reflective surfaces such that the light continues to propagate along the optical axis of the optical reso- nator. The optical axis of the resonator forms a closed path, along which the light continuous- ly traverses. Thereby, the light can be confined to the optical resonator, in which it can prop- agates in a cyclic fashion. In order to couple light into the optical resonator, the optical reso- nator can comprise at least one partially reflective surface, through which light can be partial- ly transmitted. In turn, a fraction of the light propagating in the optical resonator can be transmitted through the partially reflective surface in each cycle and thus can leave the opti- cal resonator along an outgoing optical axis of the optical resonator. Each photon coupled into the optical resonator can thus perform a number of round trips before being emitted from the resonator such that the effective optical path length of the resonator, i.e. the optical path length that the photon travels within the resonator, can be enhanced compared to the physical length and optical path length of the resonator. Here, the physical length of the res- onator refers to the length of the closed path formed by the optical axis of the resonator, whereas the optical path length additionally takes into account the refractive index along the optical axis of the resonator. The enhancement of the optical path length can be characterized by a finesse of the optical resonator. As detailed below, the finesse is defined as the ratio of the free spectral range of the optical resonator and the full width at half maximum (FWHM) of the peaks in the transmission spectrum of the optical resonator.

[9] By mounting the sample in the sample holder in such a way that the optical axis of the resonator intersects with the sample, light confined in the resonator passes through the sam- ple each cycle, thereby enhancing effective optical path length within the sample, leading to a stronger interaction between the light and the sample. The first imaging system is configured to image the sample with a first imaging technique by collecting light with the first wave- length that is emitted from the resonator along an outgoing optical axis. Correspondingly, the second imaging system is configured to image the sample with the second imaging technique by collecting light with the second wavelength that is emitted from the resonator along an outgoing optical axis. The outgoing optical axes used by the first and second imaging system, respectively, may be different from each other if the optical resonator has multiple outgoing optical axes, e.g. if the optical resonator comprises multiple partially reflective surfaces.

[to] At the first wavelength, the optical resonator has the first finesse, which characterizes the average number of round trips in the optical resonator for photons with the first wave- length. The first finesse is adapted to the first imaging system in order to enhance the first image quality indicator, which quantifies a quality of an image of the sample taken with the first imaging system. Here, enhancing the first image quality factor refers to a situation, wherein the first image quality factor is higher if the optical resonator is in place than if the optical resonator is removed from the device, e.g. by making the corresponding reflective surfaces perfectly transparent. The finesse can be the determining factor for the image quality and in particular may have different optimal values for different imaging systems. In one ex- ample, a very large finesse may be advantageous, e.g. to increase the strength of a signal by increasing the number of round trips through the sample. In another example, a small finesse may lead to a better image quality, e.g. to avoid phase noise or a reduction in sharpness due to the overlapping of signals originating from parts of the light that have performed different numbers of round trips. In yet another example, an intermediate value of the finesse may provide the best image quality due to an enhanced effective optical path length through the sample while avoiding a deterioration of the image quality at larger finesse values e.g. due to the aforementioned effects.

[n] Correspondingly, the optical resonator exhibits the second finesse at the second wavelength, which in turn characterizes the average number of round trips in the optical res- onator for photons with the second wavelength. The second finesse is adapted to the second imaging system in order to enhance the second image quality indicator, which quantifies a quality of an image of the sample taken with the second imaging system. In particular, the second finesse may be different from the first finesse e.g. if different imaging techniques are used with different requirements with respect to the finesse.

[12] By choosing the finesse of the optical resonator appropriately for both the first and the second imaging system, the image quality of the two systems can be enhanced simultaneous- ly. This facilitates the implementation of different imaging techniques within the same de- vice, while taking advantage of the improvement in image quality that the optical resonator can offer due to the enhancement of the effective optical path length within the sample. Thereby, the amount of information that can be extracted from the imaging can be increased, in particular when combining the information from measurements taken with the two differ- ent imaging systems.

[13] The first image quality indicator and the second image quality indicator can for exam- ple be a signal strength, a signal-to-noise ratio, a contrast ratio, an edge contrast or a combi- nation thereof. The signal strength can be an absolute strength of a measured signal, e.g. a measured intensity, phase shift or polarization angle, or a relative strength of a measured signal, for example a ratio between measured intensities, e.g. for different polarizations, at different wavelengths and/or at different positions in the sample. Correspondingly, a signal- to-noise ratio can be defined as the ratio of the signal strength and a measure for the noise on the signal, e.g. a time-averaged, sample-averaged and/or measurement-averaged amplitude of fluctuations of the signal. For a spectroscopic imaging technique, a signal strength can for example be defined as the amplitude of a signal like an absorption or fluorescence peak. A signal-to-noise ratio for spectroscopic imaging techniques may e.g. be defined as the signal strength divided by an average noise on the signal at resonance, wherein the noise can e.g. be averaged over multiple measurements and/or over time. Similarly, for microscopic images a signal-to-noise ratio can e.g. be defined as the ratio of contributions to the image with long correlation lengths and contributions to the image with short correlation lengths, for example by defining a lower and an upper cut-off for spatial Fourier components of an image, wherein the lower and the upper cut-off are determined relative to a characteristic wave vector associ- ated with a characteristic length scale of features in the sample. In one example, the lower cut-off may be 20% to 50% of the characteristic wave vector, whereas the upper cut-off may 2 to 5 times the characteristic wave vector. A contrast ratio of an image can e.g. be defined as the ratio of the highest and lowest intensity in the image, wherein the intensity may be aver- aged over a region comprising for example 4 to 25 pixels, e.g. to minimize the influence of noise. An edge contrast may for example be defined as a local contrast ratio in a subregion of an image or a ratio of an intensity at two adjacent points.

[14] Enhancing the effective optical path length through the sample may be beneficial for a variety of imaging techniques, in particular microscopic imaging and spectroscopy, wherein microscopic imaging refers to spatially resolved measurements and spectroscopy refers to spectrally resolved measurements, which may or may not be spatially resolved. Each of the first and second imaging system can be configured to perform at least one of the following imaging techniques: absorption imaging, phase contrast imaging, fluorescence imaging, po- larization imaging, photoacoustic imaging, absorption spectroscopy, fluorescence spectros- copy or Raman spectroscopy. In absorption imaging, spatial variations in the absorption of light by the sample are measured, which may e.g. result from resonant absorption or off- resonant scattering. Phase contrast imaging relies on a measurement of a phase, in particular a spatial variation of a phase difference, that light acquires when passing through the sample. For fluorescence imaging and spectroscopy, the sample is excited by light at a wavelength that is different from the wavelength at which the measurement is performed, wherein the measurement can e.g. be a spatially resolved image of the fluorescence intensity or a meas- urement of a spatially averaged fluorescence intensity, each of which may be performed at a fixed wavelength or spectrally resolved. Polarization imaging techniques probe the change of the polarization of light passing through the sample, e.g. using polarization optics like polar- izers. In photoacoustic imaging, the sample is excited by an optical pulse, which induces fast heating of the sample, resulting in a jump in temperature and/or pressure. This can excite acoustic waves in the sample and can alter the refractive index of the sample, thereby chang- ing the optical length of the optical resonator. This change can e.g. be detected by a change in the transmission at a probe wavelength, i.e. the first or second wavelength, which may be identical to the wavelength of the excitation pulse. Absorption spectroscopy measures spec- tral variations in the absorption of light by the sample or a part of the sample, which may e.g. result from resonant absorption or off-resonant scattering. Raman spectroscopy are spectral- ly resolved measurements of inelastic scattering processes involving low-energy modes in the sample, e.g. rotational and vibrational degrees of freedom.

[15] In a preferred embodiment, the first imaging technique is different from the second imaging technique. In one example, the first imaging technique may be fluorescence spec- troscopy and the second imaging technique may be phase contrast imaging. In other embod- iments, the first and the second imaging techniques may be the same, but are performed at different wavelengths. For example, the first and second imaging techniques may both be absorption imaging, but the first wavelength is in the ultraviolet spectrum, e.g. 350 nm, whereas the second wavelength is in the near-infrared spectrum, e.g. 1064 nm.

[16] Preferably, the first finesse and the second finesse are chosen such that a combination of the first image quality indicator of the imaging with the first imaging system and the sec- ond image quality indicator of the imaging with the second imaging system is enhanced corn- pared to an optical resonator with the second finesse at the first wavelength and the first fi- nesse at the second wavelength. The combination of the first and second image quality indi- cators may e.g. be a sum or a weighted sum of the first and second image quality indicators. For example, the image quality of both the first and the second imaging system may be im- proved by increasing the finesse, but the image quality of the second imaging system may deteriorate if the second finesse exceeds a certain threshold. In this case, it may be advanta- geous to choose a larger value for the first finesse than for the second finesse. In another ex- ample, the image quality of both the first and the second imaging system may be improved by increasing the finesse, but the image quality of the second imaging system may improve more slowly. In this case, it may also be advantageous to increase the first finesse at the expense of the second finesse.

[17] The first finesse can for example be larger than 50, preferably larger than 100. A large finesse may in particular advantageous for spectroscopic imaging techniques, e.g. absorption or fluorescence spectroscopy, as the signal can be enhanced by a larger number of round trips of the light through the sample, while the overlapping of signals originating from parts of the light that have performed different numbers of round trips may have no detrimental effects, e.g. when measuring with a point-like detector like a photodiode.

[18] In one example, the second finesse may be smaller than 10, preferably smaller than 5. This may for example be advantageous for spatially resolved imaging, in particular phase contrast imaging, as the image quality may deteriorate for larger values of the finesse as a result of the overlapping of signals originating from parts of the light that have performed different numbers of round trips, which may e.g. reduce the edge contrast and introduce phase noise.

[19] In another example, the first finesse and the second finesse can be larger than 50, preferably larger than 100, and the first and second wavelengths can be separated by more than 50 nm, preferably more than 100 nm. Thereby, the imaging quality may e.g. be im- proved for spectroscopic measurements at two wavelengths with a large separation as the effective optical path length through the sample is enhanced strongly for both wavelengths simultaneously.

[20] The finesse of the optical resonator is determined by loss processes in the optical res- onator, which lead to a loss of light from the resonator, e.g. absorption and scattering pro- cesses. In particular, the reflectivity of the reflective surfaces forming the resonator can be of great relevance and may be chosen appropriately to achieve the desired finesse values. In one example, the optical resonator comprises a first and a second reflective surface. A reflectivity spectrum of the first and/or second reflective surface can be chosen such that the optical res- onator exhibits the first and second finesse at the first and second wavelength, respectively. For example, the first mirror may exhibit a reflectivity greater than 99.9% at the first and second wavelength. If the first finesse is to be large, e.g. larger than 100, the reflectivity of the second mirror at the first wavelength may e.g. be chosen to be greater than 98%. If the sec- ond finesse is to be small, e.g. less than 5, the reflectivity of the second mirror at the second wavelength may e.g. be chosen to be smaller than 40%. [21] In one example, the effective optical path length of the optical resonator at the first and/or second wavelength can be smaller than five times, preferably smaller than two times the depth of field of the first and/or second imaging system, respectively. Here, the depth of field of an imaging system is defined as the Rayleigh length of a Gaussian laser beam at the respective wavelength focused onto the sample, wherein the waist of the laser beam at the focus is chosen such that the waist of the laser beam at a first aperture of the imaging system as seen from the sample, in particular a first lens of the imaging system is equal to the radius of the first aperture, e.g. the radius of the first lens. The effective optical path length of the optical resonator in turn is defined as the product of the finesse of the optical resonator at the respective wavelength and the length of the optical resonator. To achieve such an effective optical path length, the length of the optical resonator has to be sufficiently small. In one ex- ample, the length of the optical resonator may be smaller than 100 pm, preferably smaller than 10 pm.

[22] The optical resonator can comprise at least one focusing element, wherein the at least one focusing element is configured to focus light propagating along the optical axis of the optical resonator such that a plane perpendicular to the optical axis of the optical resonator is imaged onto itself. For this, the at least one focusing element together with the reflective sur- faces and other optical elements within the optical resonator can form an imaging system such that the intensity distribution in this plane perpendicular to the optical axis is the same after an integer number of round trips - up to a constant factor arising from loss processes. The at least one focusing element can for example be any number of refractive or diffractive focusing elements such as any number of convex and/or concave lenses. The at least one fo- cusing element may e.g. be a pair of convex lenses, wherein the length of the optical resonator equals two times the sum of the focal lengths of the lenses. In other examples, at least one of the focusing elements may comprise at least one curved reflective surface, e.g. a curved mir- ror. The optical resonator may for example be a concentric, confocal, hemispherical or con- cave-convex two-mirror optical resonator. The optical resonator may in particular be config- ured to image a plane through the sample onto itself.

[23] The device can further comprise at least one light source for generating light at the first and second wavelengths. The at least one light source may be formed by one or more coherent and/or incoherent light sources. The device may for example comprise a single light source with a broadband spectrum, e.g. a white LED or a supercontinuum laser, which may e.g. be filtered to obtain light at the first and second wavelengths. In another example, the device may comprise two narrowband light sources, e.g. lasers or narrowband LEDs, one of which emits light at the first wavelength and the other one emits light at the second wave- length. The at least one light source may be a pulsed laser, in particular a pulsed laser with a pulse length that is smaller than a round trip time of the optical resonator.

[24] An outgoing optical axis of the optical resonator can be parallel to an input optical axis for the imaging with the first and/or second imaging system, wherein the input optical axis is an axis of propagation of light that is to be coupled into the optical resonator, e.g. from the at least one light source. If the input optical axis is parallel to the outgoing optical axis, the light coupled into the optical resonator can propagate along the optical axis of the resona- tor, e.g. to ensure that the light propagates along the same path in each round trip. In other examples, the outgoing optical axis of the optical resonator can be tilted relative to an optical axis for the imaging with the first and/or second imaging system, e.g. by an angle of less than 15°, preferably less than 5 ⁰ .

[25] In a preferred embodiment, the sample holder comprises a fluid chamber configured to hold a liquid medium. The fluid chamber can be configured to hold the liquid medium such that the optical axis of the optical resonator intersects with the liquid medium in the fluid chamber. In one example, the fluid chamber may be a cylinder with flat top and bottom surfaces that are oriented perpendicular to the optical axis of the resonator. The cylinder may e.g. have an interior diameter between 10 pm and 5 mm and an interior height between 10 pm and 2 mm. The fluid chamber may for example consist of glass or plastic. The fluid cham- ber may be transparent at the first and second wavelength and thus may comprise an antire- flection coating. Alternatively, the optical resonator may be formed by two opposing surfaces of the fluid chamber, in particular by two opposing interior surfaces of the fluid chamber, e.g. the inner top and bottom surfaces of a cylindrical fluid chamber. This may be advantageous to achieve a short length of the optical resonator. For this, the respective surfaces may be coated with a reflective coating, e.g. a metallic coating or a dielectric coating. To reduce the length of the optical resonator, the distance between the opposing surfaces, e.g. the inner top and bottom surfaces of a cylindrical fluid chamber, may be less than 100 pm, preferably less than 10 pm.

[26] The device can further comprise a pump system, wherein the pump system is config- ured to create a flow of a liquid medium through the fluid chamber. To this end, the fluid chamber may e.g. comprise an input port and an output port, which are connected with the pump system. The fluid chamber and the pump system may be configured to create a laminar flow of the liquid medium through the fluid chamber. In a preferred embodiment, the fluid chamber and the pump system are configured to create a sheath flow through the fluid chamber, e.g. for hydrodynamic focusing. A sheath flow can be created by a parallel laminar flow of the liquid medium and a sheath medium, wherein the viscosity of the sheath medium is adapted to the viscosity of the liquid medium such that the two media do not mix. The two media can flow with different velocities, e.g. the sheath medium may be faster than the liquid medium. In another example, a viscoelastic liquid medium may be used, e.g. for viscoelastic flow focusing.

[27] The device can also comprise a wavelength tuning apparatus for adjusting the first wavelength and/or the second wavelength. The wavelength tuning apparatus may for exam- ple be a tunable laser source or a tunable optical filter, e.g. a rotatable etalon filter. The wave- length tuning apparatus may comprise a feedback unit configured to stabilize the first and/or the second wavelength relative to a transmission peak of the optical resonator, e.g. via a Pound- Drever-Hall locking scheme.

[28] Furthermore, the device can comprise an adjusting mechanism for adjusting a reso- nance frequency of the optical resonator. Here, a resonance frequency is a frequency at which the transmission spectrum of the optical resonator exhibits a local maximum. The adjusting mechanism may for example comprise an actuator, e.g. a piezo actuator, or a translation stage for moving one or more of the reflective surfaces forming the resonator in order to change the length of the resonator. Alternatively or additionally, the adjusting mechanism may comprise a refractive element for changing an optical length of the resonator, e.g. by changing an index of refraction of the refractive element or a propagation length through the refractive element.

[29] The device can also comprise a third imaging system for imaging the sample with a third imaging technique, wherein the imaging is performed along an axis different from an outgoing optical axis of the optical resonator, in particular along an axis perpendicular to the optical axis of the optical resonator. The third imaging system can be configured such that it collects light from the resonator that does not leave through one of the reflective surfaces forming the resonator. For this, the third imaging system may e.g. be configured to image the sample under an angle relative to the optical axis of the optical resonator. The third imaging system may be configured to image at a third wavelength, which may be different from the first and second wavelengths, and may be configured to perform at least one of the following imaging techniques: absorption imaging, phase contrast imaging, fluorescence imaging, po- larization imaging, photoacoustic imaging, absorption spectroscopy, fluorescence spectros- copy or Raman spectroscopy.

[30] The device may also comprise a fourth imaging system for imaging the sample at a fourth wavelength with a fourth imaging technique, wherein the imaging is performed along an outgoing optical axis of the optical resonator. The fourth imaging system can be config- ured to perform at least one of the following imaging techniques: absorption imaging, phase contrast imaging, fluorescence imaging, polarization imaging, photoacoustic imaging, ab- sorption spectroscopy, fluorescence spectroscopy or Raman spectroscopy. A finesse of the optical resonator at the fourth wavelength is chosen such that a fourth image quality indica- tor of the imaging with the fourth imaging system does not decrease due to the presence of the optical resonator. The fourth imaging technique may be different from the first and sec- ond imaging techniques.

[31] The invention also provides a method for multicolor optical imaging of a sample with wavelength-dependent optical path length enhancement using a device according to any of the aforementioned embodiments. The method comprises (1) placing the sample in the opti- cal resonator, wherein the sample is placed such that the optical axis of the optical resonator intersects with the sample; (2) imaging the sample at the first wavelength with the first imag- ing technique, wherein the imaging is performed along an outgoing optical axis of the optical resonator; and (3) imaging the sample at the second wavelength with the second imaging technique, wherein the imaging is performed along an outgoing optical axis of the optical resonator and wherein the second wavelength is different from the first wavelength. The first finesse and the second finesse are chosen such that the optical resonator enhances the first image quality indicator of the imaging with the first imaging technique and the second image quality indicator of the imaging with the second imaging technique. The numbering of the steps above is for clarity only and does not indicate a certain order of execution. As far as technically feasible, the steps can be permuted and the method and any embodiment thereof can be performed in an arbitraiy order of these steps.

[32] Each of the first and second imaging technique can be any one of the following imag- ing techniques: absorption imaging, phase contrast imaging, fluorescence imaging, polariza- tion imaging, photoacoustic imaging, absorption spectroscopy, fluorescence spectroscopy or Raman spectroscopy. The first imaging technique can be different from the second imaging technique.

[33] In a preferred embodiment, the sample comprises a liquid medium containing imag- ing objects, in particular individual biological cells or substances, and placing the sample in the optical resonator comprises providing the liquid medium in the fluid chamber. The liquid medium can for example comprise an isotonic fluid or a cell-culture medium that is config- ured to support biological cells. The liquid medium may also comprise a blood sample, in particular a diluted blood sample. The biological substances can for example be biomolecules like proteins or nucleic acids. In particular, the liquid medium may be configured to not alter optical and/or chemical properties of the imaging objects. [34] Providing the liquid medium in the fluid chamber can comprise creating a flow of the liquid medium through the fluid chamber. In particular, the flow of the liquid medium may be created and/or maintained while performing the imaging with the first and/or second im- aging technique. Alternatively, the flow may be created to provide the liquid medium in the fluid chamber and may be interrupted while performing the imaging. The flow of the liquid medium through the fluid chamber may e.g. be a laminar flow. In one example, a sheath flow of the liquid medium and a sheath medium may be created in the fluid chamber, e.g. for hy- drodynamic focusing. In another example, the liquid medium may be a viscoelastic medium, e.g. for viscoelastic flow focusing.

[35] Placing the sample in the optical resonator can alter the optical properties of the reso- nator and in particular the finesse, the free spectral range and/or the optical length of the resonator, e.g. by scattering and/or absorption in the sample. Preferably, scattering and/or absorption by constituents of the sample other than imaging objects to be studied, e.g. the imaging object in the liquid medium, is minimized in order to not affect the intrinsic finesse of the optical resonator. For this, for example the liquid medium may for example be trans- parent and have a homogeneous index of refraction, in particular an index of refraction of less than 1.5. In one example, the first finesse of the optical resonator with the sample in place can be larger than 20, preferably larger than 50. Alternatively or additionally, the sec- ond finesse of the optical resonator with the sample in place can be smaller than 10, prefera- bly smaller than 5.

[36] In a preferred embodiment, the first and/or second wavelength is/are set to a corre- sponding peak of the transmission spectrum of the optical resonator. Thereby, the amount of light coupled into the optical resonator can be increased and be more robust against fluctua- tions in the first and/or second wavelength and/or in the transmission spectrum of the opti- cal resonator. To this end, the method may further comprise adjusting the first wavelength, the second wavelength and/or the length of the optical resonator to tune a transmission of the optical resonator at the first wavelength and/or a transmission of the optical resonator at the second wavelength.

[37] In one example, the first wavelength, the second wavelength and/or the length of the optical resonator may be adjusted to tune a transmission of the optical resonator at the first wavelength relative to a transmission of the optical resonator at the second wavelength. This may for example be used to adjust an intensity of the light used for the first and/or second imaging technique, e.g. to achieve similar intensities and/or signal strengths. In one example, the first wavelength may be set to a peak of the transmission spectrum of the optical resona- tor, whereas the second wavelength may be set to a point in the vicinity of a peak of the transmission spectrum of the optical resonator, at which the transmission is e.g. 50% of the peak transmission.

[38] The method can also comprise imaging the sample with a third imaging technique, wherein the imaging is performed along an axis different from the outgoing optical axes of the optical resonator, in particular along an axis perpendicular to the optical axis of the opti- cal resonator.

LIST OF FIGURES

[39] In the following, a detailed description of the invention and exemplary embodiments thereof is given with reference to the figures. The figures show schematic illustrations of

[40] Fig. 1: a device for multicolor optical imaging of a sample according to an exem- plary embodiment of the invention;

[41] Fig. 2: an example of a transmission spectrum of an optical resonator in a device in accordance with an embodiment of the invention;

[42] Fig. 3a: an optical resonator with a small effective optical path length according to an embodiment of the invention;

[43] Fig· 3b: an optical resonator with focusing elements in accordance with an em bodiment of the invention; and

[44] Fig. 4: a flow chart of a method for multicolor optical imaging of a sample accord- ing to an exemplary embodiment of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[45] Fig. 1 depicts a sectional view of a device 100 for multicolor optical imaging of a sam- ple 102 according to an exemplary embodiment of the invention. The device 100 comprises a sample holder 104 for mounting the sample 102, wherein the sample 102 may e.g. be placed on or mounted to a substrate like a microscopy slide or may be provided in a fluid chamber. The device 100 further comprises a resonator 106, which in this example is formed by two mirrors 108 and 110 with parallel opposing reflective surfaces. The mirrors 108, 110 may for example comprise a metallic or dielectric coating on the reflective surfaces. In other exam- ples, the reflective surfaces may be curved, e.g. convex or concave, more than two mirrors may be used, e.g. in a ring-type cavity, or the reflective surfaces may be integrated in the sample holder 104, e.g. opposing surfaces of a fluid chamber. The optical resonator 106 has an optical axis 112 forming a closed path in the resonator 106, along which light can propa- gate.

[46] The device 100 comprises a first imaging system 114, which is configured to image the sample 102 at a first wavelength with a first imaging technique. For this, light of the first wavelength 116 may be coupled into the resonator 106, e.g. through the mirror 108 along an incoming optical axis 118, which is aligned to an outgoing optical axis 120 of the optical reso- nator 106. The first imaging system 114 is configured to perform the imaging along an out- going optical axis 122 of the optical resonator 106, i.e. the outgoing optical axis 122 is parallel to an optical axis 124 of the first imaging system 114. In other examples, the optical axis 124 of the first imaging system 114 may be tilted by an angle, e.g. less than 5 ⁰ , from the outgoing optical axis 122, such that the first imaging system 114 captures at least some of the light emitted from the optical resonator 106 along the outgoing optical axis 122. In other embodi- ments, the same outgoing optical axis may be used for imaging and coupling of light into the resonator 106.

[47] The device 100 further comprises a second imaging system 126, which is configured to image the sample 102 at a second wavelength with a second imaging technique. For this, light of the second wavelength 128 maybe coupled into the resonator 106, e.g. through the mirror 108. The second imaging system 126 is configured to perform the imaging along an outgoing optical axis, e.g. the outgoing optical axis 122 of the optical resonator 106, i.e. the outgoing optical axis 122 is parallel to or tilted by a small angle, e.g. less than 5 ⁰ , relative to an optical axis of the second imaging system 126.

[48] The first 114 and second imaging system 126 may be configured to perform different imaging techniques. In the example shown in Fig. 1, the first imaging system 114 is config- ured to perform microscopy, e.g. absorption or fluorescence imaging, with a spatially resolv- ing detector 130, e.g. a CCD or CMOS camera. For this, the first imaging system 114 can corn- prise additional optical elements, e.g. a lens 132. The second imaging system 126 is config- ured to perform spectroscopic measurements with a point-like detector, e.g. a photodiode 134 configured to measure an average light intensity. The first imaging system 126 can also corn- prise additional optical elements, e.g. a lens 136, and can furthermore share optical elements with the first imaging system, e.g. an objective 138. To separate the light of the first wave- length and light of the second wavelength, a wavelength-specific optical element may be used, e.g. a dichroic mirror 140. In other examples, a prism or diffraction grating may be used for this. [49] Fig· ² shows an example of a transmission spectrum T(v) (200) as a function of the frequency v of the light for an optical resonator of a device in accordance with an embodi- ment of the invention, e.g. the optical resonator 106. Destructive interference between partial waves undergoing different numbers of round trips strongly suppresses transmission through the resonator 106 for most frequencies such that all incoming light is reflected off the resona- tor 106. If a resonance condition for constructive interference is fulfilled, e.g. if the length of the resonator 106, i.e. the length of a round trip along the optical axis 112, is an integer multi- ple of l/2, wherein l denotes the wavelength of the light, the partial waves interfere construc- tively and the optical resonator 106 becomes transmissive, i.e. transmits a large fraction of the light. This gives rise to a periodic succession of transmission peaks in the transmission spectrum 200, wherein the spacing between neighboring peaks is determined by a free spec- tral range 202 of the resonator 106. The free spectral range 202 correspondingly depends on the length of the resonator and is defined as the inverse of the round trip time of a photon in the resonator. Each of the transmission peaks has a width 204, which in turn is related to the average time a photon spends in the resonator 106 via the Fourier transform. Thus, the fi- nesse of the resonator 106, which is defined as the ratio of the free spectral range 202 and the peak width 204, e.g. the full width at half maximum, characterizes an average number of round trips that a photon performs before leaving the resonator 106.

[50] The finesse of the resonator 106 can be set to different values for the first and second wavelength. For example, the finesse may be larger at the second wavelength than at the first wavelength, i.e. the width 204 of the transmission peaks at the first wavelength compared to the free spectral range 202 is broader than the width 208 at the second wavelength compared to the free spectral range 206. In general, the free spectral range 206 is similar to the free spectral range 202 unless the resonator contains strongly dispersive elements.

[51] Fig. 3a depicts an optical resonator 106 with a small effective optical path length in a device 300 according to an embodiment of the invention. The device 300 may be similar to the device 100 shown in Fig. 1, from which the device 300 mainly differs in the design of the optical resonator 106. Correspondingly, the device 300 may comprise other elements in addi- tion to the components shown in Fig. 3a, e.g. the first imaging system 114 and the second imaging system 126. The sample 102 is placed in the resonator 106. When imaging the sam- ple 102, e.g. through the objective 138, the repeated cycling of light in the resonator 106 through the sample 102 can be interpreted as creating ghost images 302 of the sample since in each cycle a small fraction of the light leaves the resonator and creates an image of the sample e.g. on the camera 130 (not shown in Fig. 3a). Due to diffraction of the light, the ghost images 302 may not be identical such that overlapping of the ghost images 302 on the cam- era 130 can reduce the sharpness of the image. This reduction can depend on an effective separation of the ghost images, which is determined by the effective optical path length 304 of the resonator and thus its finesse and length, and the depth of field 306 of the respective imaging system. Here, the depth of field 306 of the imaging system is defined as the Rayleigh length of a Gaussian laser beam 308 at the respective wavelength focused onto the sample 106, wherein the waist of the laser beam 308 at the focus is chosen such that the waist of the laser beam 308 at a first aperture of the imaging system as seen from the sample 102, in this case the first lens of the objective 138, is equal to the radius of the first aperture, e.g. the radi- us of the first lens. To enhance the image quality, the effective optical path length 304 may be chosen to be comparable to the depth of field 306, e.g. less than a factor of 2 larger than the depth of field 306. This can in particular be achieved by reducing the size of the optical reso- nator 106, such that the effective optical path length 304 remains small even if the finesse of the optical resonator 106 at the respective wavelength is large.

[52] Fig. 3b depicts an optical resonator 106 with focusing elements in a device 310 in ac- cordance with an embodiment of the invention. The device 310 may be similar to the device 100 shown in Fig. 1, from which the device 310 mainly differs in the design of the optical res- onator 106. Correspondingly, the device 310 may comprise other elements in addition to the components shown in Fig. 3b, e.g. the first imaging system 114 and the second imaging sys- tem 126. Inside the optical resonator 106, two focusing lenses 312 and 314 are placed. The length of the resonator 106 is chosen such that it equals two times the sum of the focal lengths of the lenses 312 and 314, e.g. 4/ if/ denotes the focal length of both lenses. In the example shown in Fig. 3b, the lenses 312 and 314 are placed such that a point on a surface of the left mirror 108 is imaged onto a point on a surface of the right mirror 110. Thus, after a complete round trip, an intensity pattern on the surface is imaged onto itself. This may allow for compensating a divergence of light propagating along the optical axis 112 of the resonator 106. If the sample 102 is positioned close to the surface of the mirror 108, e.g. at a distance smaller than 10%, preferably smaller than 5% of the focal length of the lens 312, a plane through the sample 102 can hence be imaged onto itself after one round trip as a virtual im- age 316 of the sample 102 is created close to the surface of the mirror 110, which is then im- aged onto the sample 102 again after the light is reflected back by the mirror no. In other embodiments, the mirrors 108 and no may be focusing elements and may have curved sur- faces to focus the light. Furthermore, a different number of focusing element, e.g. lenses, may be placed inside the resonator 106.

[53] In Fig. 4, a flow chart of a method 400 for multicolor optical imaging of a sample ac- cording to an exemplary embodiment of the invention is shown, which can e.g. be imple- mented with the device 100 and is described in the following with reference to Fig. 1. The method 400 can, however, also be implemented with other devices in accordance with an embodiment of the invention, e.g. the devices 300 and 310.

[54] In step 402, the sample 102 is placed in the sample holder 104, e.g. by creating a flow of a liquid medium containing imaging objects through a fluid chamber of the sample holder 104. In step 404, which is preferably conducted after step 402 when the sample is already in place, the transmission through the optical resonator 106 can be tuned. In one example, the transmission spectrum 200 is measured at least in part and the frequencies V and v ₂ corre- sponding to the first and second wavelength, respectively, are set to desired points in the transmission spectrum 200, e.g. by tuning the first and/or second wavelength to shift the frequencies V and v ₂ or by changing the transmission spectrum 200 by adjusting a resonance frequency of the optical resonator 106, e.g. by changing its length. The frequencies V and v ₂ may for example both be set to different peaks in the transmission spectrum 200. In another example, v ₂ may be set to a peak in the transmission spectrum 200, whereas V may be set to a wing of another peak in the transmission spectrum 200, e.g. at a point at which the trans- mission is 1/3 of the transmission at the corresponding peak. In a third example, V may be set to the high frequency wing of a peak in the transmission spectrum 200, i.e. to the right of the peak, whereas v ₂ may be set to the low frequency wing of another peak in the transmis- sion spectrum 200, i.e. to the left of the respective peak. This may e.g. be used to distinguish between a change in absorption by the sample 102 and a change of the optical path length of the resonator 106 created by the sample 102 since a change in absorption leads to simultane- ous increase or decrease in transmission of both frequencies, whereas a change in the optical path length leads to a shift of the peaks in the transmission spectrum 200, resulting in an asymmetric change of the transmission at both frequencies.

[55] Subsequently, in steps 406 and 408, the sample is imaged with the first imaging tech- nique at the first wavelength and the second imaging technique at the second wavelength, e.g. by coupling the light of the first 116 and second wavelength 128 into the resonator 106 and measuring light transmitted through the resonator 106 via the first and second imaging sys- tems 114 and 126, e.g. on the detectors 130 and 134. If a spectroscopic measurement is per- formed in one or both of the steps 406 and 408, this may involve tuning the first and/or sec- ond wavelength during the measurements, e.g. setting the respective wavelength to different peaks in the transmission spectrum 200. Steps 406 and 408 may be performed simultane- ously or sequentially. In some embodiments, the transmission through the resonator 106 at the first and/or second wavelength may further be stabilized during steps 406 and 408, e.g. to keep an intensity at the sample or on a detector constant. [56] The embodiments of the present invention disclosed herein only constitute specific examples for illustration purposes. The present invention can be implemented in various ways and with many modifications without altering the underlying basic properties. There- fore, the present invention is only defined by the claims as stated below.

LIST OF REFERENCE SIGNS

100 - Device for multicolor optical imaging

102 - Sample

104 - Sample holder

106 - Optical resonator

108 - Mirror

no - Mirror

112 - Optical axis of the optical resonator

114 - First imaging system

116 - Light at the first wavelength

118 - Incoming optical axis

120 - Outgoing optical axis of the optical resonator

122 - Outgoing optical axis of the optical resonator

124 - Optical axis of the first imaging system

126 - Second imaging system

128 - Light at the second wavelength

130 - Spatially resolving detector

132 - Lens

134 - Photodiode

136 - Lens

138 - Objective

140 - Dichroic mirror

200 - Transmission spectrum of an optical resonator

202 - Free spectral range at the first wavelength

204 - Width of a transmission peak at the first wavelength

206 - Free spectral range at the second wavelength

208 - Width of a transmission peak at the second wavelength

300 - Device for multicolor optical imaging

302 - Ghost images of the sample

304 - Effective optical path length

306 - Depth of field

308 - Gaussian laser beam

310 - Device for multicolor optical imaging

312 - Lens

314 - Lens 316 - Virtual image of the sample

400 - Method for multicolor optical imaging

402 - Step of placing the sample in the optical resonator

404 - Step of tuning the transmission through the optical resonator

406 - Step of imaging the sample at first wavelength with first imaging technique 408 - Step of imaging the sample at second wavelength with second imaging technique