Patent Description:
Micropipette aspiration is a technique for measuring mechanical properties of a sample, in particular a biological sample, such as, for example, a cell. In this technique, the sample contacts a tip of a micropipette, which may have a radius comprised between, for example, <NUM> micron and <NUM>. A suction pressure inside the micropipette causes a portion of the sample to be drawn into the micropipette. As a result, the sample deforms to a degree that varies as a function of the suction pressure. A mechanical property of the sample, such as, for example, an elastic modulus, can be determined based on a measured stimulus-response relationship between a change in the suction pressure and a resulting displacement of the portion of the sample that has been aspired in the micropipette.

A microscope camera can be used to track the displacement of the portion of the sample that has been aspired in the micropipette as well as, more generally, deformation of the sample. Image analysis then allows quantifying this displacement and thus measuring the stimulus-response relationship with the change in suction pressure.

For example, the article entitled "<NPL> describes that in micropipette-aspiration experiments, a suction pressure is applied by connecting the micropipette (microcapillary) to an adjustable water reservoir or a pump. Cell changes are determined microscopically by means of image analysis. The suction pressure is given by the height difference between the tip of the micropipette and the top of the reservoir h and the specific weight of water. If there is a flow, the pressure drop along the microcapillary must be taken into account.

However, this image-based tracking of sample deformation presents several drawbacks. First of all, the image analysis that is required may be computationally demanding and, therefore, relatively slow. This may make that a significant amount of time is required to conduct a single experiment. Moreover, the image analysis may require relatively costly hardware and software.

Another drawback of image-based tracking is that relatively small deformations, and thus relatively small displacements, may be difficult to quantify. Deformations may be considered as being relatively small if, for example, a ratio of the displacement into the micropipette with respect to the radius of the tip of the micropipette is less than <NUM>. Quantifying these relatively small deformations may be of interest, for example, in studying the role of different internal components of a cytoskeleton on cellular mechanical properties. However, in practice, microscope cameras generally have a pixel size that is too large to capture these relatively small deformations with sufficient precision. That is, image resolution may be insufficient. What complicates matters is that subpixel detection algorithms, which may potentially increase resolution, require negligible drift and a precise parallelism between a plate supporting the sample and the micropipette to avoid projection errors.

The article by<NPL>, describes a fiber-optic pipette that couples a glass capillary, a common syringe, and a single optical fiber to provide for a facile means of achieving long-pathlength capillary spectroscopy. The fiber-optic pipette allows for the acquisition of rapid spectroscopic measurements of small-volume liquid samples while simultaneously achieving signal enhancements of the collected spectroscopic signal.

The article by <NPL>, describes an investigation of different optical configurations of a low-finesse Fabry-Perot interferometer used for displacement sensing. The different configurations of the Fabry-Perot cavity are selected in order to achieve large measurement ranges and angular alignment tolerances and to make the interferometer applicable for targets of various reflectivity ranges. Use of a confocal arrangement enables a measurement range of up to about <NUM>, or to work with an angular tolerance of more than ± <NUM>°.

The article by <NPL>, recapitulates experimental variations of micropipette aspiration. Analysis models focusing on important limitations of widely used biomechanical models are discussed.

<CIT> discloses a Fabry-Perot interferometer for measuring changes of physical quantities.

There is a need for a technique for measuring deformation of a sample, in response to a change in an environmental condition, that allows an improvement in at least one of the following aspects: resolution, speed, ease-of-use and cost.

An aspect of the invention as defined in claim <NUM> provides for a method of optically measuring a response of a sample to a change in an environmental condition to which the sample is exposed, wherein use is made of an optical sensing device comprising:.

Yet a further aspect of the invention as defined in claim <NUM> provides for an optical sensing device comprising:.

Yet a further aspect of the invention as defined in claim <NUM> provides for an optical measurement system comprising:.

In each of these aspects, the invention allows a significantly higher resolution with which deformation can be measured compared with the image-based tracking described hereinbefore. Namely, a variation in optical path length in the optical interferometric cavity can be detected with a resolution that may be several orders of magnitude higher than the resolution achievable with image-based tracking. Moreover, the change in the environmental condition, which causes the deformation of the sample, may also be detected by means of another interferometric cavity designed for that purpose. This then allows precise time matching between variations in the environmental conditions and variations in the deformation in response thereto. This contributes to measurement precision and accuracy. Moreover, it allows real-time monitoring of a stimulus-response relationship between these variations.

A further advantage of the invention is that measurements can be carried out in a simpler manner, more easily, and thus at lower cost. This is because the sample need not necessarily be precisely positioned with respect to a microscope camera, or another type of tracking device. In fact, the sample need not require a specific fixation or preparation; the sample may even be free-floating, as it were. In contrast, in the image-based tracking described hereinbefore, the sample should be perpendicular to a virtual line extending between a lens center of the microscope camera and a support holding the sample. In practice, this requirement also puts constraints on a measurement setup: the micropipette should be oriented horizontally with respect to the sample. These constraints may complicate making measurements, which may be relatively time consuming, adversely affecting throughput of measurements to be carried out.

What further contributes to lower cost and ease of use is that the invention provides measurement results that can be represented by a relatively small amount of data. The measurement results may be, for example, in the form of a table specifying respective optical path lengths measured at respective instants. A relatively small data file may comprise such a measurement table. In contrast, the image-based tracking described hereinbefore, requires storing and handling relatively large image files. Moreover, real-time monitoring of the stimulus-response relationship as mentioned hereinbefore, requires hardware and software capable of processing these image files sufficiently fast.

For the purpose of illustration, some embodiments of the invention are described in detail with reference to accompanying drawings. In this description, additional features will be presented, some of which are defined in the dependent claims, and advantages will be apparent.

<FIG> schematically illustrates an optical measurement system <NUM> that can measure deformation of a sample <NUM> in response to a change in pressure. <FIG> provides a schematic block diagram of the optical measurement system <NUM> with the sample <NUM> to be analyzed. The sample <NUM> may be biological, such as, for example, a cell, biomimetic, or synthetic. By measuring the deformation of the sample <NUM> in response to a change in pressure, information about mechanical properties of the sample <NUM> can be obtained. This information may concern, for example, an elastic modulus of the sample <NUM>.

The optical measurement system <NUM> comprises an optical interrogator <NUM>, an optical sensing device <NUM>, and a syringe pump <NUM>. In this embodiment, the optical interrogator <NUM> comprises a light source <NUM>, a depolarizer <NUM>, a circulator <NUM>, and a spectrometer-based analyzer <NUM>. A polarization-maintaining optical fiber <NUM> may optically couple the light source <NUM> to the depolarizer <NUM>. A single mode optical fiber <NUM> may couple the depolarizer <NUM> to the circulator <NUM>. Another single mode fiber <NUM> may couple the spectrometer-based analyzer <NUM> to the circulator <NUM>.

The light source <NUM> may be in the form of, for example, a super-luminescent diode. In an experimental embodiment, the super-luminescent diode had a center wavelength of <NUM>, a full-width-at-half maximum bandwidth of <NUM>, and provided <NUM> mW light power. The spectrometer-based analyzer <NUM> may comprise a spectrometer and a spectral analysis arrangement as described in patent publication <CIT>, which is also in the name of the present applicant. In the experimental embodiment, the spectrometer had a wavelength range of <NUM> to <NUM>, and an optical resolution of <NUM> pm.

The optical sensing device <NUM> may be in the form of a micropipette and will be referred to hereinafter as micropipette <NUM> for the sake of convenience and illustration. The micropipette <NUM> has a tip <NUM> with an aperture <NUM>, which will be referred to as tip aperture <NUM> hereinafter. The sample <NUM> to be analyzed is in contact with the tip <NUM> and, more precisely, with the tip aperture <NUM>. The micropipette <NUM> may comprise a hollow interior that is filled with a fluid, such as, for example, water. The hollow interior may extend to tip aperture <NUM>. Accordingly, the sample <NUM> may be in contact with the fluid.

An optical fiber <NUM> is concentrically arranged in the micropipette <NUM> and extends into the tip <NUM> thereof. A free end <NUM> of the optical fiber <NUM> may be aligned with the tip aperture <NUM>. To that end, the micropipette <NUM> may comprise a support for the optical fiber <NUM> so as to prevent the free end <NUM> thereof from significantly bending. The optical fiber in the micropipette <NUM> will be referred to hereinafter as sample measurement fiber <NUM> for the sake of convenience. The sample measurement fiber <NUM> may be an extension of an optical fiber <NUM> that provides, at least partially, optical coupling with the circulator <NUM>.

The micropipette <NUM> further comprises an optical pressure sensor <NUM>. A separate optical fiber <NUM> optically couples the optical pressure sensor <NUM> to the circulator <NUM>. The micropipette <NUM> is fluidically coupled to the syringe pump <NUM>. For that purpose, a conduit <NUM> of relatively small diameter may extend from the micropipette <NUM> to the syringe pump <NUM>.

<FIG> schematically illustrates an embodiment of the micropipette <NUM>. <FIG> provides a schematic perspective cross-sectional diagram of this embodiment, which will be referred to hereinafter as the micropipette <NUM> for reasons of convenience. The micropipette <NUM> comprises the sample measurement fiber <NUM> with its free end <NUM> and the tip <NUM> with the tip aperture <NUM> mentioned hereinbefore with reference to <FIG>. The optical pressure sensor <NUM> is not represented in <FIG> for the sake of simplicity and convenience.

The micropipette <NUM> further comprises a housing <NUM>, a ferrule <NUM> , a mating sleeve <NUM>, a mounting gasket <NUM>, two sealing gaskets <NUM>, <NUM>, and a tubing member <NUM>. The housing <NUM> may be made of, for example, resin material. The ferrule <NUM> and the mating sleeve <NUM> may be made of, for example, ceramic material. The tip <NUM> may be made of, for example, glass. Specifically, the tip <NUM> of the micropipette <NUM> may be a glass suction capillary.

The housing <NUM> has a center opening, which is relatively small, and two lateral openings. The sample measurement fiber <NUM> passes through the relatively small center opening of the housing <NUM>. The sample measurement fiber <NUM> may be secured to the housing <NUM> by means of, for example, glue at the center opening. The tubing member <NUM>, which is exterior of the housing <NUM> at the center opening thereof, envelopes the sample measurement fiber <NUM>. The tubing member <NUM> may provide strain relief for the sample measurement fiber <NUM> and sealing additional to that of the glue.

The housing <NUM> has a hollow interior that is shaped to accurately position the ferrule <NUM> and the mating sleeve <NUM> within the housing <NUM>. The sample measurement fiber <NUM> concentrically passes through the ferrule <NUM> and the mating sleeve <NUM>. The ferrule <NUM> constitutes a support for the sample measurement fiber <NUM>. A portion of the mating sleeve <NUM> overlaps a portion of the ferrule <NUM> as illustrated in <FIG>. Another, complementary portion of the mating sleeve <NUM> overlaps a portion of the tip <NUM> of the micropipette <NUM>. The tip <NUM> of the micropipette <NUM> abuts against a chamfered end of the ferrule <NUM>. The mounting gasket <NUM> secures the tip <NUM> to the housing <NUM>.

The tip <NUM> of the micropipette <NUM> may have a largest outer diameter that is slightly less than an inner diameter of the mating sleeve <NUM>. For example, the largest outer diameter of the tip <NUM> of the micropipette <NUM> may be <NUM> whereas the inner diameter of the mating sleeve <NUM> may be <NUM>. In such a case, there is a cylindrical clearance gap between the mating sleeve <NUM> and the tip <NUM>. The portion of the tip <NUM> that is enveloped by the mating sleeve <NUM> may then be at least partially provided with a bridging sleeve. The bridging sleeve fills the cylindrical clearance. The bridging sleeve may comprise elastic material such as, for example, silicone. This contributes to a satisfactory co-centricity between the sample measurement fiber <NUM> in the micropipette <NUM> and the tip <NUM> of the micropipette <NUM>.

An end portion <NUM> of the sample measurement fiber <NUM> pokes out of the ferule This end portion <NUM> may thus bend to a certain extent. Such bending adversely affects concentricity and may thus cause misalignment of the free end <NUM> of the sample measurement fiber <NUM> with respect to the tip aperture <NUM>. The micropipette <NUM> illustrated in <FIG> has a structure that allows the tip <NUM> to be relatively short. The end portion <NUM> of the sample measurement fiber <NUM> that pokes out of the ferrule <NUM> may thus also be relatively short. This contributes to satisfactory co-centricity between the end portion <NUM> of the sample measurement fiber <NUM> and the tip aperture <NUM> and counters misalignment. In an experimental embodiment, bending deformation was found to be smaller than <NUM> for a fiber curl radius of <NUM>.

The ferrule <NUM> comprises longitudinal slits on an outer surface. These slits are not represented in <FIG> for the sake of simplicity. The slits provide adequate fluid communication between the tip <NUM> of the micropipette <NUM> and a back section of its hollow interior where the two lateral openings are located. The slits may be approximately <NUM> deep, for example. The slits may be engraved by means of, for example, a diamond wire cutter.

A lateral opening of the housing <NUM> may receive the conduit <NUM> illustrated in <FIG>, which fluidically couples the micropipette <NUM> to the syringe pump <NUM>. The conduit <NUM> may be held by a sealing gasket <NUM> arranged in this opening. Another lateral opening of the housing <NUM> may receive the optical pressure sensor <NUM> illustrated in <FIG>. A sealing gasket <NUM> arranged in this lateral opening may serve as a sleeve in which the optical pressure sensor <NUM> may be inserted.

The free end <NUM> of the sample measurement fiber <NUM>, which faces the tip aperture <NUM>, may comprise an optical lens. The optical lens may be formed by a graduated refractive index at the free end <NUM> of the sample measurement fiber <NUM>. The optical lens may focus a light beam emanating from the sample measurement fiber <NUM> on the tip aperture <NUM> and thus on a portion of a sample that is within the tip aperture <NUM>. The light beam may have a profile having a narrow portion, which is commonly referred to as beam waist, that is proximate to the tip aperture <NUM>. This contributes to achieving a satisfactory signal-to-noise ratio with the optical interrogator <NUM> illustrated in <FIG>. In practice, a compromise may need to be made between achieving a beam waist with a relatively small diameter, on the one hand, and locating the beam waist sufficiently close to the tip aperture <NUM>, on the other hand.

The sample measurement fiber <NUM> may be an assembly of various optical fiber sections that have been joined to each other by splicing. For example, in an experimental embodiment, the sample measurement fiber <NUM> comprised three sections that were spliced together: a back fiber section, which is furthest away from the tip aperture <NUM>, a middle fiber section, and a front fiber section, which comprises the free end <NUM> that faces the tip aperture <NUM>. The back fiber may be formed by a <NUM>/<NUM> single mode optical fiber. This optical fiber may poke out from the micropipette <NUM> and be provided with a connector that allows coupling the optical fiber to the optical interrogator <NUM> illustrated in <FIG>. That is, the back section may be an extension of the optical fiber <NUM> illustrated in <FIG>, which optically couples the micropipette <NUM> with the optical interrogator <NUM>. The middle section may be formed by a <NUM> long coreless fiber of that also has a diameter of <NUM>. The front section may be formed by a <NUM>/<NUM>, <NUM>-µm long GRIN multimode fiber, GRIN being an acronym for gradient index. The sample measurement fiber <NUM> thus formed provided <NUM> beam waist at <NUM> in water.

The micropipette <NUM> presented hereinbefore with reference to <FIG> has various advantageous properties. First of all, the micropipette <NUM> allows sufficiently good concentricity between the sample measurement fiber <NUM> therein and the tip <NUM>, which also acts as a waveguide for light that enters the micropipette <NUM> through the sample measurement fiber <NUM>. This allows efficient transfer of this entering light to the sample <NUM> that is in contact with the tip aperture <NUM> as illustrated in <FIG>. The efficient transfer of the entering light contributes to achieving a satisfactory signal-to-noise ratio and thus contributes to achieving sufficiently accurate and precise measurement results, which may be obtained in a manner that will be described hereinafter. In addition, the micropipette <NUM> is sufficiently mechanically stable for obtaining consistent measurement results, allowing reliable comparison of these obtained with, for example, various samples. Moreover, the micropipette <NUM> allows a sufficiently efficient transfer of fluid pressure from the back section of its hollow interior, which is fluidly coupled to the syringe pump <NUM>, to the tip <NUM> of the micropipette <NUM>.

<FIG> schematically illustrates an embodiment of the optical pressure sensor <NUM>. <FIG> provides a very schematic cross-sectional view of this embodiment, which will be referred to hereinafter as the optical pressure sensor <NUM> for reasons of convenience. The optical pressure sensor <NUM> comprises a cap-like element <NUM> that is fitted on an end portion of an optical fiber <NUM>. The cap-like element <NUM> may be, for example, a monolithic semiconductor device described in patent publication <CIT>, which is also in the name of the present applicant. The optical fiber <NUM> on which the cap-like element <NUM> is fitted may be the optical fiber <NUM> that optically couples the optical pressure sensor <NUM> to the circulator <NUM>; as illustrated in <FIG>.

The cap-like element <NUM> comprises a light reflecting membrane <NUM> that faces an end <NUM> of the optical fiber <NUM>. There is a refractive index discontinuity at the end <NUM> of the optical fiber <NUM>. The light reflecting membrane <NUM> and the refractive index discontinuity at the end <NUM> of the optical fiber <NUM> jointly define a Fabry-Perot cavity <NUM>. This Fabry-Perot cavity will be referred to hereinafter as the pressure-sensing Fabry-Perot cavity <NUM> for the sake of convenience. The pressure-sensing Fabry-Perot cavity <NUM> has an optical path length "a" that varies as a function of pressure exerted by the fluid in the micropipette <NUM> on the light reflecting membrane <NUM>, which may bend inwardly and outwardly. This pressure is indicated by the reference "P" in <FIG>. The optical path length "a" is thus indicative of the fluid pressure in the micropipette <NUM>. Variations in the optical path length "a" represent pressure variations in the fluid in the micropipette <NUM>.

<FIG> schematically illustrates in greater detail the sample <NUM> in contact with the tip <NUM> of the micropipette <NUM>. <FIG> provides a very schematic cross-sectional diagram of the sample <NUM> in contact with the tip <NUM> of the micropipette <NUM>. In this illustration, the sample <NUM> has undergone a deformation: a portion of the sample <NUM> has been drawn into the tip <NUM> of the micropipette <NUM> through the tip aperture <NUM>. This portion of the sample <NUM> will be referred to hereinafter as the aspirated portion for the sake of convenience. The free end <NUM> of the sample measurement fiber <NUM>, which is concentrically arranged in the micropipette <NUM>, is near to and faces the tip aperture <NUM>. Consequently, the free end <NUM> of the sample measurement fiber <NUM> also faces the aspirated portion of the sample <NUM>.

There are several refractive index discontinuities when the sample <NUM> is in contact with tip <NUM> of the micropipette <NUM> as illustrated in <FIG>. There is a refractive index discontinuity at the free end <NUM> of the sample measurement fiber <NUM>. There is a further refractive index discontinuity at a front surface <NUM> on the aspirated portion of the sample <NUM>. There is yet another refractive index discontinuity at a back surface <NUM> of the sample <NUM>, which is opposite to the front surface <NUM> on the aspirated portion of the sample <NUM>.

The aforementioned refractive index discontinuities define several Fabry-Perot cavities. The refractive index discontinuity at the front surface <NUM> on the aspirated portion of the sample <NUM>, on the one hand, and the refractive index discontinuity at the back surface <NUM> of the sample <NUM>, on the other hand, jointly define a Fabry-Perrot cavity <NUM>. This Fabry-Perot cavity will hereinafter be referred to as the cross sample Fabry-Perot cavity <NUM> hereinafter for the sake of convenience. The cross sample Fabry-Perot cavity <NUM> has an optical path length "b" that is indicative of a spacing between the front surface <NUM> on the aspirated portion of the sample <NUM> and the back surface <NUM> of the sample <NUM>. This spacing will be referred to hereinafter as the cross sample spacing for the sake of convenience. The optical path length "b" corresponds with the cross sample spacing times an effective refractive index of substances in the sample <NUM> along a path between the front surface <NUM> on the aspirated portion and the back surface <NUM>. Variations in the optical path length "b" thus represent variations in the cross sample spacing.

The refractive index discontinuity at the free end <NUM> of the sample measurement fiber <NUM>, on the one hand, and the refractive index discontinuity at the front surface <NUM> on the aspirated portion of the sample <NUM>, on the other hand, jointly define another Fabry-Perrot cavity <NUM>. This Fabry-Perot cavity will hereinafter be referred to as the aspiration Fabry-Perot cavity <NUM> hereinafter for the sake of convenience. The aspiration Fabry-Perot cavity <NUM> has an optical path length "c" that is indicative of a spacing between the free end <NUM> of the sample measurement fiber <NUM> and the front surface <NUM> on the aspirated portion of the sample <NUM>. This spacing will be referred to hereinafter as the aspiration spacing for the sake of convenience. The optical path length "c" corresponds with the aspiration spacing times a refractive index of the fluid in the tip <NUM>. Variations in the optical path length "c" thus represent variations in the aspiration spacing.

The refractive index discontinuity at the free end <NUM> of the sample measurement fiber <NUM>, on the one hand, and the refractive index discontinuity at the back surface <NUM> of the sample <NUM>, on the other hand, jointly define yet another Fabry-Perrot cavity <NUM>. This Fabry-Perot cavity will hereinafter be referred to as the sample back Fabry-Perot cavity <NUM> hereinafter for the sake of convenience. The sample back Fabry-Perot cavity <NUM> has an optical path length "d" that is indicative of a spacing between the free end <NUM> of the sample measurement fiber <NUM> and the back surface <NUM> of the sample <NUM>. This spacing will be referred to hereinafter as the sample back spacing for the sake of convenience. The optical path length "d" corresponds with the sample back spacing times an effective refractive index of the aforementioned substances in the sample <NUM> and the fluid in the tip <NUM>, which are present along a path between the free end <NUM> of the sample measurement fiber <NUM> and the back surface <NUM> of the sample <NUM>. Variations in the optical path length "d" thus represent variations in the sample back spacing.

It should be noted that <FIG> is a very schematic representation that is not drawn to scale. Proportions between the aforementioned optical path lengths "b", "c", and "d" do not necessarily correspond with what <FIG> suggests. For example, the optical path length "b" of the cross sample Fabry-Perot cavity <NUM> may be longer than the optical path length "c" the aspiration Fabry-Perot cavity <NUM> although <FIG> suggests otherwise.

The optical measurement system <NUM> illustrated in <FIG> basically operates as follows. The syringe pump <NUM> makes that the fluid in the micropipette <NUM> has an under-pressure: a pressure that is lower than that exerted on a major part of the sample <NUM>, which is not within the tip aperture <NUM> and thus not in contact with the fluid in the micropipette <NUM>. As a result, a portion of the sample <NUM> that is within the tip aperture <NUM>, and thus in contact with the fluid, is drawn into the tip <NUM> of the micropipette <NUM> as illustrated in <FIG>. The portion of the sample <NUM> that is drawn into the tip <NUM> will be referred to hereinafter as the aspired portion for the safe of convenience. The aspired portion is thus a deformation of the sample <NUM> in response to the under-pressure.

In addition, the syringe pump <NUM> may induce variations in the under pressure, which is the pressure of the fluid in the micropipette <NUM>. These pressure variations may have an oscillating character. The aspired portion of the sample <NUM> undergoes the pressure variations. In response, this may cause variations in the deformation of the sample <NUM>: the aspired portion varies in length. Consequently, the aforementioned aspiration spacing and, therefore, the optical path length "c" will vary with the pressure variations. The same may apply to the aforementioned cross sample spacing, and the sample back spacing and, therefore, the optical path lengths "b" and "c", respectively.

The optical interrogator <NUM> measures the aforementioned optical path lengths "a", "b", "c" and "d", as well as the variations therein. The optical path length "a" of the pressure-sensing Fabry-Perot cavity <NUM>, which is represented in <FIG>, indicates the under-pressure, as well as the variations therein. The optical path lengths "b", "c", and "d", of the cross sample Fabry-Perot cavity <NUM>, of the aspiration Fabry-Perot cavity <NUM>, and of the sample back Fabry-Perot cavity <NUM>, respectively, which are represented in <FIG>, indicate the deformation that the sample <NUM> undergoes in response to the under-pressure, as well as, the variations therein.

In order to measure the aforementioned optical path lengths "a", "b", "c" and "d", as well as the variations therein, the optical interrogator <NUM> may operate in a manner similar to that described in the patent publication <CIT>, mentioned hereinbefore. This operation will be summarily described hereinafter in the context of the optical measurement system <NUM> illustrated in <FIG>.

The optical interrogator <NUM> injects light into the sample measurement fiber <NUM> and into the optical pressure sensor <NUM>, both incorporated in the micropipette <NUM>. The light may have a center wavelength of <NUM>, and be relatively broad band having a full-width-at-half maximum bandwidth of <NUM>, as mentioned hereinbefore. A larger portion of the light may be injected into the sample measurement fiber <NUM>, whereas a smaller portion of the light may be injected into the optical pressure sensor <NUM>. In <FIG>, this is indicated by means of percentages, <NUM>% and <NUM>%, respectively, which are presented by way of illustration only; different percentages may be suitable.

In response, the optical interrogator <NUM> receives reflected light from the micropipette <NUM>. The reflected light is a combination of reflected light from the sample measurement fiber <NUM> and reflected light from the optical pressure sensor <NUM>. The circulator <NUM> directs the reflected light to the spectrometer-based analyzer <NUM>. The spectrometer-based analyzer measures a wavelength spectrum of the reflected light in a wavelength band of interest. The wavelength band of interest may be centered on the aforementioned center wavelength of <NUM> and have a width similar to the aforementioned full-width-at-half maximum bandwidth of <NUM>.

The wavelength spectrum of the reflected light reveals a combined spectral response, which is a combination of respective spectral responses of respective Fabry-Perot cavities in the micropipette <NUM>. These respective Fabry-Perot cavities comprise the pressure-sensing Fabry-Perot cavity <NUM>, represented in <FIG>, the cross sample Fabry-Perot cavity <NUM>, the aspiration Fabry-Perot cavity <NUM>, and the sample back Fabry-Perot cavity <NUM>, represented in <FIG>. A spectral response of a Fabry-Perot cavity, which has an optical path length, is typically a sinusoidal curve of amplitude versus wavelength. This sinusoidal curve has a periodicity that is determined by the optical path length of the Fabry-Perot cavity.

The spectrometer-based analyzer <NUM> applies a Fourier transform to the wavelength spectrum that has been measured. The Fourier transform thus provides a Fourier-transformed wavelength spectrum. The Fourier transform may be complex so that an amplitude representation of the Fourier-transformed wavelength spectrum is provided, as well as a phase representation of the Fourier-transformed wavelength spectrum. In each of these representations, periodicity is linearly related to optical path length. Thus, periodicity and optical path length are interchangeable in the Fourier-transformed wavelength spectrum.

<FIG> illustrates an amplitude representation of a Fourier-transformed wavelength spectrum <NUM>, which may be obtained by a spectral response measurement described hereinbefore. <FIG> is a graph having a horizontal axis, which represents optical path length in units of micron [µm], and a vertical axis, which represents amplitude (V). A curve in the graph represents the amplitude representation of the Fourier-transformed wavelength spectrum <NUM>. The Fourier-transformed wavelength spectrum <NUM> comprises four peaks: a first peak <NUM> at approximately <NUM> on the horizontal axis, a second peak <NUM> at approximately <NUM>, a third peak <NUM> at approximately <NUM>, and a fourth peak <NUM> at approximately <NUM>.

The first peak <NUM> is related to the pressure-sensing Fabry-Perot cavity <NUM> of which the optical path length "a" is nominally approximately <NUM>. The second peak <NUM> is related to the cross sample Fabry-Perot cavity <NUM> of which the optical path length "b" is nominally approximately <NUM>. The third peak <NUM> is related to the aspiration Fabry-Perot cavity <NUM> of which the optical path length "c" is nominally approximately <NUM>. The fourth peak <NUM> is related to the sample back Fabry-Perot cavity <NUM> of which the optical path length "d" is nominally approximately <NUM>. For each of these peaks, it holds that the peak has a position on the horizontal axis that represents a measured optical path length of the Fabry-Perot cavity to which the peak is related.

The optical interrogator <NUM> can repeatedly carry out spectral response measurements, each as previously described, in successive time intervals. Accordingly, the spectrometer-based analyzer <NUM> then measures successive wavelength spectra in these successive time intervals. As explained hereinbefore, a wavelength spectrum comprises a spectral response of a Fabry-Perot cavity, which has an optical path length. The spectral response of the Fabry-Perot cavity manifests itself as a periodicity in the wavelength spectrum, whereby the periodicity depends on the optical path length. The spectrometer-based analyzer <NUM> may detect a change in the periodicity over the successive time intervals in which the spectral response measurements have been carried out. The optical interrogator <NUM> may then derive a change in the optical path length from this change in the periodicity.

High resolutions measurements of optical path length variations are possible by detecting a phase evolution of the periodicity in the successive wavelength spectra that have been measured. This technique is described in patent publication <CIT>, mentioned hereinbefore. In the optical measurement system <NUM> illustrated in <FIG>, the spectrometer-based analyzer <NUM> may use successive phase representations of successive Fourier-transformed wavelength spectra that have been measured. The spectrometer-based analyzer <NUM> may extract local phase data from each of the successive phase representations at a location corresponding to a location in an amplitude representation of a Fourier-transformed wavelength spectrum where a peak occurs. Accordingly, a series of local phase date is obtained for the peak concerned, which are related in time to the successive time intervals in which the successive spectral response measurements have been carried out. The series of local phase date represents the phase evolution of the periodicity with relatively great precision, which, in turn, represents optical path length variations with relatively great precision.

Accordingly, the optical interrogator <NUM> may measure with relatively great precision variations in the optical path length "a" of the pressure-sensing Fabry-Perot cavity <NUM> illustrated in <FIG>, as well as variations in the optical path length "c" of the aspiration Fabry-Perot cavity <NUM> illustrated in <FIG>. The thus measured variations in the optical path length "a" represent pressure variations with relatively great precision. The thus measured variations in the optical path length "c" represent variations in the aspiration spacing, and thus deformation of the sample <NUM>, with relatively great precision. Moreover the optical interrogator <NUM> detects these variations in an inherently synchronized manner. This allows measuring with relatively great precision a stimulus-response relationship between the pressure variations, which constitute the stimulus, and the variations in the aspiration spacing, which constitutes the response.

The optical interrogator <NUM> may further measure variations in the optical path length "b" of the cross sample Fabry-Perot cavity <NUM> and variations in the optical path length "d" of the sample back Fabry-Perot cavity <NUM> illustrated in <FIG>. The aforementioned remarks equally apply to these measured variations, which may further characterize sample deformation.

As illustrated in <FIG>, the third peak <NUM> and the fourth peak <NUM>, which are related to the optical path lengths "c" and "d", respectively, are less pronounced, having a smaller amplitude compared with the first peak <NUM> and the second peak <NUM>. This is related to a lower signal-to-noise ratio of reflected light from the related cavities: the aspiration Fabry-Perot cavity <NUM> and the sample back Fabry-Perot cavity <NUM>, respectively, illustrated in <FIG>. The signal-to-noise ratio depends on optical and geometrical properties of the sample <NUM>, as well as on the profile of the light beam emanating from the sample measurement fiber <NUM> in the micropipette <NUM>.

<FIG> illustrates a measured stimulus-response relationship <NUM> between relative pressure in the micropipette <NUM> and displacement of the aspired portion of the sample <NUM> in the micropipette <NUM>. <FIG> is a composite graph having a horizontal axis, which represents time in units of seconds [s]. A right-hand vertical axis represents the relative pressure in units of detection signal level. A left-hand vertical axis represents the displacement in units of nanometer [nm].

<FIG> comprises two curves <NUM>, <NUM>. A first <NUM> curve represents a measurement of the relative pressure in the micropipette <NUM>, which varies with time due to a pressure variation imposed by the syringe pump <NUM>. This is the stimulus. A second curve <NUM> represents a measurement of the displacement of the aspired portion of the sample <NUM> in the micropipette <NUM>. The displacement of the aspired portion of the sample <NUM> in the micropipette <NUM> varies with the relative pressure in the micropipette <NUM>. This is the response. The relative pressure and the displacement are inherently concomitantly measured. The relative pressure is derived from a measured variation in the optical path length "a" of the pressure-sensing Fabry-Perot cavity <NUM> that is measured. The displacement is derived from a measured variation in the optical path length "c" of the aspiration Fabry-Perot cavity <NUM> and the refractive index of the fluid in the tip <NUM>.

<FIG> illustrates the same stimulus-response relationship <NUM> in a different manner. <FIG> is a graph having a horizontal axis, which represents the displacement of in units of nanometer [nm]. A vertical axis represents the relative pressure ΔP expressed in units of Pascal [Pa]. <FIG> comprises a solid line curve <NUM> that represents the stimulus-response relationship <NUM> that is also represented, but differently, in <FIG> further comprises a straight broken line curve <NUM> that represents a linearized version of a socalled Zhou model for determining an elastic modulus E. The elastic modulus E can be determined on the basis of the following equation: <MAT> in which ΔP represents a differential pressure, Lp the displacement of the aspired portion of the sample <NUM> in the micropipette <NUM>, Rp the radius of the tip aperture <NUM>, Rc the radius of the sample <NUM>, β<NUM> = <NUM>, and β<NUM> = <NUM>.

The optical measurement system <NUM> described hereinbefore with reference to <FIG> allows studying frequency-dependent sample viscoelasticity. To that end, the syringe pump <NUM> may impose sinusoidal pressure variations of different frequencies in the micropipette <NUM> as stimuli. In response to a sinusoidally varying pressure, the sample <NUM> will exhibit a sinusoidally varying deformation, such as, for example a sinusoidally varying displacement of the aspired portion of the sample <NUM> in the micropipette <NUM>. Due to viscous characteristics of the sample <NUM>, the sinusoidally varying displacement will be out of phase with the sinusoidally varying pressure. There will be a phase lag between these two, which may be frequency dependent.

<FIG> illustrates a measured stimulus-response relationship <NUM> between a sinusoidally varying pressure in the micropipette <NUM> at a frequency of <NUM> and a sinusoidally varying displacement of the aspired portion of the sample <NUM> in the micropipette <NUM>. <FIG> is a composite graph having a horizontal axis, which represents time in units of seconds [s]. A left-hand vertical axis represents the relative pressure ΔP in units of Pascal [Pa]. A left-hand vertical axis represents displacement Lp in units of nanometer [nm].

<FIG> comprises two sinusoidal curves: an upper sinusoidal curve <NUM> and a lower sinusoidal curve <NUM>. The upper sinusoidal curve represents <NUM> a measurement of the sinusoidally varying pressure in the micropipette <NUM>. The lower sinusoidal curve <NUM> represents a measurement of the sinusoidally varying displacement of the aspired portion of the sample <NUM> in the micropipette <NUM>. <FIG> indicates a phase lag <NUM> between these two curves <NUM>, <NUM>. The phase lag is <NUM> thus measured for the aforementioned frequency of <NUM>. Different phase lags may be measured for different frequencies due to frequency-dependent sample viscoelasticity as mentioned hereinbefore.

The phase lag <NUM> that is measured allows determining a storage modulus E' and a loss modulus E", which are viscoelastic properties the sample <NUM>. The storage modulus E' may be determined on the basis of the following equation: <MAT> in which δ represents the phase lag <NUM> discussed hereinbefore and illustrated in <FIG>, P<NUM> represents the average pressure of the fluid in the tip <NUM> of the micropipette <NUM>, and L<NUM> represents the average length of the aspired portion of the sample <NUM> in the micropipette <NUM>, the other factors being as defined hereinbefore with respect to the equation relating to the elastic modulus.

The loss modulus E" may be determined on the basis of the following equation: <MAT>.

<FIG> illustrates a measured frequency-dependency <NUM> of the storage modulus E' and the loss modulus E" based on measured stimulus-response relationships as the one illustrated in <FIG> is a graph having a horizontal axis, which represents frequency in units of Hertz [Hz]. A vertical axis represents pressure in units of kilopascal [kPa]. <FIG> comprises two sinusoidal curves: an upper curve <NUM> and a lower curve <NUM>. The upper curve <NUM> represents the storage modules E', which is frequency dependent. The lower curve <NUM> represents the loss modulus E", which is also frequency dependent. The aforementioned curves <NUM>, <NUM> have been obtained on the basis of different phase lags measured at different frequencies with which sinusoidally varying pressure variation are imposed on the fluid in the tip <NUM> of the micropipette <NUM>. The different frequencies comprise <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> as indicated in <FIG>.

The measured stimulus-response relationship <NUM> illustrated in <FIG> and the measured frequency-dependency <NUM> of the storage modulus E' and the loss modulus E" illustrated in <FIG> were obtained with the sample <NUM> being a bovine oocyte to assess the frequency-dependent rheological properties of the Zona Pellucida. at a fixed temperature of <NUM>. A preload suction pressure of <NUM> Pa was applied to the micropipette <NUM>. After a delay of <NUM> seconds, a series of <NUM> different sinusoidal pressure variations at the aforementioned frequencies of <NUM> <NUM>, <NUM>, <NUM>, and <NUM>, respectively. Each sinusoidal pressure variation comprised <NUM> oscillatory periods of <NUM> Pa amplitude, A delay of <NUM> seconds was provided between two succussive sinusoidal pressure. The storage modulus E' and the loss modulus E" thus measured, illustrated in <FIG>, show a power-law rheological behavior, in accordance with what has been observed in several other biological systems.

The embodiments described hereinbefore with reference to the drawings are presented by way of illustration. The invention may be implemented in numerous different ways. In order to illustrate this, some alternatives are briefly indicated.

The invention may be applied in numerous types of products or methods related to optically measuring deformation of a sample in response to a change in an environmental condition. In the embodiments presented hereinbefore, a sample deforms in response to a change in pressure. In other embodiments, an environmental condition other than pressure may change and cause deformation, such as, for example, temperature or radiation, including light. well as other environmental conditions.

There are numerous different ways of implementing a support for receiving a sample and for removably holding the sample in an optical sensing device in accordance with the invention. In the embodiments presented hereinbefore, the support is in the form of a tip of a micropipette. In other embodiments, the support may have a different form, such as, for example, a microfluidic channel or a MEMS device with an opening, MEMS being an acronym for Micro Electro-Mechanical System.

What is more, a method of optically measuring a response of a sample to a change in an environmental condition in accordance with the invention, need not make use of an optical sensing device having a support in the form of a micropipette. The sample may be free floating or may be retained by an element separate from an optical waveguide used for optically measuring a response of the sample to a change in an environmental condition. There are numerous setups that allow placing a sample so that a refractive index discontinuity at a surface of the sample faces an end of an optical waveguide, whereby the end of the optical waveguide and the refractive index discontinuity form an optical interferometric cavity.

For example, a miniature device that is functionally equivalent to a micropipette may be manufactured by means of optical lithography. Such a miniature device may be more suited for measuring relatively small samples than a micropipette. For example, a relatively small sample may require a micropipette having a tip aperture of a few microns only and thus a relatively narrow tip. In such a case, the relatively narrow tip may make that an end of an optical fiber, which is fitted therein, is relatively distant from the sample. This relatively large distance may complicate achieving a satisfactory signa-of-noise, and thus satisfactory accuracy and precision, or may even make this impossible. The aforementioned miniature device may provide a solution to this problem.

There are numerous different ways of implementing an optical waveguide in an optical sensing device in accordance with the invention. In the embodiments presented hereinbefore, the optical waveguide is an optical fiber. In other embodiments, the optical waveguide may be comprised in an integrated photonics system.

There are numerous different ways of implementing an optical measurement system in accordance with the invention. In the embodiments presented hereinbefore, an optical measurement system comprising a single optical sensing device only was described for the sake of simplicity, the optical sensing device being in the form of a micropipette. In other embodiments, an optical measurement system may comprise multiple optical sensing devices jointly coupled to an optical interrogator. In such an embodiment, respective optical interferometric cavities formed with respective samples may have respective nominal optical path lengths that are sufficiently different so as to allow making a distinction between these in the optical interrogator. Patent publication <CIT> describes such a multiplex optical interrogation technique. This allows for more elaborate experiments, such as cell-cell adhesion studies.

There are numerous different ways of measuring a spectral response of an optical interferometric cavity in an optical measurement system in accordance with the invention. In the embodiments presented hereinbefore, an interrogator comprises a relatively broad band light source and a spectrometer-based analyzer. In other embodiments, an interrogator may comprise a relatively narrow band tunable light source, which is made to sweep throughout a wavelength band of interest. Furthermore, the spectral response may be analyzed using a technique different from that described in patent publication <CIT>, although the technique described in this patent publication allows relatively high accuracy and precision in measuring variations in optical path length.

Claim 1:
A method of optically measuring a response of a sample (<NUM>) to a change in an environmental condition to which the sample is exposed, wherein use is made of an optical sensing device (<NUM>) comprising:
- a support (<NUM>) with an aperture (<NUM>), the optical sensing device being adapted to removably hold the sample against the support around the aperture so that a portion of the sample is free to deform through the aperture; and
- an optical waveguide (<NUM>) fixedly arranged with respect to the support whereby an end (<NUM>) of the optical waveguide faces the aperture,
the method comprising:
- forming an optical interferometric cavity (<NUM>) between, one the one hand, the end (<NUM>) of the optical waveguide (<NUM>) and, on the other hand, a refractive index discontinuity at a surface (<NUM>) of the portion of the sample, which is free to protrude through the aperture (<NUM>), the optical interferometric cavity having an optical path length ("c");
- changing the environmental condition to which the sample is exposed so as to cause deformation of the portion of the sample through the aperture; and
- measuring a variation in the optical path length of the optical interferometric cavity in response to the change in the environmental condition.