OPTOACOUSTIC MONITORING DEVICE FOR CELL CHARACTERIZATION

Abstract

A monitoring device of biological cells including: a fluidic channel in which a fluid including biological cells is made to flow, including a first and second glass walls placed in parallel and a reflective surface adjoined to the second wall; and a piezoelectric transducer, the piezoelectric transducer emitting acoustic waves creating a force acting on the biological cells, making them flow substantially along a single motion plane. The external interface between the first wall and the exterior of the fluidic channel and the reflective surface are configured to function as a Fabry-Perot interferometer, such that the motion planes sensibly perpendicular to the optical axis of the Fabry-Perot interferometer. The finesse of the interferometer is lower than 10. The monitoring device further includes a detecting unit detecting a fringe pattern stemming from the Fabry-Perot interferometer and an analysing device, analysing the fringe pattern and configured to output data on physical properties of the cells based on the pattern.

Claims

1-15. (canceled)

16. A monitoring device of biological cells comprising: a fluidic channel in which a fluid comprising biological cells is made to flow, comprising a first and second glass walls placed in parallel and a reflective surface adjoined to the second wall, and a piezoelectric transducer, said piezoelectric transducer emitting acoustic waves creating a force acting on the biological cells, making them flow substantially along a single motion plane, wherein the external interface of the first glass wall in contact with the exterior of the fluidic channel, for example air, and the reflective surface are configured to function as a Fabry-Perot interferometer, the finesse of which is lower than 10, such that the motion plane is sensibly perpendicular to the optical axis of the Fabry-Perot interferometer, the monitoring device further comprising a detection unit detecting a fringe pattern stemming from the Fabry-Perot interferometer, and an analysing device, analysing said fringe pattern and configured to output data on physical properties of the cells based on said pattern.

17. The monitoring device according to claim 16, wherein the frequency of the piezoelectric transducer is set at the fundamental resonance frequency of the fluidic channel.

18. The monitoring device according to claim 16, wherein the analysing device retrieves, from the detected fringe pattern, the distances between the optical axis of the Fabry-Perot interferometer and at least two fringes of the fringe pattern.

19. The monitoring device according to claim 16, wherein the Finesse of the Fabry-Perot interferometer is lower or equal to 1, preferably comprised between 0.5 and 0.7, even more preferably substantially equal to 0.6.

20. The monitoring device according to claim 16, wherein the physical properties of the cells are taken from at least one of the following, alone or in combination: mechanical properties of the cells, shape of the cells, refractive index of the cells, and deformability of the cells.

21. The monitoring device according to claim 16, wherein the reflective surface is sandwiched between the second wall and the piezoelectric transducer.

22. The monitoring device according to claim 16, wherein the optical reflectivity of the reflective surface is above 90%, preferably substantially equal to 99%, and the optical reflectivity of the interface between the glass wall and the exterior of the fluidic channel, for example air, is above 2%, preferably above 4%.

23. The monitoring device according to claim 16, wherein it also comprises an acoustic reflector for reflecting the acoustic waves emitted by the piezoelectric transducer so that the acoustic waves become stationary within the fluidic channel.

24. The monitoring device according to claim 23, wherein the first wall functions as the acoustic reflector for the acoustic waves emitted by the piezoelectric transducer.

25. The monitoring device according to claim 16, also comprising an acoustic wave frequency modulator configured to modulate the amplitude of the acoustic waves generated by the piezoelectric transducer.

26. The monitoring device according to claim 25, wherein the frequency of the acoustic waves generated by the piezoelectric transducer is set at the resonant frequency of fluidic channel.

27. The monitoring device according to claim 16, also comprising an acoustic wave amplitude modulator configured to modulate the amplitude of the acoustic waves generated by the piezoelectric transducer.

28. The monitoring device according to claim 16, wherein the detection unit comprises at least two optical detectors recording time traces at different points along the motion plane of the biological cells and one camera for calibration.

29. The monitoring device according to claim 16, wherein the optical arrangement comprises a laser-emitting device emitting a laser beam focused by a focus lens, for example a microscope objective, into a zone of the Fabry-Perot interferometer, wherein the laser beam preferably is aligned perpendicularly with motion plane.

30. The monitoring device according to claim 29, wherein the focus lens is also used to retrieve a reflected laser beam carrying the detected fringe pattern.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0046] The invention will be better understood in view of the following description, referring to the annexed Figures in which:

[0047] FIG. 1 is a schematic view in perspective of a monitoring device according to the invention,

[0048] FIG. 2 is a cut view along plane II-II of the fluidic channel of the monitoring device according to the invention,

[0049] FIG. 3 is a schematic view of the reflected and exiting wavefronts interfering at the plane of the microscope objective of the monitoring device according to the invention.

DETAILED DESCRIPTION

[0050] FIG. 1 illustrates a monitoring device 8 of biological cells according to the invention.

[0051] Monitoring device 8 comprises a fluidic channel 10, better shown on FIG. 2, in which a fluid F comprising biological cells C is made to flow. The cell fluid F is introduced through syringe channels 12, one syringe channel 12 being placed at one side of the fluidic channel 10 to introduce the fluid F in the fluidic channel 10, and the other at the other side of the fluidic channel 10 to evacuate the fluid F from the fluidic channel 10. Cell fluid F may be water.

[0052] Fluidic channel 10 comprises a first and second parallel glass walls 14a, 14b. A reflective surface 15 is adjoined to the second wall 14b. The external interface of the glass wall 14a in contact with the exterior of the fluidic channel 10, here air, and the reflective surface 15 are configured to function as a Fabry-Perot interferometer 16.

[0053] Preferably, the optical reflectivity of the reflective surface 15 is above 90%, preferably substantially equal to 99%. Reflective surface 15 may be a mirror. The optical reflectivity of the interface between the glass wall 14a and air is above 2%, preferably above 4%. Having an optical reflectivity under 2% would give rise to too much background noise. A value of 4% optical reflectivity corresponds to the natural reflectivity of glass in air, which is found to give satisfactory results. A partially transparent mirror can also be used for channel wall instead of a glass wall. This would allow for obtaining an optical reflectivity of 10% for wall 14a, for example.

[0054] Such reflectivities lead to coefficient of finesse around 0.15, corresponding to a finesse of 0.6.

[0055] Monitoring device 10 also includes a piezoelectric transducer 18, that is placed proximate to one of the walls 14b of the fluidic channel 10 and emits acoustic waves W towards the fluidic channel.

[0056] Acoustic waves W create a force acting on the biological cells C, making them flow substantially along a single motion plane P, shown on FIG. 2.

[0057] Monitoring device 10 also comprises an acoustic reflector for reflecting the acoustic waves W emitted by the piezoelectric transducer 18 so that the acoustic waves W become stationary within the fluidic channel 10.

[0058] In the preferred embodiment shown on the Figures, wall 14a functions as an acoustic wave reflector for acoustic waves W emitted by piezoelectric transducer 18.

[0059] Owing to this particular arrangement, motion plane P is parallel to the walls 14a, 14b of the fluidic channel 10. Hence, the optical axis of the Fabry-Perot interferometer 16 is perpendicular to motion plane P. More specifically, the acoustic waves W generated by the piezoelectric transducer 18 aligns the cells C onto the plane P corresponding to the wave's node at the centre of the microfluidic channel 10, i.e. substantially at an equal distance from each wall 14a, 14b.

[0060] Preferably, the monitoring device also comprises an acoustic wave frequency modulator (not shown on the Figures) configured to modulate the frequency of the acoustic waves W generated by the piezoelectric transducer 18.

[0061] The acoustic frequency is preferably set to the resonance of the fluidic channel 10, while the amplitude is selected to ensure good focusing. The acoustic focus confines the cells C in the laminar flow region.

[0062] In addition to the function of making the cells C flow along a motion plane P, if the amplitude of the standing wave is large enough, the cells C are deformed.

[0063] Hence, in order to induce further significant control and deformation of the cells C, monitoring device 10 may also comprise an acoustic wave amplitude modulator (not shown on the Figures), configured to modulate the amplitude of the acoustic waves W generated by the piezoelectric transducer 18. The amplitude of the acoustic waves W generated by the piezoelectric transducer 18 is chosen depending on the size, shape and type of the cells C.

[0064] Monitoring device also includes a detection unit 21, shown on FIG. 1, detecting a fringe pattern stemming from the Fabry-Perot interferometer 16.

[0065] In particular, according to the preferred embodiment of the invention illustrated in the Figures, the detection unit 21 comprises a focus lens 22, for example a microscope objective, placed so that its focal plane matches the cell plane of movement P.

[0066] Monitoring device 8 also includes a laser-emitting device 26 emitting a beam L illuminating a zone of the optical cavity 16. Laser beam L is aligned perpendicularly to motion plane P. Hence, laser beam is aligned in a direction that is parallel to the optical axis of the Fabry-Perot interferometer 16. The laser beam is preferably a Helium-Neon laser beam which provides for a satisfactory fringe pattern from the Fabry-Perot interferometer 16 results, however other laser beams can be used.

[0067] The laser beam L introduced in the optical cavity 16 is reflected by the glass wall 14a and mirror 15 acting as the Fabry Perot resonator. Said reflected laser beam L is retrieved by the detection unit 21.

[0068] In this particular embodiment, focus lens 22 is used to retrieve the reflected laser beam L.

[0069] In order to increase the quality of the measurements, the detection unit 21 also comprises at least two optical detectors 24 recording time traces at different points along the motion plane P of the biological cells C. This facilitates the retrieval of data about the physical properties of the cells C. A camera 28 is also included in the detection unit 21 for calibration.

[0070] Preferably, the detection unit also includes a plurality of beam splitters BS and mirrors M used to redirect the reflected laser beam L towards detectors 24 and camera 28. The arrangement of mirrors M and beam splitters BS will naturally vary according to the positioning of the optical detectors 24, laser emitting device 26 and camera 28 in the monitoring device 10 setup.

[0071] Monitoring device 8 also comprises an analysing device (not shown on the Figures) analysing the fringe pattern stemming from the Fabry-Perot interferometer 16 and configured to output data on the cells' C physical properties based on said pattern.

[0072] In the present embodiment shown in the Figures, the analysis of the fringe pattern when the cells C flow along the motion plane P provides direct information about their shape and optical thickness.

[0073] However, other physical properties of the cells C can be obtained using the monitoring device 10, such as: [0074] mechanical properties of the cells, [0075] shape of the cells, [0076] refractive index of the cells, [0077] deformability of the cells.

[0078] When an acoustically focussed cell crosses the Fabry-Perot cavity formed by the channel, it perturbs the resonator fringe pattern (mathematically described by the Airy transmission function). Said cell-induced fringe pattern perturbations can be modelled as a modification of the Fabry-Perot's resonator fringe pattern induced by a thin lens. The mathematical model used to describe the cell-resonator interaction is developed using propagation ray-matrices and is based on the distance from the optical axis of the interferometer to the n-th intensity fringe of the pattern. These distances are used to compute the radius of curvature of the wavefront exiting the resonator which interferes with the incoming light beam.

[0079] The width of the fluidic channel L, is much larger than the cells C diameter :L≥10×. The broad channel width is chosen to enable the establishment of an acoustic standing wave at frequencies which can be attained with standard piezotransducers (size and cost issues). In addition, it ensures the avoidance of edge effects (channel sides) both in the velocity and in the pressure fields.

[0080] The resulting large-gap interferometer possesses the additional advantage of permitting the observation of changes in the Resonator Fringe Pattern (RFP) induced by the presence of an intracavity cell, as opposed to the simple phase-shift typical of small-gap (L≈) Fabry-Perot resonators.

[0081] A thin-lens model may be used to reconstruct the fringe pattern perturbation induced by an intracavity cell. First, a ray-matrix approach describes the light propagation in the low-Finesse Fabry-Perot resonator (with the inclusion of a generic thin lens—as cell proxy). Next, the radius of curvature of the reflected wave is computed to retrieve the Cell Focal Length (CFL).

[0082] Taking into account the refractive index of each medium, the optical path, denoted by a tilde over the corresponding symbol, becomes:

{tilde over (L)}=n.sub.wL  (1)

{tilde over (t)}.sub.c=n.sub.gt.sub.c  (2)

{tilde over (t)}.sub.m=n.sub.gt.sub.m  (3)

[0083] Where n.sub.w and n.sub.g are the fluid F (here water) and glass refractive indices, respectively, and:

.sub.1={tilde over (t)}.sub.c+½{tilde over (L)}  (4)

.sub.2={tilde over (L)}.sub.c+2{tilde over (t)}.sub.m  (5)

[0084] are the optical paths measured on either side of the lens. A spherical lens, placed in the center of the microfluidic channel (acoustic focusing plane), approximates the cell with a radius of curvature r.sub.c and refractive index n.sub.c.

[0085] Using the thin lens formula (1), one can obtain its equivalent focal lens:

[00001]$\begin{array}{cc}{f}_c=\frac{r_c}{2{\left(n_c-n_w\right)}_C}& \left(6\right)\end{array}$

[0086] The focal distance is assumed to be very long, f.sub.c>>r.sub.c, justifying the thin lens approximation. In the system according to the invention,

[00002]$\frac{f_c}{r_c}=\frac{1}{2\left(n_c-n_w\right)}1$

, due to the closeness of the medium's (fluid within the fluidic channel) and cells' C refractive indices (typically within a few percent).

[0087] Thus, the focal length is always much larger than the cell radius. It should however be noted that the focal length is not required to be small compared to the resonator's length (and is not even necessarily expected to be so).

[0088] The beam evolution inside the resonator can be computed with the beam propagation matrices:

{tilde over (M)}=.Math..Math..Math..Math.  (7)

[0089] where the expressions for the various matrices are (3):

[00003]$\begin{array}{cc}{M}_{{ℒ}_1}=\left(\begin{array}{cc}1& {ℒ}_1\\ 0& 1\end{array}\right)& \left(8\right)\end{array}$$\begin{array}{cc}{M}_{{ℒ}_2}=\left(\begin{array}{cc}1& {ℒ}_2\\ 0& 1\end{array}\right)& \left(9\right)\end{array}$$\begin{array}{cc}{M}_{\ell }=\left(\begin{array}{cc}1& 0\\ -\frac{1}{f_c}& 1\end{array}\right)& \left(10\right)\end{array}$

[0090] f.sub.c is the cell's focal length defined above in equation. (6), represents the propagation between the resonator entrance (considered as the first wall glass-air interface) and the thin lens.

[0091] The thin lens, represented by , is symmetrically placed inside the acoustic channel and is the roundtrip from the thin lens, to the mirror and back to the lens.

[0092] At this level of approximation, the reflection at the glass-fluid interface of the walls 14a, 14b is ignored given that the reflection coefficient at normal incidence, computed with:

[00004]$\begin{array}{cc}r={\left(\frac{n_1-n_2}{n_1+n_2}\right)}^2& \left(11\right)\end{array}$

[0093] where n.sub.1 and n.sub.2 are the refractive indices for the two media, indicates that the fluid-glass interface contributes ten times less than the air-glass one (R.sub.air-glass≈0.04 and R.sub.glass-water≈0.004, respectively).

[0094] The combined reflectivity R.sub.air-glass and R.sub.mirror=0.9 produce for the empty resonator (no cell, thus no thin lens) a Finesse=0.61, whence the denomination “low-Finesse” Fabry-Perot resonator.

[0095] Expanding the algebra in equation. (7), a transfer matrix is obtained:

[00005]$\begin{array}{cc}\tilde{M}=\left(\begin{array}{cc}A& B\\ C& D\end{array}\right)& \left(12\right)\end{array}$

[0096] with coefficients:

[00006]$\begin{array}{cc}A=\frac{f_C^2-\left[L_2+2{ℒ}_1\right]f_C+{ℒ}_1{ℒ}_2}{f_C^2}& \left(13\right)\end{array}$$\begin{array}{cc}B=\frac{\left[L_2+2{ℒ}_1\right]f_C^2-2{ℒ}_1\left[L_2+2{ℒ}_1\right]f_C+{ℒ}_1^2{ℒ}_2}{f_C^2}& \left(14\right)\end{array}$$\begin{array}{cc}C=\frac{{ℒ}_2-2_{f_C}}{f_C^2}& \left(15\right)\end{array}$$\begin{array}{cc}D=\frac{f_C^2-\left[2L_1+{ℒ}_2\right]f_C+{ℒ}_1{ℒ}_2}{f_C^2}& \left(16\right)\end{array}$

[0097] In particular, the expression:

2+=2({tilde over (t)}.sub.m+{tilde over (L)}+{tilde over (t)}.sub.c)  (17)

[0098] corresponds to one full roundtrip (optical length) inside the resonator.

[0099] The multiple reflections inside the Fabry-Perot resonator have been ignored and have considered just one roundtrip in the context of the invention. The reason for this, at is the very weak amount of light making a second (or more) roundtrip(s).

[0100] The first glass-air interface (with first wall 14a) transmits approximately 0.96I.sub.in, (I represent the Poynting vector of each component). Accounting for the finite reflectivity of the reflective surface (here a mirror) R.sub.b and for the losses at the glass-fluid interface, the intensity exiting the resonator after the first roundtrip is ≈0.91R.sub.bI.sub.in, while the component reflected back into the resonator after the first roundtrip will be 0.04×0.95R.sub.bI.sub.in≈0.038R.sub.bI.sub.in. Thus only approximately 3.8% of the intensity goes for a second roundtrip, thus justifying its removal from the computation of the low-finesse resonator's response.

[0101] The radius of curvature R.sub.out of the wavefront exiting the resonator after one roundtrip, as a function of the entering wavefront curvature, is (3):

[00007]$\begin{array}{cc}{R}_{out}=\frac{A.Math.R_{\mathrm{in}}+B}{C.Math.R_{\mathrm{in}}+D}& \left(18\right)\end{array}$

[0102] hence, the two wavefronts R.sub.in coupled into the resonator by the microscope objective and R.sub.out exiting the resonator, can be superposed to compute their circular interference fringes.

[0103] The analysing device identifies the values of the distance ρ between a fringe and the optical axis, where constructive interference fringes (bright rings) are expected.

[0104] The positions of the two origins for the two emerging waves is arbitrary (the two could be exchanged). The problem is solved in cylindrical coordinates. Intrinsic symmetry considerations justify neglecting the azimuthal angle ϕ. The distance from the axis is ρ.

[0105] The distance from the axis ρ.sub.1 is defined as the one for which the detection unit detects the first bright fringe:

[00008]$\begin{array}{cc}\sqrt{R_{out}^2+{\rho }_1^2}-\sqrt{R_{in}^2+{\rho }_1^2}=m\lambda & \left(19\right)\end{array}$

[0106] where the integer m is defined such that:

[00009]$\begin{array}{cc}{R}_{out}=R_{in}+\left(m-1\right)\lambda +ϵ& \left(20\right)\end{array}$$\begin{array}{cc}0\le ϵ<\lambda & \left(21\right)\end{array}$$\begin{array}{cc}m=1+\mathrm{Int}\left[\frac{R_{out}-R_{\mathrm{in}}}{\lambda }\right]& \left(22\right)\end{array}$

[0107] FIG. 3 is a schematics of the exiting R.sub.out and reflected R.sub.in wavefronts curvature radii interfering at the plane of the microscope objective. The respective origins for the two curved wavefronts match the radii of curvature. The relative positions are arbitrarily marked, as the value of R.sub.out relative to R.sub.in, depends on experimental parameters. ρ is the coordinate measuring distances from the optical axis.

[0108] Solving for R.sub.out for two consecutive bright fringes ρ.sub.1 and ρ.sub.2 (equation (19)):

[00010]$\begin{array}{cc}\sqrt{R_{out}^2+{\rho }_1^2}-\sqrt{R_{in}^2+{\rho }_1^2}=m\lambda & \left(23\right)\end{array}$$\begin{array}{cc}\sqrt{R_{out}^2+{\rho }_2^2}-\sqrt{R_{in}^2+{\rho }_2^2}=\left(m+1\right)\lambda & \left(24\right)\end{array}$

[0109] we obtain:

[00011]$\begin{array}{cc}{R}_{out}=\pm \sqrt{{\frac{1}{4k^2}\left[{\rho }_2^2-{\rho }_1^2-k^2\right]}^2-{\rho }_1^2}& \left(25\right)\end{array}$$\begin{array}{cc}k=\lambda +\sqrt{R_{in}^2+{\rho }_2^2}-\sqrt{R_{in}^2+{\rho }_1^2}& \left(26\right)\end{array}$

[0110] Substituting equations. (13) to (16) into equation. (18), a quadratic equation for f.sub.c (equation. (27)), a physical quantity of experimental interest, can be obtained:

d.sub.2f.sub.c.sup.2+d.sub.1f.sub.c+d.sub.0=0  (27)

d.sub.2=(R.sub.in−R.sub.out)+[+2]  (28)

d.sub.1=2R.sub.inR.sub.out+(R.sub.out−R.sub.in)[.sub.2+2.sub.1]−2L.sub.1[.sub.2+.sub.1]  (29)

d.sub.0=(R.sub.in−R.sub.out+.sub.1).sub.2.sub.1−(R.sub.inR.sub.out).sub.2  (30)

[0111] The cell focal length (CFL) f.sub.c is determined by computing the two roots of the second degree algebraic equation (27):

[00012]$\begin{array}{cc}{f}_c+=\frac{-d_1+\sqrt{d_1^2-4d_2{d}_0}}{2d_2}& \left(31\right)\end{array}$$\begin{array}{cc}{f}_c-=\frac{-d_1-\sqrt{d_1^2-4d_2{d}_0}}{2d_2}& \left(32\right)\end{array}$

[0112] where we make use of the expression for R.sub.out, eq. (25) in the computation of the coefficients d.sub.j.

[0113] The cell radius, connected to the focal length of the associated thin lens, is obtained by inverting eq. (6), once the cell refractive index is known:

r.sub.c=2(n.sub.c−n.sub.w)f.sub.c  (33)

[0114] For example, in order to obtain physical properties of the cell linked to its deformation, the flow inside the channel may be stopped when a cell is aligned with the resonator's axis (while acoustically focussed). Then, the interference pattern resulting from the immobile and levitated cell is then acquired, obtaining the information in the absence of deformation. The acoustic wave's amplitude is increased to induce a cell deformation and a new image of the interference pattern is acquired. The cycle is repeated to obtain 20 pairs of patterns for each cell in the absence and in the presence of deformation. The primary acoustic field is predicted to be of order 500 kPA, with associated (oscillating) acoustic displacements of a few nm. These displacements are thus small (and insignificant) in comparison to the cell deformation likely to be created from the non-zero time averaged acoustic radiation force. Hence the Young's Modulus of the cells may be obtained.

[0115] Naturally, other embodiments or variants of the invention will appear to a skilled person in the art, such as the use of other lasers or various configurations of the fluidic channel and Fabry-Perot interferometer.

LIST OF REFERENCES

[0116] 8: Monitoring device [0117] 10: Fluidic channel [0118] 12: Syringe tubing [0119] 14a, 14b: Channel walls [0120] 15: Reflective surface [0121] 16: Fabry-Perot interferometer [0122] 18: Piezoelectric transducer [0123] 21: Detection unit [0124] 22: Focus lens [0125] 24: Optical detectors [0126] 26: Laser-emitting device [0127] 28: Camera [0128] B: Beam splitter [0129] C: Biological cells [0130] F: Fluid [0131] L: Laser beam [0132] M: Mirrors [0133] P: motion plane of the cells [0134] W: Acoustic waves