WO2014133451A1

WO2014133451A1 - System and method to analyze non-spherical cells

Info

Publication number: WO2014133451A1
Application number: PCT/SE2014/050251
Authority: WO
Inventors: Ola JAKOBSSON; Carl Johan Grenvall; Thomas Laurell
Original assignee: Acousort AB
Current assignee: Acousort AB
Priority date: 2013-02-28
Filing date: 2014-02-28
Publication date: 2014-09-04
Anticipated expiration: 2015-08-28

Abstract

The invention relates to a method and system to orient non-spherical cells or particles present in a suspension wherein said suspension is exposed to an acoustic force, acting in one or two dimensions, wherein the cells or particles in the suspension will be oriented such that their smallest dimension is parallel with the strongest acoustic force and wherein changing the frequency or amplitude of the acoustic actuation allows reorientation of the cells 90° with respect to their previous orientation

Description

SYSTEM AND METHOD TO ANALYZE NON-SPHERICAL CELLS

FIELD OF THE INVENTION

The invention relates to a method and system to orient non-spherical cells or particles present in a suspension wherein said suspension is exposed to an acoustic force, acting in one or two dimensions, wherein the cells or particles in the suspension will orient themselves such that the net acoustic force acting on them is minimized. Non-spherical but axis-symmetrical cells or particles will then be oriented such that their smallest dimension is parallel with the strongest acoustic force and wherein changing the frequency or amplitude of the acoustic actuation allows reorientation of the cells or particles 90° with respect to their previous orientation

BACKGROUND OF THE INVENTION

Microfluidics is inherently a domain where high performance cell and particle handling has proven to be very successful. Some of the ruling technology platforms, which are industrial and clinical standards for high quality cell processing, are found in the fluorescence activated cell sorter (FACS) (Fluorescent detection and sorting) and in the Coulter Counter (size distribution measurements). The combination of fluorescently labeled cell specific antibodies and the FACS technique opened the route to a revolution in modern cell biology.

The Fluorescence Activated Cell sorter (FACS) remains a major workhorse in cell biology laboratories today. After more than 40 years of development, the FACS excels at analyzing and sorting cells at very high speed. However, some cell types are still very challenging to analyze with the conventional FACS. It was early realized in the field of cytometry that the scattered light from non-spherical cells is both a function of their relative position and orientation to the illuminating laser axis beam.

One of the greatest challenges in the history of flow cytometry has been X and Y chromosome detection and sorting of mammalian sperm cells. The fact that there is a difference in the amount of DNA content between the X and Y chromosome bearing cells can be utilized by staining the DNA inside the cells with a fluorescent dye. The intensity of fluorescent light emitted from such a labeled cell is proportional to the amount of DNA inside. The emitted light can be collected and the difference in signal intensity can be measured to distinguish between the two chromosome types. However, due to the fact that sperm cells are non spherical cells with a flat head and a tail, there is a much larger variation in signal intensity produced by the uncertainty of the relative orientation of the cell to the illuminating laser axis. The problem was eventually partially solved by the invention of asymmetric sheath flow nozzles, using a hydrodynamic flow focusing technique to achieve partial orientation. Orientation efficiencies of about 60% has been reported using such nozzles and today such nozzles are being used in specialized cell sorters dedicated for sperm sorting.

The upcoming field of imaging cytometry is a promising tool for collecting more information from cells. Shape, size and morphology are properties that can be measured by taking snapshots of cells passing a camera and using image analysis software. However, the uncertainty of the relative orientation of the cells to the optical axis of the camera may also cause artifacts in the software based image analysis, making the method unreliable for analyzing non spherical cell types.

In many studies performed on non-spherical cells there is a clear need of being able to control the orientation of the cells, to be able to identify the shape or content of such cells. One example being erythrocytes (a non-spherical but axis-symmetrical cell). Erythrocytes exhibit a biconcave, disc-like shape. When performing flow cytometry analysis, light scattering from such cells is dependent on both their position and orientation and may cause artifacts or inconsistency in the collected signal if not taken into account. While co-axial flow focusing typically is used for positioning, it does not normally control the orientation of non-spherical particles.

Methods to compensate for this problem have been developed such as smarter gating algorithms or simply by sphericalisation of the discs by the addition of chemical substances to the sample. Although these solutions offer satisfactory results in some cases, it may be preferred to actually control the orientation of cells prior to imaging, especially for the upcoming field of imaging cytometry, rather than changing their actual physical shape using chemicals.

Techniques that exist for controlling the orientation of non-spherical cells in continuous flow include the use of magnetic and electric forces, non-spherical nozzle geometries, inertial focusing or fabrication of obstructions within the fluidic channels.

Acoustic focusing has also been shown to orientate non spherical particles in cylindrical capillaries. Cylindrical acoustic resonators will in most cases induce shape specific orientation of non-spherical particles/cells. However, the orientation in such a resonator cannot be predicted. SUM MARY OF THE INVENTION

Acoustic standing wave force fields have been used extensively to focus/concentrate or separate cells in microfluidic system, utilizing the fact that the flow channel serves as the fluid conduit at the same time as it acts as an acoustic resonator defined by the channel dimension.

Typically an acoustic resonance is obtained across the channel perpendicular to the flow direction where a λ/2 standing wave or an integer multiple of λ /2 standing waves are obtained. In a λ /2 resonance particles or cells with a positive acoustic contrast factor Φ will experience an acoustic radiation force F_ax according to the fundamental radiation force equation for a 1-dimensional acoustic standing wave, Eq. 1.

However, so far the acoustic standing wave focusing has shown limited success in studying non- spherical cells or particles or managed to study different sides of non-spherical cells such as erythrocytes in a predictable manner.

By using an essentially square or rectangular micro-channel as resonator, the orientation induced by the acoustic force can always be predicted and controlled. A non-spherical cell/particle exposed to an acoustic force in such a resonator will be orientated so that it will orient itself such that net acoustic force acting on the cell/particle is minimized and is perpendicular to either the sidewalls or the bottom of such a channel. By changing the mode of actuation, either by changing frequency or amplitude of the acoustic actuation, the orientation can be chosen so that in the case of non-spherical and axis-symmetrical cells/particles the smallest dimension of the cells or particles is either perpendicular to the sidewalls, or the top/bottom of the channel. The current invention discloses an adjustable method for both alignment and orientation of non-spherical cells or particles, such as red blood cells and other non-spherical cell types or particles in a continuous flow stream. By actuating a microfluidic structure with ultrasound at its resonance frequency, an acoustic radiation force will both align and orient the cells.

In one aspect the invention relates to a method to orient non-spherical cells or particles in a suspension, which comprises the steps of;

i) subjecting the suspension to pressure, wherein said pressure forces said suspension into one inlet and into at least one channel e.g. present on a microfluidic chip,

ii) subjecting said suspension to one or two dimensional acoustic force(s) directed perpendicular to the length direction of the channel, wherein the channel have an essentially square or rectangular shape, wherein at least one frequency and/or the amplitude and/or FSK

(frequency shift keying) is set so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles these will be aligned and oriented such that its smallest dimension is parallel to the strongest acoustic force along a first axis,

iii) changing the frequency to excite the non-spherical cell or particle orientation to be perpendicular to the initial orientation and

iv) analyzing the non-spherical cells or particles.

Thus it is for the first time possible to study different orientations of non-spherical cells or particles and collect information of the shape of the cells or particles from two orthogonal projections in a controlled and predetermined manner. By controlling the orientation of the cells or particles it is possible to reduce the amount of artifacts in the collected signal produced by the uncertainty of the orientation. By changing the orientation 90° of the cells or particles during an experiment it is possible to collect information from both projections of the cells with a single camera in an imaging cytometer. This is achieved by utilizing different forces that orient the cells or particles into different positions, wherein the difference of the orientation normally is 90 ° from each other. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 Schematic presentation of a microchannel on a chip.

Fig. 2 Schematic presentation of an essentially square/rectangular microchannel on a chip, which typically is obtained by isotropic etching of eg. glass or silicon. Fig. 3 Schematic presentation of a microchannel on a chip.

Fig. 4 Schematic presentation of orientation of asymmetric cells or particles in a square micro- channel or capillary using one frequency. The larger arrows indicate the strongest acoustic force.

Fig. 5 Schematic presentation of orientation of asymmetric cells or particles in a rectangular micro-channel or capillary using multiple frequencies and transducers. The larger arrows indicate the strongest acoustic force.

Fig. 6 An illustration of the microfluidic chip used in the experiments.

Fig. 7 Classification of orientation. The cells are classified as either flat, semi tilted or upended. Care was taken to obtain images with some time apart to avoid analyzing the same cell in more than one image.

Fig. 8 Top left: The horizontal acoustic radiation force F_horizonta| is stronger than the vertical acoustic radiation force F_vett and BCs are presenting ther smallest dimension to the camera, top right. Bottom left: F_vett > F_h0rizontai and RBCs are presenting their flat side to the camera, bottom right.

Fig. 9 Percentage of flat, semi tilted, and upended red blood cells of total observations.

When the voltage of the 2 MHz transducer was varied between 0-2 V the majority of the cells, more than 75 %, were still horizontally oriented with their flat side facing the top of the channel.

DETAILED DESCRIPTION OF THE INVENTION

Letters (x), (y) and (z) refers to the spatial position along the length (I), width (w), and the height (h) of the microchannel, respectively. Letters (Q,), (v), and (p,) refers to volume flow rate, flow velocity and pressure, respectively where subscript (i) indicate multiple instances of a property.

The word suspension refers to a fluid containing solid particles or cells that are sufficiently large for sedimentation. The term "aspect ratio" is intended to mean the correlation between the height:width of a cross section of the channel.

The term "FSK" is intended to mean frequency shift keying, wherein the transducer is actuated with two or more alternating frequencies.

The term "transducers" is intended to mean piezoelectric elements that convert electricity to vibrations.

Methods

The invention relates to methods to orient non-spherical cells or particles in a suspension, wherein the cells or particles are exposed to one or two dimensional acoustic forces which forces the cells or particles to be aligned and oriented so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles the smallest dimension of the cell/particle is parallel with the strongest acoustic force. The cells or particles can then be reoriented 90 degrees, with respect to the previous orientation by altering the resonance conditions in the channel. The resonance conditions are changed by altering either the amplitude, frequency or FSK rate of the actuation transducers. By such an alteration the orientation of the cells or particles can be predicted and shape of the cells can be analysed.

i) subjecting the suspension to pressure, wherein said pressure forces said suspension into one inlet and into at least one channel present on a microfluidic chip,

ii) subjecting said suspension to one or two dimensional acoustic force(s) directed perpendicular to the length direction of the channel, wherein the channel have square rectangular (Figure 1), or an essentially square or rectangular (Figure 1) shape, wherein at least one frequency and/or the amplitude and/or FSK is set so that the cells or particles will be aligned and oriented such that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles the smallest dimension is parallel to the strongest acoustic force along a first axis,

iii) changing the frequency to excite orientation along a second axis perpendicular to the first axis and

iv) analyzing the non-spherical cells or particles. The non-spherical cells or particles may be banana shaped, cubical, rod shaped, rod like flat, rod like bent, spherical with buds, or disc shaped. Thereby cells, including bacterial cells, as well as most of the cells derived from mammals or plants are included. The cells may be any kind of eukaryotic to prokaryotic cells and examples includes both mammalian cells as well as bacterial cells. Specific examples are yeast cells, cancer cells, platelets, red blood cells, white blood cells (such as: neutrophils, eosinophils, basophils, lymphocytes, monocytes and macrophages), adipocytes, Escherichia coli and other bacteria. Another example is sperm cells, wherein there is a need to sort the X and the Y chromosome containing sperm cells from each other prior to insemination.

The pressure that forces the suspension into the inlet of the channel may be induced by a pump or by a syringe as long as the pressure forces the suspension into the inlet of the channel and further into the channel.

The acoustic forces, which force the cells or particles to be aligned and oriented may be induced by the use of one or more piezoelectric transducers as defined above. The transducers may be placed at the same position, at 90° for each other or at arbitrary angles from each other depending on the purpose of the analysis.

The shape of the channel is either square or rectangular or essentially square or rectangular. The aspect ratio may be 1:1, 1:1,5, 1:2,5 or 1:3,5. One example being 1:1.

Examples of aspect ratio between the height and the width can be found in the table below.

Table. 1. Explains different transducer setups and strategies to achieve controlled orientation

• Number of frequencies needed to achieve orientation

The channel may have a width and/or height ranging from 10 μιη to 1000 μιη, 75 μιη to 800 μιη, such as from 75 μιη to 200 μιη, or ranging from 200 μιη to 375 μιη, or ranging from 300 μιη to 400 μιη, or ranging from 400 μιη to 700 μιη, or ranging from 700 μιη to 800 μιη, or being 150 μιτι, 300 μη% 188 μη% 375 μιη or 750 μηη.

Predicting the resonance frequency

The resonance frequency for a channel with a square or rectangular cross-section is determined by the dimensions of the channel, and the speed of sound for the liquid inside. The width and height of the channel may be related such that the width w divided by an integer number n equals the height h divided by an integer number m. In this case a single frequency of vibration may be chosen to fulfill a resonance condition simultaneously for the height and width dimension, such that where c is the speed of sound in the suspending fluid.

If the width and height of the channel are not related such that the width w divided by an integer number n equals the height h divided by an integer number m, the resonance conditions in the channel may be controlled individually/selectively by using two separate frequencies, f_x and f₂ , of vibration for width and height respectively. The frequencies are chosen such that

_ cn _ cm

2w _and ² 2h

where n and m may be any integer number

The frequency of vibration may vary in a range from 1 MHz to 10 MHz and is implicitly dictated by the dimensions of the channel as mentioned above. One example being when the frequency in a first step is l,88Mhz and said frequency then is altered in a next step to l,89Mhz.

In one embodiment the invention relates to a method to orient non-spherical cells or particles in a rectangular microchannel using multiple frequencies and transducer.

The cell or particle suspension containing non spherical cells or particles is continuously injected into the microchannel with rectangular cross-section at a given flow rate. The flow rate may vary between Ο,ΐμί- 20mL/min per channel. The cells or particles are observed through a microscope with high magnification and a high speed camera with sufficiently short shutter time to capture sharp images of cells or particles.

Two ultrasonic (piezoelectric) transducers are attached to the microchannel and are actuated by individual electric signals that can be sine, square, triangle or of any other periodic shape. One transducer is typically actuated at a frequency that gives a vertical resonance at λ/2 in the channel, but can also be multiples of this. The other transducer is typically actuated at a frequency that gives a horizontal resonance at λ/2 in the channel, but can also be multiples of this.

Once a resonance is obtained in one dimension, particles with positive acoustic contrast factor will start to migrate towards the pressure node in the system. Once an asymmetric particle reaches the pressure node, it will stop migration and orient itself according to the direction of the acoustic radiation force, F_rad. The particle will be oriented so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles the smallest dimension is parallel with the strongest acoustic radiation force. Controlling the orientation.

Non-spherical and axis-symmetrical cells or particles will always be oriented so that their smallest dimension is parallel with the strongest acoustic force. In a microchannel with a rectangular cross-section, two main acoustic, half wavelength, resonances can be found, one vertical and one horizontal. The resonances can be actuated by two individual transducers and frequencies. The acoustic pressure amplitude of the individual resonances is controlled by the amplitude of the electric signal actuating the individual transducer. If the vertical resonance induces the strongest acoustic force, cell exposed to this acoustic field will be oriented so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles their smallest dimension is oriented vertically. In this case, by increasing amplitude of the transducer actuating the horizontal resonance the orientation can be changed from vertical to horizontal. By controlling the amplitude of the individual transducers, the orientation can be changed from vertical to horizontal and vice versa.

The width may be chosen such that the frequency of vibration f is

_ cn

2w

there c is the speed of sound in the suspending fluid.

The frequency of vibration may vary in a range from 1 MHz to 10 MHz and is implicitly dictated by the specified dimensions of the channel as mentioned above and by choosing n = 1 and c = 1500 m/s. One example is given when the frequency in a first step is l,88Mhz and said frequency then is altered in a next step to l,89Mhz.

The size of the microchannel constitutes an upper limit of the size of the non-spherical cells or particles to be analysed. The cells or particles may be spherical, cubical, rod shaped, rod like flat, rod like bent or disc shaped The cells may vary in shape and size ranging from 1 μιη to 50 μιτι, such as 1-5 μη% 1-25 μη% 5-50 μη% 5-40 μη% 5-30 μη% 5-25 μη% 8-25 μη% or 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μη% or from 10-20 μιη or 10-15 μιη. The cells or particles may have a volume ranging from 0.0005 to 70 x 10 ¹⁵ m³, such as 0.0005-0.003 x 10 ¹⁵ 0.0005-0.07 x 10 ¹⁵ m³, 0.0005-8 x 10 ¹⁵ m³, 0.05-0.10 x 10 ¹⁵ m³' 0.07-70 x 10 ¹⁵ m³, 0.07-35 x 10 ¹⁵ m³, 0.07-14 x 10 ¹⁵ m³, 0.07-8 x 10 ¹⁵ m³, 0.25-6 x 10 ¹⁵ m³0.3-8 x 10 ¹⁵ m³, 0.07-35 x 10 ¹⁵ m³ or 0.5-15 x 10 ¹⁵ m³.

Once all cells are aligned, either by the acoustic radiation force or the hydrodynamic focusing, the orientation effects from the acoustic radiation force can be observed. In order to minimize the acoustic force potential, the cells will be rotated until their smallest dimension is in parallel with the strongest acoustic radiation force. While still keeping the cells focused in two dimensions by the two perpendicular acoustic fields, the acoustic energy (and thus the acoustic force) in either the vertical or horizontal resonance mode can be controlled by adjusting the driving voltage amplitude of the respectively actuating transducer. This leads the cells to be aligned and rotated into two different angles (Figure 8).

Following examples are intended to illustrate but not to limit the invention in any manner, shape, or form, either explicitly or implicitly. EXAMPLES

Experimental setting for example 1 and 2.

A microchannel structure and holes for inlets and outlets is KOH etched in a <100> silicon wafer of thickness 350 μιη and cut to the dimensions 40 mm by 3 mm. A piece of borosilica glass (40 mm by 3 mm by 1 mm) is anodically bonded to seal the channel. Inlets and outlets comprises of pieces of silicone tubing, which are glued to the backside of the chip to connect tubing for external fluidics. Two piezoceramic actuators, one ~5 M Hz transducer (5 mm by 5 mm) positioned below the microchannel, and one ~2 MHz transducer (15 mm by 5 mm) placed under the microchannel,

Alternatively, the square or rectangular microchannel is made of glass, quartz, metal, ceramic or polymer.

The piezoceramic transducers are driven by two signal-generators equipped with signal power amplifiers, depending on the need power requirements. The acoustic resonances can be controlled by tuning the frequency and voltage driving the transducers.

Example 1. Orientation of non-spherical cells in a rectangular or essentially rectangular silicon/glass microchannel using multiple frequencies and transducer. Experiment setup

The cell suspension containing non-spherical cells is continuously injected into the microchannel with rectangular cross-section at a given flow rate. The flow rate may vary between Ο,ΐμί- 20mL/min per microchannel. The cells are observed through a microscope with high magnification and a high speed camera with sufficiently short shutter time to capture sharp images of cells.

Two ultrasonic (piezoelectric) transducers are attached to the microchannel and are actuated by individual electric signals that can be sine, square, triangle shaped or a combination of sine functions of the fundamental excitation frequency and higher harmonics. One transducer is typically actuated at frequency that gives a vertical resonance at λ/2 in the channel, but can also be multiples of this. The other transducer is typically actuated at frequency that gives a horizontal resonance at λ/2 in the channel, but can also be multiples of this.

Once a resonance is obtained in one dimension, particles with positive acoustic contrast factor will start to migrate towards the pressure node in the system. Once a non-spherical particle reaches the pressure node, it will stop migration and orient itself according to the acoustic radiation force field. The particle will be oriented such that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles its smallest dimension becomes parallel to the acoustic radiation force.

Controlling the orientation.

Particles will always be oriented so that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis-symmetrical cells/particles their smallest dimension becomes parallel to the strongest acoustic force. In a microchannel with a rectangular cross-section or essentially rectangular cross-section, two main acoustic, half wavelength, resonances can be found, one vertical and one horizontal. The resonances are actuated by individual transducers and frequencies. The acoustic pressure amplitude of the individual resonances is controlled by the amplitude of the electric signal actuating the individual transducer. By controlling the amplitude of the individual transducers, the orientation can be changed from vertical to horizontal and vice versa. Example 2 Orientation of non-spherical cells in an essentially square glass microchannel/capillary using a single frequency with minor tuning. Experiment setup

The cell suspension containing non-spherical cells is continuously injected, at a given flow rate, into the microchannel with an essentially square cross-section, having vertical and horizontal channel dimensions only differing a few percent, Channel dimensions can vary between 10-lOOOum. The flow rate may vary between Ο,ΐμί- 20mL/min per channel. The cells are observed through a microscope with high magnification and a high-speed camera with sufficiently short shutter time to capture sharp images of cells.

An ultrasonic (piezoelectric) transducer is attached to the microchannel and is actuated by an electric signal that can be sine, square or triangle shaped or a combination of sine functions of the fundamental excitation frequency and higher harmonics. The actuation frequency is typically set to achieve a λ/2 resonance in either the vertical or the horizontal direction of the channel, but can also be multiples of this. Once a resonance is obtained, particles with positive contrast factor will start to migrate towards a pressure node in the system. Once a non-spherical particle reaches the pressure node in a λ/2 resonance set-up, it will stop the migration and orient itself according to the acoustic radiation force field. The particle will be oriented such that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis- symmetrical cells/particles its smallest dimension becomes parallel to the acoustic radiation force.

Controlling the orientation

Non-spherical and axis-symmetrical cells/particles will always be oriented so that the smallest dimension is parallel to the strongest acoustic force. In a microchannel with an approximately square cross section, two acoustic resonances (one vertical and one horizontal resonance) may be found at almost the same frequency. One of these resonances will always dominate significantly over the other mode, thus orientating the non-spherical cells/particles according to either a horizontal or vertical direction. By fine tuning the frequency, it is possible to find a resonance mode that excites orientation along the other axis rotated 90°, this usually occurs at 10-100 KHz from the first resonance mode depending on the vertical and horizontal dimensions of the essentially square cross-section microchannel. One method to achieve this is by scanning the frequency and observe the orientation of the non-spherical cells. The frequency is scanned until the orientation angle changes 90°. Once the 2 frequencies that give vertical and horizontal orientation are determined, the orientation can be controlled as horizontal or vertical by choosing one of these frequencies as the actuation frequency. By having two resonance modes close to each other both modes are actuated although the driving frequency is chosen to have either of the modes to dominate. At the same time the weaker actuation mode assists in driving the cells/particles into the center of the channel such that cells/particles are acoustically focused in two dimensions and localized in the center of the channel and hence in the optical focal line of the imaging system. The switching between the resonance modes subsequently only decides on a horizontal or vertical orientation of the cell/particle. Example 3.

Chip fabrication

The glass chip, with channel dimensions 375 by 150 μιη and length 4 cm, was fabricated in borosilicate chromium blanks (Telic Company, Valencia, CA) precoated with positive photoresist and fabricated by the means of photolithography and wet etching using a mixture of HF/HN0₃/H₂0. Holes for inlets and outlets were drilled using a diamond glass drill and a glass lid was then thermally bonded to the chip to seal it. The chip had one trifurcation inlet and a single outlet (Figure 6), Two piezoceramic transducers (PZT), (PZ26, Ferroperm piezoceramics, Kvistgaard, Denmark), 2 and 5 MHz respectively, were used to actuate the chip. The two actuators were glued underneath the chip using cyanoacrylate glue (Loctite Super Glue, Henkel Norden AB, Stockholm, Sweden).

System setup

A dual channel function generator (AFG 3022B, Tektronix, UK Ltd., Bracknell, UK) was used to actuate the transducers and the signals were amplified using in-house built amplifiers based on a LT 1012 power amplifier (Linear Technology Corp., Milpitas, USA). The applied voltages were monitored using an oscilloscope (TDS 2120, Tektronix, UK Ltd., Bracknell, USA). Syringe pumps (Nemesys, Cetoni GmbH, Korbussen, Germany) with mounted plastic syringes (BD Plastipak, BD Bioscience, San Jose, USA) were used to control the flow rates. Images were obtained using a high-speed camera (EoSens mini MC-1370, Mikrotron GmbH, Uterschleissheim, Germany). Experiments

Blood samples were collected from healthy donors after acquiring their consent. The blood was then diluted in PBS to appropriate concentrations. A mixture of 7μιη polystyrene beads (Fluka/Sigma Aldrich, Buchs, Switzerland) was prepared as a size and shape reference.

The flow rate was kept constant during all experiments at a total of 12 μί min^"1 distributed as 2 μί min^"1 for the centre inlet (blood sample) and 5 μί min^"1 from each side inlet (sheath flow, PBS). The side inlets were used to hydrodynamically position cells in order to facilitate imaging when the actuator driving the horizontal resonance was turned off. Images of the cells were obtained at a fixed position in the channel, and care was taken so that no picture contained the same cell twice.

The 5M Hz transducer was driven at 10.2 Vpp with a 5.76 M Hz sine signal during all experiments. This transducer actuated a vertical resonance in the chip, forcing all cells to the vertical (cross-sectional) center of the microfluidic, putting them all in focus of the microscope lens and orientating them with their flat side orthogonal to the camera axis. The 2 M Hz transducer was operated at 2.26M Hz sine, and the voltage amplitude was varied between 0-10 Vpp in order to obtain the rotation of the cells. This transducer induced a horizontal resonance in the chip, driving all cells to the horizontal (cross-sectional) center of the chip when actuated

Classification of orientation

From the images obtained 200 cells were counted and classified for each voltage setting. The cells were classified as either flat, horizontally oriented in the channel, semi tilted, or upended, vertically oriented in the channel, seen from above. (Figure 7) Orientation efficiency

The orientation efficiency of the system was measured by analyzing images obtained of the cells at a fixed part of the channel. The cells were classified as either flat, semi tilted, or upended, seen from above. While keeping the voltage of 5 MHz transducer constant, focusing the cells in the horizontal dimension, the voltage of the 2 MHz transducer was varied to in an interval from 0-10 V to get the cells more and more vertically oriented instead. From figure 9 three different orientation stages can be observed.

The maximum percentage of cells that could be horizontally oriented was 87 %. Between 2.5-3 V the cells were starting to become turned to be vertically oriented. For 2.5 V the percentage of cells that were horizontally oriented or semi tilted were about the same at 40 % and 40.8 %, respectively while the vertically oriented were 18.9 %. At 3 V this had changed in favor of the vertically oriented cells that were about the same percentage as the semi tilted (39 % and 46.3 %, respectively, while the percentage of horizontally oriented cells had decreased to 15 %. When the voltage of the transducer was varied between 3.5-10 V the vertically oriented cells were well in majority with more than 86.1 % of the cells oriented in this way. The maximum percentage of cells that could be vertically oriented was 98.1 %, while the rest 1.9 % were semi tilted.

Claims

1. A method to orient non-spherical cells or particles in a suspension, which comprises the steps of;

ii) subjecting said suspension to one or two dimensional acoustic force(s) directed perpendicular to the length direction of the channel, wherein the channel have square or rectangular cross-section shape, wherein at least one frequency and/or the amplitude and/or frequency shift keying is set so that the cells will be aligned and oriented such that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis- symmetrical cells/particles its smallest dimension is parallel to the strongest acoustic force along a first axis,

iii) changing the frequency to excite the non-spherical cell or particle orientation to be perpendicular to the first axis and

iv) analyzing the non-spherical cells or particles.

2 The method according to claim 1, wherein the channel in i) is either rectangular, square or essentially rectangular or square.

3. The method according to claim 2, wherein the channel is rectangular or essentially rectangular.

4. The method according to claims 1-3, wherein the channel have the aspect ratio of 1:1, 1:1,5, 1:2,5 or 1:3,5.

5. The method according to any of preceding claims, wherein two frequencies and either the amplitude or FSK is set so that the cells will be aligned and oriented such that the net acoustic force acting on the cell/particle is minimized and in the case of non-spherical and axis- symmetrical cells/particles its smallest dimension is parallel to the strongest acoustic force.

6. The method according to any of the preceding claims, wherein the height or the width of channel is from 10 um to 1000 um.

7. The method according to any of the preceding claims, wherein said acoustic force is induced by an excitation frequency ranging from 1 to 10 MHz.

8. The method according to any of the preceding claims, wherein said frequency in step ii) is l,88Mhz and said frequency in step iii) is l,89Mhz.

9. The method according to any of preceding claims, wherein said non-spherical cells or particles are banana shaped, cubical, rod shaped, rod like flat, rod like bent or disc shaped.

10. The method according to any of the preceding claims, wherein said cells are red blood, bacteria or sperm cells.