Cell adhesion measurement

Abstract

The invention relates to an optical nanomotion detection (ONMD) method for determining adhesion of one or more single living cells.

Description

[0001] CELL ADHESION MEASUREMENT

[0002] FIELD OF THE INVENTION

[0003] The invention relates to an optical nanomotion detection (ONMD) method for determining adhesion of one or more single living cells.

[0004] BACKGROUND OF THE INVENTION

[0005] Adhesion is a fundamental property of living cells that permits their attachment to other organisms or various organic and inorganic substrates. It plays a fundamental role in numerous physiological and pathological processes such as cell growth, migration, immune response, pathogen-host interaction, and tumor cell growth and spreading. Despite the existence of different techniques to measure adhesion, its quantification is still a challenge. A comprehensive review of cellular adhesion's importance in physiological and pathological processes as well as measurement techniques can be found in Ungai-Salanki, R. et al, "A Practical Review on the Measurement Tools for Cellular Adhesion Force", ADVANCES IN COLLOID AND INTERFACE SCIENCE, 2019, 269, 309-333. Adhesion measurement is relatively complex, requires sophisticated equipment, and in most cases cannot be carried out without breaking the links between the studied cell and its target. Most of the existing techniques rely on the detachment of the cells upon an applied force. Liquid flow (shear stress), centrifugal acceleration, micropipette manipulation, optical tweezers, atomic force microscopy or FRET force sensors can generate this force. For instance, US patent application 2022 / 0118453 discloses a method of manipulating and / or investigating cellular bodies adhesion onto a functionalised surface, wherein a sample comprising one or more cellular bodies in a fluid medium inside a holding space is subjected to a force generated by an acoustic wave.

[0006] However, applying it with a force in the range of nano-newton on single cells is not a trivial task, and, therefore, the setup of such measurements is relatively complex and relies on expensive equipment. In addition, detachment-based measurements do not permit gaining information about the adhesion process as a function of time at a single-cell level. An alternative to the force-applying devices is the use of planar optical waveguides that monitor the contact surface between the living organism and the substrate. This last approach requires specially treated surfaces and only informs about the surface of contact between the cell and its substrate. US patent application 2022 / 0283074 discloses a method for deriving particle characteristics comprising imaging the movement of at least one free-floating particle in a liquid environment at at least one moment in time, determining for at least one moment in time a movement parameter based on the imaged movement of the free-floating particles in the liquid environment, and deriving from the movement parameter a characteristic of the at least one particle. This is the first report on the use of optical means for visualizing so-called cell nanomotion (NM), a methodology named optical nanomotion detection (ONMD). The document also reports the use of ONMD to perform Antifungal Susceptibility Testing (AFST) of surface-attached cells, but remains silent regarding the possibility to evaluate the adhesion strength of a living cell onto a support surface.

[0007] Thus, there is a significant unmet need for analysing and determining adhesion of cells.

[0008] SUMMARY OF THE INVENTION

[0009] An aspect of the present invention provides an optical nanomotion detection (ONMD) method for determining adhesion of one or more single living cells to a substrate, the method comprising

[0010] • contacting the substrate with the one or more single living cells;

[0011] • recording nanomotion of the one or more single living cells;

[0012] • processing the recorded nanomotion of the one or more single living cells to determine the adhesion of the one or more single living cells to the substrate and / or evolution of the adhesion of the one or more single living cells to the substrate over time, wherein the processing of the recorded nanomotion comprises determining area covered by nanomotion of the one or more single living cells during the duration of the recoding, wherein the area covered by nanomotion corresponds to a number of pixels covered by the nanomotion, and wherein o the larger area and / or an increase of the area during the recording indicates a weak adhesion of the one or more single living cells to the substrate, and o the smaller area and / or a decrease of the area during the recording indicates a strong adhesion of the one or more single living cells to the substrate.

[0013] Another aspect of the present invention provides a computer implemented method for determining adhesion of one or more single living cells according to the method of the invention, the method comprising • receiving video data comprising video record of nanomotion of the one or more single living cells during at least 5 seconds, preferably during at least 10 seconds; and

[0014] • processing the video data to determine the adhesion of the one or more single living cells to the substrate, wherein the processing the video data comprises determining area of nanomotion of the one or more single living cells during the duration of the recoding, and wherein the area of nanomotion corresponds to a number of pixels covered by the nanomotion.

[0015] A further aspect of the present invention provides a device adapted to carry out the method for determining adhesion of one or more single living cells according to the method of the invention, wherein the device comprises an optical microscope unit coupled to a camera and a processing unit, wherein the optical microscope unit coupled to a camera is configured to record nanomotion of the one or more single living cells during at least 5 seconds, preferably during at least 10 seconds and the processing unit is configured to process the recorded nanomotion of the one or more single living cells to determine area of nanomotion of the one or more single living cells during the duration of the recoding, and wherein the area of nanomotion corresponds to a number of pixels covered by the nanomotion.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] Figure 1 shows two different techniques are used to measure adhesion: 1-4 Classical technique based on the ratio between floating and adhering cells and 5. nanomotion-based measurement. The classical method ( 1 -4b) consists in depositing yeast cells onto a fibronectin-coat Petri dish for 40 min (1). The supernatant is than removed (2a), deposited into a new Petri dish and covered with agar (3a). The cells eventually divide and form colony -forming units (CFU) that permits to estimate their number. The cells that remained attached to the fibronectin covered Petri dish (2b) were also covered with agar (3b) and developed CFUs too (4b). The nanomotion based measurement consists in depositing the yeast cells onto a fibronectin covered sample holder glass and in recording their nanometer scale displacements (nanomotion) during 10 s. A dedicated software processes the recorded movies of nanomotion to compute every cell displacement.

[0018] Figure 2 shows random walk simulations of a constrained (A) and not constrained (B) particle during 50,000 time steps. The “rope” that constrained the particle in the frame A. had a length of 80 a.u. and is attached at the center of the graph. The number of pixels (gray squares) visited by the particle is higher in the non-constrained case (n=13) than in the constrained one (n=4).

[0019] Figure 3 shows adhesion results for 7 different C. albicans strains. The upper panel represents traditional adhesion test results whereas the lower panel shows the corresponding ONMD data. The upper horizontal blue dashed line indicates the average displacement value of the cells at the beginning of the ONMD experiments (t=0 min). The lower brown dashed horizontal line indicates that 50% of the cells are attached to the fibronectin-coated surface in the adhesion tests. * p<0.05; **p, 0.01; *** p<0.001.

[0020] Figure 4 shows linear fit of the traditional (percentage of adhesion) versus the ONMD adhesion measurements results. The fitting equation has a slope-intercept form (y = a + b*x), with intercept (a): 133.72 ± 19.88 and slope (b): -82.90 ± 20.47.

[0021] Figure 5 shows evolution of the nanomotion of different C. albicans strains as a function of time. As it can be noticed, the different strains behave differently upon exposure to fibronectin- coated surfaces.

[0022] Figure 6 shows typical path of single (A) CEC3672 and (B) 101 C. albicans cells. As depicted in this figure, the lower adherent cell (101) moves to larger distances than the higher adherent one (CEC3672). The dashed circles correspond to the maximal distance reached by both cell types. In (B), both embedding circles are displayed for comparison.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0025] In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0026] The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Also as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of “ and / or "consisting essentially of’. The terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as "including", "comprising" or "having" certain elements are also contemplated as "consisting essentially of" and "consisting of those certain elements.

[0027] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0028] As used in the specification and claims, the term "and / or" used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A", and "B".

[0029] An aspect of the present invention provides an optical nanomotion detection (ONMD) method for determining adhesion of one or more single living cells to a substrate, the method comprising

[0030] • contacting the substrate with the one or more single living cells;

[0031] • recording nanomotion of the one or more single living cells;

[0032] • processing the recorded nanomotion of the one or more single living cells to determine the adhesion of the one or more single living cells to the substrate and / or evolution of the adhesion of the one or more single living cells to the substrate over time, wherein the processing of the recorded nanomotion comprises determining area covered by nanomotion of the one or more single living cells during the duration of the recoding, wherein the area covered by nanomotion corresponds to a number of pixels covered by the nanomotion, and wherein o the larger area and / or an increase of the area during the recording indicates a weak adhesion of the one or more single living cells to the substrate, and o the smaller area and / or a decrease of the area during the recording indicates a strong adhesion of the one or more single living cells to the substrate. In some embodiments of the method of the invention, the one or more single living cells are selected from the group comprising human cells, animal cells, plant cells, bacteria, fungi, and yeast. In some embodiments, the human cells are selected from the group comprising stem cells, bone cells, blood cells, muscle cells, fat cells, skin cells, nerve cells, epithelial cells, sex cells, and cancer cells.

[0033] According to an embodiment of the method of the invention, contacting the substrate with the one or more single living cells is carried out under suitable incubation conditions that allows the single living cell to remain alive and to adhere (attach), preferably initially adhere (initially attach) to the substrate. The suitable incubation conditions are known in the art and can be adapted to each type of cells (bacteria, fungi, yeast, animal cells, human cells, plant cells).

[0034] In preferred embodiment of the method of the invention, recording nanomotion of the one or more single living cells is recording nanomotion of the one or more single living cells attached (adhered) to the substrate.

[0035] In some embodiments, the method of the invention further comprises a comparison step between the recorded nanomotion of the one or more single living cells with nanomotion of a reference standard, wherein the step of processing the recorded nanomotion of the one or more single living cells comprises a step of comparing the area covered by nanomotion of the one or more single living cells during the duration of the recoding with the area covered by nanomotion of a reference standard during the same duration, and wherein

[0036] • the larger area and / or an increase of the area during the recording indicates a weaker adhesion of the one or more single living cells to the substrate compared to the adhesion of the reference standard, and

[0037] • the smaller area and / or a decrease of the area during the recording indicates a stronger adhesion of the one or more single living cells to the substrate compared to the adhesion of the reference standard.

[0038] As herein used, "a reference standard" is the same one or more single living cell(s) used for determining adhesion (i.e. studied one or more single living cell), but which is not attached to the substrate, i.e the single living cell is free and has free movement. The reference standard can be for instance a cell freely floating inside a liquid culture medium, or a mutated cell compared to the cell under analysis, or a non-mutated cell compared to the cell under analysis and the like. The area covered by nanomotion of the reference standard is typically recorded during the first few seconds (1 to 5 seconds) after contacting the substrate with the one or more single living cells, i.e. before adhesion of the one or more single living cells to the substrate starts. Alternatively, "a reference standard" is one or more single living cell(s) of the same or different type / strain of the studied (target) single living cell for which a nanomotion is recorded and stored as a reference in predetermined conditions. Suitable conditions may be linked to temperature, pressure, type of adhesion surface and / or any other parameter adapted to permit to a user to establish a correlation between the recorded nanomotion and a condition of interest.

[0039] In preferred embodiments, the method of the invention is a computer implemented method.

[0040] In an embodiment of the method of the invention, the substrate is a solid surface, preferably a flat solid surface. In a preferred embodiment, the solid surface is selected from a glass surface, metal surface, plastic surface, or polymer surface. In some embodiments, the solid surface is coated with an adhesive material. In some embodiments, the adhesive material is selected from the group comprising fibronectin, collagen, polyvinyl chloride (PVC), polyurethane, silicone, Teflon, nylon polyethylene, titanium, cobalt-chromium alloys, ceramics (alumina, zirconia), and acrylics. The above are typical test conditions used to determine and analyze cell adhesion.

[0041] In another embodiment of the method of the invention, the nanomotion of the one or more single living cells is recorded typically during the following recording time (period) : during at least 5 seconds, during at least 10 seconds, during at least 60 seconds, during at least one hour or during at least 1 day (24 hours). In a further embodiment of the method of the invention, the nanomotion of the one or more single living cells is recorded during 5 seconds, during 10 seconds, during 60 seconds, during 1 hour or during one day (24 hours). In a further embodiment of the method of the invention, the nanomotion of the one or more single living cells is recorded during 5 seconds to 10 seconds, during 5 seconds to 60 seconds, during 10 seconds to 60 seconds, during 5 seconds to 1 hour, during 10 seconds to 1 hour, or during one day (24 hours). In another embodiment of the method of the invention, the recording of the nanomotion of the one or more single living cells can be repeated two, three, four, five, six, seven, eight, nine or times every 5, 10, 20, 30, or 40 minutes. In another embodiment of the method of the invention, the determination of evolution of the adhesion of the one or more single living cells to the substrate over time is typically determined during the recording time (period).

[0042] In a further embodiment of the method of the invention, the recording of nanomotion of the one or more single living cells is carried out by suitable means for recording nanomotion of the one or more single living cells; preferably the recording of the nanomotion carried out by an optical microscope coupled to a camera, such as an inverted optical microscope (Zeiss Observer Z. l) with for instance a 63x oil immersion objective, coupled for instance to a PCO Edge 5.5 camera. The camera is configured to typically record at least 5 seconds or preferably at least 10 seconds long videos (e.g. avi format) at a frame rate of typically 30 frames per second (fps).

[0043] In a further embodiment of the method of the invention, the processing of the recorded nanomotion of the one or more single living cells is carried out by an image-processing software running on a computer device operatively connected to means for recording nanomotion of the one or more single living cells. The image-processing software allows tracking displacements of the one or more single living cells (such as bacteria, yeast or fungi) with a subpixel resolution. The image-processing software analyzes the number of pixels covered by nanomotion of the one or more single living cells during the duration of the recording. According to a preferred embodiment, the method of the invention analyzes the evolution (modification) of nanomotion of the one or more single living cells during the duration of the recording and compares the nanomotion of the one or more single living cells just after its exposure (contact) to the substrate with the nanomotion of the one or more single living cells during at least 5 seconds, preferably at least 10 seconds.

[0044] According to a typical embodiment of the software, a Matlab (R2023a) computer program simulates a massless particle submitted to a random walk during 50’000 time cycles. Two simulation rounds were carried out: one with a “rope” of 80 arbitrary length units that restrains the distance to which the particle can diffuse and a second round that simulates a free, “ropeless” particle diffusion. The random walk simulation consisted of randomly choosing a displacement direction at every simulation step and displacing the particle in that direction with an arbitrary unit length. In the “particle attached to a rope” simulation, the software checked the distance between the spot from which the simulation started (in the present case x=0 and y=0) and the particle's actual position. If it exceeds that of the rope another random displacement direction is tested and “refused” unless it decreases the distance between the particle and its starting position.

[0045] The method of the invention provides a single cell sensitivity, wherein a single cell is contacted to a substrate and wherein a single cell is spatially separated from other single cells. Thus, the nanomotion of one or more single cells is detected and analysed, rather than nanomotion of clumps of cells.

[0046] A single cell is obtained or isolated from a tissue, a multicell organism, a clump of cells, a population of cells or colony of cells by methods known in the art, such as microfluidic dissociation, ultrasound dissociation (sonication), enzymatic dissociation (with Trypsine and / or Collagenase), chemical dissociation (e.g. EDTA), mechanical dissociation (e.g. pipetting and / or mechanical shaking).

[0047] According to some embodiments of the optical nanomotion detection method of the present invention, the larger area and / or an increase of the area during the recording further indicates a low risk of contamination of the substrate by the one or more living cells, and wherein the smaller area and / or a decrease of the area during the recording further indicates an increased risk of contamination of the substrate by the one or more living cells.

[0048] In some embodiments, the low risk of contamination also encompasses reduced risk, delayed (slowed) contamination, and / or no contamination of the substrate by the one or more living cells. In some instances, the low risk of contamination indicates the resistance of the substrate to be contaminated by the one or more living cells.

[0049] In further embodiments the contamination refers to the ability of the one or more living cells to colonize and / or to permanently adhere (permanently attach) to the substrate.

[0050] In other embodiments, the increased risk of contamination of the substrate by the one or more living cells includes the ability of the one or more living cells to contaminate the substrate and / or indicates increased and / or important invasive ability of the one or more single living cells to contaminate the substrate. The method of the invention relies on the detection of cellular nanomotion and, is, therefore, essentially limited to living cells or organisms. Indeed, all living cells or organisms oscillate at a nanometric scale as long as they are alive. These oscillations, referred to as nanomotion since the displacements are in the nanometer-micrometers range, such as 10 nm to 10 pm, and stops as soon as the cell or the organism is dead. Nanomotion exists in virtually all living organisms on earth. Nanomotion was initially highlighted by atomic force microscopy (AFM) but later it appeared that classical optical microscopes equipped with a camera also detect nanomotion. Thus the method of the invention mainly consists of recording a movie of the living cell or organism and processing it with motion detection dedicated software that allows tracking cell or organism displacements (such as bacterial or fungal displacements) with a subpixel resolution; therefore, the method is referred to as an optical nanomotion detection (ONMD).

[0051] The nanomotion-based adhesion measurement of the invention relies on the position of the studied cell or organism over time (as a function of time), wherein the studied cell or organism adheres to a substrate and it is therefore in condition of constraint movement - i.e., the studied cell or organism cannot freely move and displace itself along the adhesion substrate. The method of the invention is based on observing the cellular nanomotion of a single cell, group of cells or organism by optical microscopy during the adhesion process. Depending on the attachment force and the length of the link between the studied cell or organism and the substrate, the displacements of the cell or the organism will be more or less constrained, i.e., the displacement envelope of a cell or an organism is inversely proportional to the adhesive force and the length of the anchoring linker. A strongly attached cell or organism will be constrained to a smaller area than a loosely (weakly) attached one. To confirm this, the nanomotion amplitude of different C. albicans strains deposited on optical quality petri dish coated with fibronectin was examined. The amplitude of nanomotion of the different strains was measured and compared it to the values obtained by classical adhesion tests. The obtained results confirmed that strongly attached cells had a lower nanomotion amplitude than those that were loosely (weakly) attached. Since adhesion between living cells and a substrate is mediated through adhesive or linker molecules that have a given length and a well-defined stiffness, the constrained nanomotion of the living cell will be a function of the very nature and amount of the adhesive or linker molecules decorating its surface membrane. The length (L) of these molecules determines to which distance the cell can move. The ability (probability) to move further than the distance L can be used to determine the linker molecule stiffness. Both these parameters can be assessed by looking at the nanomotion pattern of the attached cell. As a way of example, laminin rest length is 150 nm. A cell strongly attached to a substrate by a spacer molecule can freely move in a radius determined by the length of the linker (high movability) but might not be able to detach if the bond between the linker and the molecule and the link between the linker and the substrate is too strong (high adhesion). Accordingly, because of its constrained nature, the cell will experience a sort of “spring force” bringing the same back and forth from a starting point to the maximum length of the linker molecules, defining on a 2- dimensional plane a surface (also referred to herein as area) that is measurable through optical means.

[0052] The adhesion of an "object" to another is measured, in methods according to the state of the art, by applying an external force on one of the objects and in monitoring its displacement under the effect of this force. The stronger the force, the further the object will move. Importantly, in such measurements the displacement of the object is uni-directional, i.e. in the direction of the applied force. To the contrary, the method of the present invention is devoid of external forces expressly imparted to measure cell adhesion, and exploits on the other hand a force that is generated by the object itself i.e. by its nanomotion. Nanomotion is defined as oscillations at the nanometric scale that exist in all living organisms and that makes living organisms "shaking" as long they are alive. The force generated by the nanomotion induces displacements of the object in random directions. By considering the nanomotion-generated force constant, the object displacements are inversely proportional to its adhesion.

[0053] According to the present invention, the optical nanomotion detection (ONMD) is used to determine the adhesion of one or more single living cells (such as prokaryotes and eukaryotes) or organisms, such as bacteria, fungi, vegetal cells and animal cells, onto a substrate, such as solid flat surfaces, in a very rapid, cost-effective, and experimentally very simple way. Recording by classical optical microscopy the nanomotion of living cells or organisms and processing the movies of recorded nanomotion with a dedicated software permits not only to determine the adhesion but also following the adhesion evolution (adhesion modifications) over time (a function of time). The technique is very straightforward and does not require any forceapplying devices. The living cells spontaneous nanomotion serves as a force generator and the cellular attachment to a substrate constrains cellular displacements. Importantly, the measurement does not break the cell-substrate bond and permits, therefore, measuring the adhesion over time (as a function of time) or as a function of various chemical compounds that are put into contact with the studied cell or organism at different experimental time points to determine whether the chemical compounds increase or decrease cells or organisms adhesion onto the substrate, such as a medical device. The evolution of the adhesion over time (as a function of time) is a poorly known parameter that is relatively difficult to measure with classical adhesion tests. It very probably reflects the speed at which different adhesive molecules that are present on the cell wall bind to the substrate and could be a pertinent parameter to identify a given cell or organism (strain) or inform about the invasive potential of the studied cell or organism (strain).

[0054] In an embodiment, the adhesion force is extrapolated by evaluating the area of ONMD, and not a trajectory, because it is assumed that the movement of a living cell or an organism is constrained. In other words, the cell or the organism can only move back and forth from a central point up to a generally round perimeter to describe an area around the starting point, based on the adhesion force that links the cell or the organism to the substrate.

[0055] The method of the invention has numerous advantages as compared to traditional adhesion tests: it is extremely rapid, does not require any force-applying device, does not destroy the cell-substrate bond during the measurement, and can provide information on the adhesion evolution over time (as a function of time). Indeed, importantly the method of the invention offers the possibility of monitoring the evolution of the adhesion over time (as a function of time) without the need to destroy the link between the living cell or organism and a substrate. The experimental setup is also very simple and is limited to a traditional optical microscope equipped with a camera and dedicated image-processing software. The method of the invention is also cost effective, since the very same cell type can be employed in the very same experimental conditions to compare different adhesive surfaces.

[0056] The method of the invention is particularly suitable for providing relative values of adhesion (relative measurement / determination of adhesion), i.e. comparing the adhesion between a given (studied) cell type (the one or more single living cells) and two or more different substrates or the adhesion of different cell types (different one or more single living cells) to a given (studied) substrate. In another embodiment, the method of the invention can provide relative values of adhesion (relative measurement / determination of adhesion), i.e. the method of the invention can provide indication if one cell type (the first one or more single living cells) adheres more or less than another cell type (the second one or more single living cells) on a given (studied) substrate. In a further embodiment, absolute values for adhesion force can be obtained by calibration the nanomotion method with well-established techniques.

[0057] According to some embodiments, determining adhesion (or measuring adhesion) of living cells or the microorganisms is important to determine which surface treatment of the substrate or which material / composition of the substrate is the less adhesive to the cells or the microorganisms. Several industrial and scientific domains are interested in determining (measuring) or evaluating the cells adhesion or the microorganisms on manufactured or modified substates, such as surfaces. The following engineering / scientific domains could benefit of such adherence measurement and / or evaluation:

[0058] • Healthcare (medical devices, catheters, implants, prostheses, dental research, hospital hygiene, etc...).

[0059] • Food industry (food processing equipment, food packaging, beverage processing, fermentation in bioreactors, etc...).

[0060] • Pharmaceutical industry (development of antibiotics, antimicrobial coatings, sterilization testing, etc...).

[0061] • Water treatment (water pipelines and storage tanks, wastewater treatment, etc...).

[0062] • Material sciences (antimicrobial coatings, biomaterials, corrosion prevention, etc..).

[0063] • Environmental biology research (microbial adhesion on natural ecosystems, biofilm formation).

[0064] • Civil engineering (evaluation of biodeterioration of buildings, bridges and historical structures).

[0065] A rapid, simple to implement and label-label free adhesion measurement could dramatically accelerate the selection of the less adherent substrates (such as surfaces) in all the above- mentioned engineering / scientific fields.

[0066] Another aspect of the present invention provides a computer implemented method for determining adhesion of one or more single living cells according to the method of the present invention, the method comprising • receiving video data comprising video record of nanomotion of the one or more single living cells during at least 5 seconds, preferably during at least 10 seconds; and

[0067] • processing the video data to determine the adhesion of the one or more single living cells to the substrate, wherein the processing the video data comprises determining area of nanomotion of the one or more single living cells during the duration of the recoding, and wherein the area of nanomotion corresponds to a number of pixels covered by the nanomotion.

[0068] A further aspect of the present invention provides a device adapted to carry out the method for determining adhesion of one or more single living cells according to the method of the present invention, wherein the device comprising suitable means for recording nanomotion of the one or more single living cells, preferably an optical microscope unit coupled to a camera, and a processing unit, wherein the means for recording nanomotion of the one or more single living cells, preferably an optical microscope unit coupled to a camera, is configured to record nanomotion of the one or more single living cells during at least 5 seconds, preferably during at least 10 seconds and the processing unit is configured to process the recorded nanomotion of the one or more single living cells to determine area of nanomotion of the one or more single living cells during the duration of the recoding, and wherein the area of nanomotion corresponds to a number of pixels covered by the nanomotion.

[0069] In some embodiments, the optical microscope unit coupled to a camera is for example an inverted optical microscope (Zeiss Observer Z. l) with a 63x oil immersion objective, coupled to a PCO Edge 5.5 camera. The camera is configured to typically record at least 5 seconds or preferably at least 10 seconds long videos (e.g. avi format) at a frame rate of typically 30 frames per second (fps).

[0070] In some embodiments, the nanomotion of the one or more single living cells is recorded typically during at least 5 seconds, during at least 10 seconds, during at least 60 seconds, during at least one hour or during at least 1 day. In another embodiment of the method of the invention, the recording of the nanomotion of the one or more single living cells can be repeated two, three, four, five, six, seven, eight, nine or times every 5, 10, 20, 30, or 40 minutes.

[0071] The method of the invention can be used to measure the ability of pathogenic organisms (such as bacteria, fungi or yeast) to adhere (i.e. to contaminate) to solid surfaces of medical devices, such as catheters surfaces. The method of the invention is therefore very useful to rapidly evaluate adhesive properties of pathogenic organisms on surfaces of different medical devices. Thus the method of the invention can be also used in medical material development or antibacterial surface treatments, wherein the measuring of adhesion of pathogenic organisms is of great importance. Indeed, the material and surfaces used in the medical filed must avoid or minimize adhesion of pathogens, such as bacteria or fungi, on medical devices, such as catheters, prothesis, surgical tools, in order to avoid or minimize adherence and / or replication of these pathogens on the medical devices and ultimately avoid pathogenic infection of patients.

[0072] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[0073] The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the application and the scope of the invention.

[0074] EXAMPLES

[0075] Yeast cell culture

[0076] In this study, C. albicans strains CEC3672[9], CEC3609

[0010] , CEC3678[9], CEC3621

[0011] , SC5314

[0012] , 101

[0013] and CEC3675[9] were used. The yeasts were plated from cryo stocks on yeast-extract peptone-dextrose (YPD) agar and cultured for 24 h at 30°C. YPD was composed of D-glucose (Gibco™ 15023021) 40 gr / 1, Peptone (Millipore 82303) 10 g / 1, Yeast extract 10 g / 1, agar (plates only) 15 g / 1 diluted in distilled water. Single colonies from the agar plates were then grown in 4 ml YPD liquid medium at 30°C shaking at 160 rpm overnight.

[0077] Adhesion assay and Optical Nanomotion methodologies Two different methods were used to measure C. albicans adhesion as illustrated in Figure 1. The first method consisted of incubating freely floating yeast cells in wells coated with fibronectin. A 6-well culture plate was incubated for an hour with fibronectin (50 pg / ml) (fibronectin human plasma, lyophilized powder, BioReagent, F2006). The liquid was gently removed, and 250 cells suspended in 500 pl of YPD medium were added to each well. After 25 minutes, the supernatant containing the freely floating cells was removed (Figure 1, 2a) and inoculated in a new YPD agar Petri dish (3a); attached cells were immersed in Sabourand Dextrose (SDA) agar (3b), by adding melted agar at 40°C. SDA was composed of d-glucose (Gibco™ 15023021) 40 gr / 1, peptone (Millipore 82303) 10 g / 1, agar 15g / 1 diluted in distilled water. After an incubation of 48 h the yeast cells in both Petri dishes formed colonies (4a, 4b). The % adhesion was calculated by dividing the number of grown cells in SDA agar by the total number of grown cells (in both YPD and SDA agar) for each strain.

[0078] The second method, i.e., ONMD, involved recording with a microscope 10 seconds long movies of the attachment of C. albicans cells. Petri dishes (p-Dish 35 mm, low uncoated, 80131 IBIDI) were incubated for an hour with fibronectin (50 pg / ml) (BioReagent, F2006) according to the protocol recommended at www.ibidi.com. The liquid was removed and 1.5 ml of YPD liquid medium with 20 pl of overnight culture was added to the Petri dish with the fibronectin layer. Two minutes after placing the plates under the microscope, the first video (time 0) was recorded, followed by videos at 5, 10, 20, 30, and 40 minutes. The movies were then processed with a dedicated Matlab (R2023a) software that tracked their position with a sub-pixel resolution.

[0079] Constrained random walk simulation

[0080] A Matlab (R2023a) computer program simulates a massless particle submitted to a random walk during 50’000 time cycles. Two simulation rounds were carried out: one with a “rope” of 80 arbitrary length units that restrains the distance to which the particle can diffuse and a second round that simulates a free, “ropeless” particle diffusion. The random walk simulation consisted of randomly choosing a displacement direction at every simulation step and displacing the particle in that direction with an arbitrary unit length. In the “particle attached to a rope” simulation, the software checked the distance between the spot from which the simulation started (in the present case x=0 and y=0) and the particle's actual position. If it exceeds that of the rope another random displacement direction is tested and “refused” unless it decreases the distance between the particle and its starting position. Video recording and data processing

[0081] For these experiments, an inverted optical microscope (Zeiss Observer Z.l) with a 63x oil immersion objective, coupled to a PCO Edge 5.5 camera, was used. The camera typically recorded 10 seconds long videos (avi format) at a frame rate of 30 frames per second (fps). All the measurements were carried out at room temperature without phase contrast nor fluorescent staining. A custom-made tracking algorithm implemented in Matlab was used, that tracked individual cells in 2D along 100-200 frames. The algorithm reached a sub-pixel resolution.

[0082] Statistical analysis

[0083] Statistical analysis was done using OriginPro, version 2021. The T-test was used to analyze the significance of the results (* p < 0.05). Each experiment was replicated at least three times. Optical nanomotion was analyzed for each case using more than 30 individual cells. For the adhesion assay, more than eight replicates were conducted.

[0084] Results

[0085] To confirm that a low adhesion force corresponds to a higher displacement freedom, on constrained and non-constrained random walk simulations were carried out. If assuming that the grid lines of the graphs delimit single pixels, Figure 2 clearly shows that a constrained particle “visits” (gray color squares) much less pixels than a free one. In the present simulation, only 4 pixels were visited by the attached particle whereas 13 by the free one.

[0086] Seven different C. albicans strains were compared by both the classical cell attachment assays and ONMD. Figure 3 displays the results obtained by the two methods after a 40 min-long attachment period. These results confirm that strains whose cells have a low attachment capacity (located below the 50% adhesion line) have cells that display more freedom in their displacements (located above the blue dashed horizontal line). Inversely, C. albicans strains that show strong cell binding (located above the brown dashed line) display a smaller ONMD (are situated below the blue dashed line of the upper graph).

[0087] To highlight a putative correlation between classical adhesion tests and ONMD measurements, both data sets were displayed onto the same graph and fitted it with a first order polynomial as depicted in Figure 4. The R-square of the linear fit is 0.77 which indicates a high correlation. As mentioned previously, ONMD permits to monitor the adhesion force as a function of time. Figure 5 shows the evolution of the nanomotion of different C. albicans strains during 40 min. As visible on this graph, the different strains behave differently upon exposure to fibronectin- coated surfaces. Certain strains increase their adhesion as a function of time such as strain CEC3609 whereas others such as CEC3678, reduce it. This type of information is relatively easily accessible by ONMD. It provides valuable information about yeast attachment dynamics and subtle differences in the attachment processes of the different strains.

[0088] The free and constrained random walk simulations demonstrated that free cells cover a larger distance than attached ones. To confirm this behaviour with attached living cells, the displacement trajectories of a strongly (3672) and a poorly attached (101) cell were displayed. Figure 6 displays the trajectories of the two cells during 200 frames (i.e., about 6.7 s).

Claims

CLAIMS1. An optical nanomotion detection (ONMD) method for determining adhesion of one or more single living cells to a substrate, the method comprising• contacting the substrate with the one or more single living cells;• recording nanomotion of the one or more single living cells;• processing the recorded nanomotion of the one or more single living cells to determine the adhesion of the one or more single living cells to the substrate and / or evolution of the adhesion of the one or more single living cells to the substrate over time, wherein the processing of the recorded nanomotion comprises determining area covered by nanomotion of the one or more single living cells during the duration of the recoding, wherein the area covered by nanomotion corresponds to a number of pixels covered by the nanomotion, and wherein o the larger area and / or an increase of the area during the recording indicates a weak adhesion of the one or more single living cells to the substrate, and o the smaller area and / or a decrease of the area during the recording indicates a strong adhesion of the one or more single living cells to the substrate.

2. The optical nanomotion detection method of claim 1, wherein the substrate is a solid surface, preferably a flat solid surface.

3. The optical nanomotion detection method of claim 1 or claim 2, wherein the nanomotion of the one or more single living cells is recorded during at least 5 seconds, preferably during at least 10 seconds.

4. The optical nanomotion detection method of any one of claims 1 to 3, wherein the recording nanomotion of the one or more single living cells is carried out by an optical microscope coupled to a camera.

5. The optical nanomotion detection method of any one of claims 1 to 4, wherein the processing of the recorded nanomotion of the one or more single living cells is carried out by an image-processing software.

6. The optical nanomotion detection method of any one of claims 1 to 5, wherein the larger area and / or an increase of the area during the recording further indicates a low risk of contamination of the substrate by the one or more living cells, and wherein the smaller area and / or a decrease of the area during the recording further indicates an increased risk of contamination of the substrate by the one or more living cells.

7. A computer implemented method for determining adhesion of one or more single living cells according to the method of any one of claims 1 to 6, the method comprising• receiving video data comprising video record of nanomotion of the one or more single living cells during at least 5 seconds, preferably during at least 10 seconds; and• processing the video data to determine the adhesion of the one or more single living cells to the substrate, wherein the processing the video data comprises determining area of nanomotion of the one or more single living cells during the duration of the recoding, and wherein the area of nanomotion corresponds to a number of pixels covered by the nanomotion.

8. A device adapted to carry out the method for determining adhesion of one or more single living cells according to any one of claims 1 to 6, wherein the device comprises an optical microscope unit coupled to a camera and a processing unit, wherein the optical microscope unit coupled to a camera is configured to record nanomotion of the one or more single living cells during at least 5 seconds, preferably during at least 10 seconds and the processing unit is configured to process the recorded nanomotion of the one or more single living cells to determine area of nanomotion of the one or more single living cells during the duration of the recoding, and wherein the area of nanomotion corresponds to a number of pixels covered by the nanomotion.

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