WO2014006145A1

WO2014006145A1 - Device and method for characterising cells

Info

Publication number: WO2014006145A1
Application number: PCT/EP2013/064163
Authority: WO
Inventors: Josef Alfons KÄS; Tobias Kiessling; Anatol FRITSCH; Roland Stange
Original assignee: Universität Leipzig
Priority date: 2012-07-05
Filing date: 2013-07-04
Publication date: 2014-01-09
Also published as: DE102012211735A1; DE102012211735B4

Abstract

The invention relates to a method and a device for characterising cells in terms of the temperature dependency of the mechanical properties thereof. Due to the partial decoupling of mechanical deformation and heating in the method, the temperature dependency of the mechanical properties has become experimentally accessible for the first time. According to the invention, deformation occurs by means of a microrheological device, and heating takes place locally at the cell by at least one heating laser with a beam course distanced from the sample.

Description

Apparatus and method for characterizing cells

The invention relates to a device and a method for characterizing cells based on the temperature dependence of their mechanical properties.

From biotechnology, numerous possibilities for the characterization of cells are already known. Thus, for example, the multitude of imaging methods known today enables differentiated differentiation of cells based on their phenotype. But other properties of cells, such as e.g. The membrane proteins present on the cell surface can be exploited with the help of biochemical essays to differentiate between cells. A delimitation of the cells is possible not only on the basis of their type, but partly also on the basis of their condition.

In addition, there are known ways of distinguishing cells based on their biomechanical properties from the state of the art. Prerequisite for this are micro-rheological devices that allow a defined and reproducible mechanical manipulation of cells, cell organelles or tissue samples. In addition to AFM force microscopes or micromanipulators, in which the cells are physically contacted and thereby deformed, it is now also possible to deform biological samples without contact.

EP 1 059 871 B1 discloses an optical straightener which enables contactless positioning and deformation of dielectric particles in a two-beam laser trap. The two lasers have, in contrast to an optical tweezer, a slightly divergent beam path. The laser beams have different propagation speeds due to the different refractive indices of the organic material and the surrounding solution, inside and outside the sample. As a result, an impulse is transmitted to both when entering and exiting the laser beams from the sample. This results in forces acting perpendicular to the surface of the sample which, when the refractive index of the sample is higher than that of the solution, cause the sample to stretch along the optical axis of the laser beams.

The deformation of cells in the optical Strecker leads to a strong heating of the sample in periods of a few milliseconds [Wetzel et al. This intrinsic coupling of deformation and heating of the cell is found in all laser-based microrheological measuring devices known from the prior art. An isolated investigation of the temperature dependence of biomechanical properties of cells was therefore not possible. From DE 10 2010 041 912 B3 a method for the diagnosis and / or prognosis of cancers by the analysis of the mechanical properties of tumor cells using an optical extender is known. It is exploited that a certain proportion of tumor cells under tension has an extension against the direction of stress. Based on this behavior, tumor cells can be differentiated from healthy tissue. However, tumor cells show this behavior only in a range of mechanical stresses in which a linear deformation of the cell is to be expected. However, this limitation on the mechanical stresses to be exerted on the cell is difficult to realize given ignorance of the temperature dependence of the biomechanical properties of cancer cells. This results in the risk of unreliable or false statements in the differentiation of diseased and healthy tissue.

The object of the invention is therefore to provide an apparatus and a method for determining the temperature dependence of the mechanical properties of cells, tissue samples or particles. Furthermore, a new method for the reproducible characterization and differentiation of cells is proposed, which allows the diagnosis of cancers and the optimization of methods for the screening of substances as potential medicinal agents.

The object is achieved by a device according to claim 1 and a method according to claim 7. Preferred developments of the invention are subject of the dependent claims.

The temperature dependence of the mechanical properties of cells, tissue samples and particles can be achieved according to the invention with a device comprising:

a) a measuring chamber for receiving a sample contained in a liquid, preferably in an aqueous solution,

b) a tempering device for adjusting the temperature of the measuring chamber and liquid,

c) a micro-rheological device for generating targeted stress states in the sample,

d) at least one heating laser with a jet path which is objected to by the positioned sample,

e) at least one detector for determining the geometric shape and / or the mechanical properties of the sample and

f) at least one detector for determining the temperature of the sample, be determined. Surprisingly, the temperature dependence of biomechanical sizes is a conserved size in cells of the same cell type. The device according to the invention can thus be used for the characterization of cells, tissue samples or particles, for example subcellular structures.

The device according to the invention has a measuring chamber for receiving a sample present in a liquid (hereinafter also solution), preferably an aqueous solution. Since the samples are preferably biological samples, particularly preferably cells, in particular living cells, the measuring chamber is preferably composed of non-toxic, chemically inert materials and is suitable for receiving the sample and a nutrient solution (cell culture medium) suitable for the sample. suitable. Particularly preferably, the measuring chamber is made of plastic or glass. Since the measurement technique is often based on optical principles in the investigation of living biological systems, the measuring chamber is also preferably transparent, and has over a wide spectral range sufficient for the detection of high-contrast signals transmission.

The measuring chamber is further connected to a tempering device, which allows adjustment of the temperature of the measuring chamber and liquid. The temperature control device is preferably a thermostat with which the system of measuring chamber, liquid and sample can be heated to a specific target temperature and kept at a constant temperature. In order to counteract environmental influences and temperature fluctuations, the tempering device preferably has a feedback mechanism in order to be able to react quickly and accurately to changes in the temperature of the system.

The device according to the invention also has a micro-rheological device, this term designating in the context of this application all devices which make it possible to generate targeted stress states in the sample or to selectively deform the sample. Further preferably, the micro-rheological device is capable of positioning the sample at a defined location of the measuring chamber. The micro-rheological device is for this purpose preferably able to exert forces on the sample in order to position it within the measuring chamber. It is sufficient if a spatial area of the measuring chamber is present at which a sample, which is transported by suitable means into this area, is spatially fixed by means of the micro-rheological device. In addition, the micro-rheological device is capable of producing targeted stress states in the sample. This is preferably done by generating surface forces without a resultant force in the center of mass system of the sample. The micro-rheological device is preferably a device in which a physical contacting of the sample occurs, for example a magnetic forceps, an AFM force microscope or a micropipette for the micropipette aspiration method. However, the micro-rheological device preferably allows non-contact deformation of the sample and comprises an optical tweezer or an ultrasound generator. The micro-rheological device is particularly preferably an optical straightener, in accordance with EP 1 059 871 B1, to the content of which reference is made in its entirety. Likewise preferably, the generation of specific stress states in a sample can take place through the interaction of a microfluidic flow and a passive microfluidic system, for example by deformation of samples on obstacles in the flow. In the case of such a microfluidic flow obstacle, the microrheological device preferably comprises means for generating microfluidic flows as well as suitable microfluidic flow channels.

In order to enable the characterization of the temperature dependence of the mechanical properties of the sample, the device according to the invention also has at least one further tempering device for adjusting the temperature locally on the sample. This tempering device advantageously enables the heating of a sample-spaced volume of the liquid surrounding it. Further advantageously, when the temperature is set locally on the sample, no impulse is transferred to the sample by the tempering device itself or no forces are induced in the sample (apart from, for example, thermal expansion due to the heating in the sample). Particularly preferably, the device according to the invention for characterizing the temperature dependence of the mechanical properties of the sample has at least one heating laser. This heating laser has a beam path that is spaced from the positioned sample. The sample is thus located in the area in which it is fixed by means of the micro-rheological device, not within the beam path of the heating laser. When heating the sample by the heating laser thus advantageously no additional pulse is transferred to the sample, or causes no additional deformation of the sample by the heating laser. This makes it possible for the first time to study the temperature dependence of the mechanical properties of biological samples, in particular cells, whereby the influence of non-linear effects due to the coupling of deformation and heating is advantageously negligible. The heating of the sample is essentially carried out by heating the liquid in the measuring chamber in areas near the positioned sample through the heating laser and subsequent heat transfer to the sample. The device according to the invention thus advantageously allows an almost instantaneous heating of the sample of up to 100 K, preferably of up to 60 K, in a time of 1 to 5000 ms, preferably between 1 and 2500 ms and particularly preferably between 1 and 1000 ms. This advantageously allows for the first time the isolated investigation of the temperature dependence of the mechanical properties of the cell material, the influence of temperature-dependent regulatory processes in the cell being negligible. This is due to the fact that the reaction kinetics of such processes taking place in the cell, or a change in the ambient temperature, can adapt only slowly, in particular not in periods of milliseconds. The influence of altered reaction equilibrium or altered concentrations of various proteins in the cell on the mechanical properties of the cell can thus be advantageously neglected in the investigation of these properties with the device according to the invention.

To characterize the sample based on the temperature dependence of its mechanical properties, the device further comprises at least one detector to determine the geometric shape and / or the mechanical properties of the sample. Since the sample is preferably biological tissue and particularly preferably living cells, a detector is preferred in which the energy input into the sample is as low as possible. The device therefore particularly preferably has an optical structure, for example a confocal microscope and / or fluorescence microscope, for determining the geometric shape of the sample.

The device according to the invention also has a detector for determining the temperature of the sample. Depending on the structure of the measuring chamber and the micro-rheological device, the temperature can be determined with or without contacting the sample. If the device according to the invention has a micro-rheological device in which the cells are deformed by contacting, for example by the cantilever of an AFM force microscope, the determination of the temperature preferably also takes place by contacting the sample, for example by means of a temperature sensor. If the deformation of the sample by the micro-rheological device takes place without contact, for example by means of an optical extender, a non-contact determination of the temperature is preferred.

In a preferred embodiment of the device according to the invention, the measuring chamber is designed as a microfluidic channel. The channel is preferably provided with an inlet and outlet and connected to a microfluidic pump. Further preferably, the device is connected to a reservoir in which a plurality of similar or different samples are suspended in a suitable nutrient medium is. By a mediated by the microfluidic pump flow then individual samples can be detected and transported through the microfluidic channel. The samples transported by the microfluidics can be transported, in particular, into a spatial region of the channel in which they can be spatially fixed by means of the microrheological device. Particularly preferably, the device according to the invention also has a control device, with which the presence of a sample in the range of action of the micro-rheological device can be detected. If a sample is fixed by the micro-rheological device, the microfluidic elements, in particular the microfluidic pump, can be controlled so that the flow and thus the transport of the sample through the channel can be interrupted. The control device thus advantageously allows a stopped-flow operation of the measuring chamber, in which a discontinuous flow is generated in the channel. Thus, a single sample can be transported to the microrheological device by means of a microfluidic flow, where the temperature dependence of its mechanical properties can be determined, the sample subsequently transported away from the measuring region again by means of a microfluidic flow and moved in the same way with another sample.

Further preferably, the measuring chamber is equipped with a tempering device and by means of this continuously with a tempered cooling medium, for example. Water, rinsed. The cooling medium is preferably brought to a preferably freely adjustable temperature in a range between 0 and 100 ° C via a circulating thermostat. In this case, a separation between the nutrient solution in the measuring chamber and the cooling medium or water is ensured. The measuring chamber can also have another device for improving the heat transfer from the cooling medium, for example. Cooling fins or otherwise enlarged surface. In a particularly preferred embodiment, the measuring chamber is bounded on at least one side by an aluminum plate, which can be flushed through at least one inside channel with tempered cooling medium. Further preferably, the thermostat has a temperature sensor which is positioned in or near the measuring chamber. As a result, the temperature of the measuring chamber can be determined and used as an output signal for a feedback mechanism of the thermostat for temperature control in the measuring chamber. Further preferably, the measuring chamber and thus the liquid (nutrient solution) by means of electrothermal components, for example. Using the Peltier effect, tempered.

In a preferred embodiment of the device according to the invention, the micro-rheological device is formed by at least one stretch laser. The micro-rheological device is particularly preferably oriented through two optical fibers in the microfluidic channel, perpendicular to them and to each other Aligned aligned stretch laser formed. The rays emitted from the light-conducting fibers have a divergent beam profile without a focal region in the interior of the measuring chamber. These two divergent laser beams thus form an "optical straightener" in the sense of EP 1 059 871 B1, which enables a contact-free targeted deformation of the sample.

Furthermore, at least one heating laser is preferably conducted into the microfluidic channel via a separate light-conducting fiber from the stretch lasers. The fibers are preferably arranged relative to one another such that their beam paths do not overlap within the measuring chamber or the microfluidic channel. Particularly preferably, the at least one light-conducting fiber of the at least one heating laser is arranged parallel to the fibers of the stretch laser and thus also perpendicular to the microfluidic channel.

The at least one heating laser advantageously makes it possible to heat the sample by up to 100 K, preferably up to 60 K, in periods of 1 to 5000 ms, preferably between 1 and 2500 ms and particularly preferably between 1 and 1000 ms. without the transfer of an extra impulse to the sample. Thus, an almost instantaneous heating of the sample is advantageously possible, which causes no additional deformation of the sample apart from thermal effects. With the preferred embodiment of the structure according to the invention, therefore, a heating of the sample which is decoupled from its deformation is possible. By suitable choice of a heat spatter laser sequence, the temperature at the location of the sample can be kept constant throughout the measurement on a single sample by balancing the heat input of the stretch laser at the location of the sample. The beam emerging from the fiber of the heating laser preferably has a divergent beam profile. If such a divergent heating laser radiates through the microfluidic channel filled with a liquid, it is advantageous to uniformly heat the liquid in the entire volume of the beam path. In particular, the formation of a strong temperature gradient, such as in a convergent beam profile around the focal volume, can be avoided.

Furthermore, the device preferably has at least one detector for determining the geometric shape and temperature of the sample, which is arranged in or relative to the microfluidic channel. Also conceivable are other types of detectors that allow conclusions to be drawn about the mechanical properties of the sample without the determination of the geometric shape, for example about the determination of conductivity differences of samples in different stress states. Particularly preferably, optical methods, in particular light microscopic methods, are used to detect the geometric shape of the sample. Particularly preferably, the parallel and spaced arrangement of the fibers of the at least one heating laser to the fibers of the stretch laser allows to avoid an overlap of the beam paths of heating and stretch lasers. Further preferably, a plurality of heating lasers are directed into the measuring chamber such that the beam path of none of the heating lasers intersects with the beam path of the stretch laser. The heating lasers are preferably arranged on one or two sides of the microfluidic channel and on one or two sides of the fibers of the stretch laser. The distance of the at least one heating laser to the sample positioned by means of the stretch laser is between 1 and 1000 μm, preferably between 10 and 300 μm, and particularly preferably between 80 and 200 μm. This distance between the beam path of the heating laser and the sample positioned in the measuring region thus advantageously enables an almost instantaneous heating of the sample while at the same time avoiding additional deformation of the sample by the heating laser.

Also preferably, the wavelength of the stretch laser is between 800 nm and 2000 nm, more preferably between 800 nm and 1064 nm and particularly preferably 800 nm. By selecting this wavelength range, the heating of the sample by the stretch laser can be advantageously minimized. This warming is largely due to the interaction of the lasers with the water in the sample. The choice of stretch lasers with a wavelength at which the wavelength-dependent absorption coefficient of water has a minimum, therefore advantageously leads to a minimum heating of the sample by the stretch laser. The wavelength of the heating laser is preferably between 900 nm and 3000 nm, more preferably 1400 nm to 1500 nm and particularly preferably 1450 nm. Since the heating of the sample takes place primarily by heating the solution or a nutrient medium suitable for the sample and this to a consist largely of water, the wavelength of the heating laser is chosen so that in this the wavelength-dependent absorption coefficient of water has a maximum. Thus, advantageously, a maximum heating of the sample can be realized by means of the heating laser. The heating lasers used are preferably neodymium-doped yttrium-aluminum garnet lasers having a wavelength of 1064 nm. With these lasers, heating can be achieved locally at the sample by 7 ° K per watt. With a particularly preferred laser with a wavelength of 1450 nm, 1, 4 ° K per mWatt would probably be feasible.

In the device according to the invention, a light-microscopic structure is preferably arranged relative to the microfluidic channel that transmits images of sample and channel to a detector suitable for detecting optical information in a spectral range from 300 nm to 800 nm. The light microscopic structure is preferably a confocal and / or fluorescence microscope or transmitted light microscope. The images of the measuring chamber and cell are preferably bright field, dark field, phase contrast or DIC images. For fluorescence micrographs, the fluorescence of the sample is preferably excited with the stretch lasers. The detector advantageously makes it possible to convert the optical images of the measuring chamber and sample into digital and storable image information.

The invention furthermore relates to a method for characterizing cells, tissue samples or particles, wherein a) a sample to be examined is brought to a specific temperature together with a liquid, preferably an aqueous solution, in a measuring chamber,

b) in the sample by means of a micro-rheological device targeted stress states and this is deformed, c) before, simultaneously or during the deformation, but causally independent of this heating of the sample by up to 100 K in a time from 1 to 1000 ms takes place,

d) the deformation of the sample as a function of the forces generated by the micro-rheological device, the sample temperature and the time is detected, e) and from this biomechanical and thermodynamic parameters are derived.

According to the invention, in this novel method for characterizing a sample on the basis of the temperature dependence of the mechanical properties, first a sample is brought to a specific temperature together with a liquid (solution) within a measuring chamber. The temperature of the sample is preferably carried out by rinsing the measuring chamber with a tempered cooling medium, preferably water. The tempering takes place in such a way that the sample is given sufficient time to adapt to the respectively set temperature. If the sample is a cell, the slow heating makes it possible to adjust new reaction equilibria of the active processes taking place in the cell. The periods in which a sample has adapted to a new ambient temperature, ie has set a new reaction equilibrium, and in which a temperature of the measuring chamber and solution must thus take place, be between 5 s and 2 h, preferably between 1 min and 30 min and more preferably 20 min. Subsequently, by a micro-rheological device, a deformation of the sample by the generation of defined stress states in the sample. The micro-rheological device is specifically adjustable, so that the voltage applied to the sample voltage can be varied over a wide range. In the method according to the invention, such stress states are set that the sample is deformed, or a deformation of the sample is observable. Furthermore, the sample is thereby positioned by means of the micro-rheological device in a certain spatial area of the sample. For this purpose, forces are exerted on the sample by means of the micro-rheological device, which takes place in a contacting or non-contact manner.

According to the invention continues before, simultaneously or during the deformation of the sample, but causally independent of this, a heating of the sample by up to 100 K, preferably up to 60 K, in a time of 1 to 5000 ms, preferably between 1 and 2500 ms and more preferably between 1 and 1000 ms. If the sample is previously heated, it is first heated to a specific temperature in a targeted and almost instantaneous manner. Subsequently, different voltage states are set in the sample by means of the micro-rheological device. This makes it possible to examine the effect of the initial sample temperature on the mechanical response of the cell to the set voltage states. If the heating of the sample occurs simultaneously with the deformation, the same takes place during the entire duration of the generation of stress states in the sample. When the sample is heated while it is being deformed, the sample is heated after the generation of the voltage conditions and before the end of the generation. By suitable choice of a heat spatter laser sequence, the temperature at the location of the sample can be kept constant throughout the measurement on a single sample by balancing the heat input of the stretch laser at the location of the sample.

In the method according to the invention, the heating of the sample takes place in such a way that, apart from thermal effects, no additional stress states are produced in it. Excluded are stresses that arise in the sample just because of the temperature dependence of the mechanical properties of the sample to be investigated, for example, by their thermal expansion. Since the sample can nevertheless be heated by means of the microrheological device to heat the sample, this form of heating means an at least partial decoupling of deformation and heating. This decoupling advantageously makes it possible to characterize the thermo-rheological properties of the sample.

According to the invention, the deformation of the sample in response to the generated stress states depending on the means of the micro-rheological device generated forces and the sample temperature detected time-resolved. The detection of the sample preferably takes place by means of a light-microscopic structure. The deformation of the sample is then determined as relative deformation from images of the sample before and during the generation of the stress states by means of the micro-rheological device. Simultaneously with these images, the temperature of the sample is measured. The acquisition of these data advantageously makes it possible to determine the temperature dependence of the mechanical properties of the sample.

The determination of the temperature dependence of the mechanical properties of the sample advantageously allows the determination of biomechanical characteristics. Some of these parameters represent conserved values for different cell types, so that a characterization of the cells can be carried out on the basis of these variables. Furthermore, the method according to the invention advantageously makes it possible to determine thermodynamic parameters, such as, for example, the coefficient of thermal expansion and possibly a cell-specific glass transition temperature.

In a preferred embodiment of the method according to the invention, the sample is brought to a specific temperature together with a solution in a microfluidic channel. Further preferably, the microfluidic channel is connected via a suitable inflow to a reservoir in which a plurality of the samples to be examined are suspended in a nutrient solution. Further preferably, the temperature of the sample and the solution by a temperature control device, more preferably by a circulating thermostat and appropriate on the microfluidic channel mounted heat exchanger.

Furthermore, the microfluidic channel is preferably connected to a microfluidic pump, so that individual samples are transported through the channel with the aid of an adjustable flow. At one location of the microfluidic channel, at least one, preferably two, photoconductive fiber (s) are disposed perpendicular to the channel, and preferably aligned with each other, with each of the fibers emitting a divergent laser field. If the sample is transported into the laser fields by means of the microfluidic flow, it is fixed and positioned therein. This at least one stretch laser also leads to a deformation of the positioned cell. Among other things, this depends on the performance of the stretch laser. For a detailed description of the positioning and deformation of the sample in the "optical straightener" formed by the two divergent lasers, reference is made to EP 1 059 871 B1 The forces acting on the sample are, in particular, linear to the performance of the stretch for an optical straightener A control device particularly preferably detects the laser positioned between the two stretch lasers Sample and stops the flow mediated by the microfluidic pump for the duration of the measurements.

In this preferred embodiment, before, simultaneously or during the deformation of the sample by the stretch laser by means of at least one heating laser, a volume of the solution spaced from the sample by up to 100 K, preferably up to 60 K, in a time of 1 ms to 5000 ms, preferably between 1 ms to 2500 ms and more preferably between 1 ms to 1000 ms, and thus heated by heat transfer and the sample. This is done by thermal excitation of the solution or the solvent by the absorption of the laser light of the at least one heating laser. The heating of the solution surrounding the sample takes place in such a way that no additional impulse is transmitted to the sample by the heating laser or no further optical forces are induced on the cell. This is preferably achieved in that the beam profiles of stretch laser and heating laser in the microfluidic channel do not overlap. In addition, the heating lasers are controlled so that the heating of the solution takes place only when a transported through the channel to the stretch lasers sample has passed the heating laser. This form of indirect heating of the sample advantageously leads to a decoupling of deformation and heating of the sample and makes the temperature dependence of the mechanical properties of a sample experimentally accessible for the first time. Furthermore, this form of heating advantageously allows a very accurate adjustment of the temperature of the sample based on the absorption of the laser light in the solution. Since the heat transfer is realized by absorption of laser light, a precise knowledge of the absorption coefficient is necessary to achieve the desired heat input. The absorption coefficient of living cells varies from cell to cell and is also unknown. However, the absorption coefficient of the surrounding solution can be determined exactly and reproducibly. This results in an increased accuracy of the heat introduced by laser light in the measuring region. Since the cells are not directly irradiated, negative effects such as 'photodamage' or wavelength-dependent absorption of photons by proteins are also avoided.

In the preferred embodiment of the method according to the invention, the deformation of the sample is detected as a function of the power of the stretch laser, the sample temperature and the time by means of a light-microscopic structure. Preferably, images of the sample are determined at different times, at different powers of the stretch laser and at different temperatures. The time-dependent, relative deformation ε (ε) of the cell is particularly preferably determined by measuring the images of the sample according to a reproducible scheme. This quantity is essentially calculated as the quotient of any, reproducibly determinable extent of the sample to be examined at an initial time t ₀ = 0 and at later times t> t ₀ . Preferably, an expansion of the sample is selected, which is subject to strong changes due to the action of the stretch laser. Further preferably, the contour lines of the sample are traced in the images produced at different times in order to determine any drift or rotation movements of the cell as a whole. Such movements superimposed on the deformation of the cell are preferably calculated from the acquired data by means of a suitable algorithm. Furthermore, data sets in which the cells as a whole show a movement that exceeds a certain limit are not preferred for determining the temperature dependence of the mechanical properties of the cell.

In order to ensure the highest possible heating of the sample in the method according to the invention, the wavelength of the heating laser is preferably adapted to the liquid used, preferably an aqueous solution. The sample is preferably a cell that requires a specific nutrient medium to optimally maintain its vital functions. These nutrient media are usually water-based, but may show differences in the wavelength-dependent absorption of light due to the solutes. Nevertheless, in order to ensure optimal energy input for each solution through the heating laser, its wavelength is preferably selected such that the absorption coefficient has a maximum at this wavelength.

By correlating the relative deformation of the cell, the forces acting on the cell, and temperature, the temperature dependence of the mechanical properties of the sample can be quantified. In a particularly preferred embodiment of the method according to the invention, the temperature of the sample can be determined via a fluorescent dye contained in the solution surrounding the sample. This dye has a temperature-dependent intensity of the emitted and thus detectable fluorescence radiation. Thus, the temperature of the solution and the sample can be determined by measuring the intensity of the fluorescence radiation of this dye.

In a particularly preferred embodiment of the method according to the invention, as described above, for a multiplicity of similar samples, their deformation is determined as a function of the force exerted thereon and the sample temperature.

For each of the samples, the relative deformation ε (ε) is thus determined as a function of the power applied to the stretch laser and the resulting force acting on the sample as well as the temperature of the respective sample. From the data thus determined, the creep behavior of the samples can be determined according to (1) determine. The optically induced in the sample voltage is linearly proportional to the σ applied to the stretch performance lasers ₀ "P _s tretch- The samples show as a rule, at different temperatures, a different creep behavior. In a preferred embodiment of the method according to the invention, the curve of] (t) of a sample which was determined at a first temperature can be determined according to the time temperature superposition theory (TTS) known from polymer physics by means of a scaling factor of the time "Time shift factors" a _T, i _, with the, at any other sample temperature T, determined, curve J (t) of another sample according to

be brought to the overlay. The prerequisite is that the temperature of the measuring chamber and the solution as a whole, regardless of the sample temperature, during the plurality of measurements have a constant temperature.

With the data for relative deformation determined for a large number of similar samples, the creep behavior according to (1) is first characterized for each cell. Subsequently, after the selection of a reference curve determined at a reference temperature T _ref , all curves for] (t) are made to coincide according to (2). In this case, the scaling factors a _T, i are particularly preferably varied until the error squares of the superimposition fall below a specific limit value.

From the TTS polymer theory it is further known that the scaling factors a _T, i show a functional dependence on the temperature which is given by the Arrhenius equation

can be described. Here R denotes the universal gas constant and E _A the activation energy.

As experimental findings show, the activation energy E _{A is} a size that is largely conserved for individual cells of the same type, but different values for cells of different types. The determination of the activation energy E _A , as a parameter of the temperature dependence of the mechanical properties of a sample, in particular living cells, thus advantageously and surprisingly enables the characterization and differentiation of cells of different cell types. Cells of different cell types are cells of different organisms as well as cells which have different functions within the same organism. Moreover, in certain cases, it is possible to distinguish the cells of the same cell type on the basis of a specific condition, for example, cell age, stage of the cell cycle, or viability.

However, according to the TTS theory, the dependence (3) is not valid for all polymers and temperature range, for example, for certain temperatures, the dependence of the scaling factors a _T, i on the temperature is described by the William Landel-Ferry equation. For vitreous materials, for which biological samples, especially cells, are frequently counted, the Arrhenius equation describes the behavior of the scaling factors for T <T _G and T> T _G + 50 ° K, whereas the William Landel-Ferry equation describes the behavior of the scaling factors Temperature dependence of a _T, i for T _G <T <T _G + 50 ° K describes. However, the glass transition temperatures of biological samples have not been determined.

For the purposes of this patent application, the equation (3) is therefore to be understood as a rule for the numerical determination of a parameter, the activation energy E _A. This quantity is determined in the sense of this application and independently of any theory exclusively by equation (3). The applicability of equation (3) is thus determined solely by whether a value E _A can be found with which this equation (3) describes the temperature dependence of the scaling factors a _Tj found for the plurality of similar samples with an accuracy to be selected by the person skilled in the art. For example, the adaptation of the values to a graph to be determined according to (3) can be done by minimizing the error squares. If these error squares do not fall below a limit value selected by the person skilled in the art, then (3) is not applicable to the determined scaling factors a _T, i.

In the event that equation (3) is not applicable and the activation energy E _{A is} therefore not determinable, the quality of the dependence of the scaling factors a _T, i on the temperature can be used to differentiate cells of different cell types. Even if no quantitative value of the activation energy E _A can be determined, a characterization or differentiation of cells based on their cell type is advantageously possible with the method according to the invention. On the implementation of such a characterization will be discussed in more detail in the description of the uses of the invention.

In a further preferred embodiment of the method according to the invention, the deformation of a plurality of similar samples is determined as a function of the force exerted thereon and the sample temperature.

For each of the samples, in turn, the relative deformation ε (ε) depending on the power applied to the laser sweeper and the resulting, on the sample acting force and determined as a function of the temperature of each sample. From the data thus determined, the creep behavior of the samples can be determined according to (1).

The samples usually show a different creep behavior at different temperatures. In a preferred embodiment of the method according to the invention, the curve of] (t) of a sample determined at a first temperature can be determined according to the "time temperature superposition" theory (TTS) known from polymer physics by means of a scaling factor of time, " Time shift factors "a _T, i, as well as a factor for scaling the entire function, the" modulus shift factor "sb _T, i, with the, at any other sample temperature T, determined, curve J (t) of another sample according to

be brought to the overlay.

If the measuring chamber as well as the solution as a whole and independent of the sample temperature have a largely constant temperature during the entire time of the measurements, a constant is also preferably selected for the scaling factor b _T, i. Below a critical temperature T _crit , b _T, i assumes different constant values for different temperatures of the measuring chamber and the solution as a whole, which are independent of the sample temperature over a wide range. However, if the sample temperature _exceeds the critical temperature T _crit , b _T, i can no longer be set constant. Rather, the values for b _T, i for those at different temperatures T | measured curves J, a sufficient overlap of the curves should be achieved. According to the TTS theory, the samples thus show thermorheologically complex behavior for a sample temperature T> T _crit .

In order to determine the respective values of b _T, i, the scaling factors a _T, i and _d b _T, i are varied until the error squares of the superimposition fall below a specific limit value. From the thus determined values for the modulus shift factor b _T, i, the temperature T _{crit can be} determined, as the temperature above which the temperature dependence of the scaling factors b _T, i can no longer be described by at least one constant. The critical temperature T _C RIT is thus the temperature above which the totality of the individual scaling factors b _T, i have a temperature dependence. The critical temperature is determined as the mean value of the temperatures above which the temperature dependence of the individual scaling factors b _T, i, b _T, 2, b _T, 3 ... can no longer be described by a constant. As experimental findings show, the critical temperature T _{crit is} a size that is largely conserved for individual cells of the same type, but different for cells of different types. The determination of the critical temperature T _crit , as a parameter of the temperature dependence of the mechanical properties of a sample, in particular living cells, thus advantageously and surprisingly enables the characterization and differentiation of cells of different cell types. Moreover, in certain cases, it is possible to distinguish the cells of the same cell type on the basis of a specific condition, for example, cell age, stage of the cell cycle, or viability.

As already described for the scaling factors a _T j, it is also possible on the basis of the qualitative profile of the dependence of the scaling factors b _T j on the temperature to distinguish between cells of different cell types. Even if no quantitative value of the critical temperature T _C RIT can be determined, it is advantageously possible with the method according to the invention to characterize or distinguish cells on the basis of their cell type. On the implementation of such a characterization will be discussed in more detail in the description of the uses of the invention.

The invention further relates to the use of a device according to the invention comprising:

a) a measuring chamber for receiving a sample contained in a liquid, preferably an aqueous solution,

b) a tempering device for adjusting the temperature of the measuring chamber and solution,

f) at least one detector for determining the temperature of the sample for carrying out the method according to the invention for characterizing cells, tissue samples or particles, wherein

g) a sample to be examined is brought to a specific temperature together with a solution in a measuring chamber,

h) in the sample by means of a micro-rheological device targeted stress states are generated and this is deformed thereby, wherein i) before, simultaneously or during the deformation, but causally independently of this, a heating of the sample by up to 100 K takes place in a time of 1 ms to 1000 ms,

j) the deformation of the sample, as a function of the forces generated by the micro-rheological device, the sample temperature and time, is detected, and

from this biomechanical and thermodynamic parameters are derived.

The invention further relates to the use of the method according to the invention or the device according to the invention for the diagnosis of cancer.

As studies have shown, tumor cells show a different thermorheological behavior than healthy cells of the same tissue. In particular, healthy cells generally have a viscosity similar to that of a pseudoplastic fluid. In contrast, tumor cells have a viscosity similar to that of a Newtonian fluid. Thus, healthy tissue can be differentiated from tumor tissue by the qualitative thermo-rheological properties of the respective cells.

To use the method according to the invention for the diagnosis of cancerous diseases, the method according to the invention is carried out on tissue samples of a potential tumor, or of individual cells obtained therefrom, of a patient.

The determined thermorheological behavior of the samples or cells in question is then compared with reference data. The reference data are preferably obtained by means of the implementation of the method according to the invention on non-tumorous non-tumorous tissue of the patient. Also preferably, the reference data can be taken from a database from which data on the thermorheological behavior of the corresponding healthy tissue can be retrieved.

Particularly preferably, a multiplicity of individual cells are isolated from at least one tissue sample of the potential tumor of a patient. By carrying out the method according to the invention on this multiplicity of cells, the thermorheological behavior of this cell type can be determined with high accuracy. For this purpose, the mechanical response of the cells is detected at a particular voltage applied to this particular at different temperatures.

Subsequently, reference data are preferably determined by performing the method according to the invention on a plurality of healthy cells of the same tissue of the patient. Also preferred is the mechanical response of the cells detected on a specific applied to this voltage at different temperatures. Further preferably, the reference data are retrieved from a database in which the thermorheological behavior of this cell type is stored, which was previously characterized on a plurality of cells of the same healthy tissue.

From the comparison of the thermorheological behavior of the potential tumor cells with the reference data, a statement can be made as to whether the tissue taken from the patient is a sample of a tumor.

It has also been found that thermo-rheological or biomechanical parameters determined in the method according to the invention assume healthy and different values for tumor cells. This applies in particular to the activating energy E _A determinable in the method according to the invention and the critical temperature T _C RIT ZU. Thus, healthy tissue of tumor tissue can be particularly advantageously distinguished from one another by the quantitative thermorheological properties of the corresponding cells. Advantageously, it has been found that the activation energy E _A assumes different values for tumor cells of a tissue than for healthy cells of the same tissue.

As described above, the activation energy E _A and / or the critical temperature T _C RIT of the measured cell type are determined on the basis of the measured data. The data thus obtained are compared with reference data for the activation energy E _A and / or the critical temperature T _C RIT. The reference data are values determined for cells of the same, but healthy, tissue such as the potential tumor tissue or taken from a database containing such reference data.

Particularly preferably, a multiplicity of individual cells are isolated from at least one tissue sample of the potential tumor of a patient. By carrying out the method according to the invention on this multiplicity of cells, the activation energy E _A and / or the critical temperature T _C RIT of this cell type can be determined with high accuracy. For this purpose, the mechanical response of the cells is detected at a particular voltage applied to this particular at different temperatures.

Subsequently, reference data are preferably determined by performing the method according to the invention on a plurality of healthy cells of the same tissue of the patient. Also preferred is the mechanical response of the cells detected on a specific applied to this voltage at different temperatures. Furthermore, the reference data are preferably retrieved from a database in which the values of the activation energy E _A and / or the critical temperature T _C RIT of this cell type are stored, which were previously determined for a multiplicity of cells of the same healthy tissue.

From the comparison of values of the activation energy E _A and / or the critical temperature TCRIT of the potential tumor cells with the corresponding reference data, it is possible to make a statement as to whether the tissue taken from the patient is a sample of a tumor. Preferably, the value of the activation energy of the potential tumor cells is compared with the value of the activation energy of the same healthy tissue. If the cells of the potential tumor tissue have a clearly differentiable activation energy than the healthy cells of the same tissue, then it is very likely that the tumor cells taken from the patient are potentially tumor cells.

The method according to the invention can also be used for the classification of tumors, for the isolation, identification and characterization of stem cells, rare cells and extracellular fluids, for the diagnosis of sepsis or for blood analysis in general and for reproductive medicine, based on the use described here become.

The invention further relates to the use of the method according to the invention or the device according to the invention for screening substances as potential medicinal agents.

The invention therefore also encompasses a method for screening substances as potential active substances for oncology (screening method), in which the influence of the substances on biomechanical properties of tumor cells, in particular on the values of the activation energy E _A and / or the critical temperature T _C RIT , is examined by analyzing the elongation of a variety of tumor cells under mechanical stress and at different temperatures. In this case, i) at least one tumor cell is contacted with a substance and subsequently a deforming mechanical stress is exerted on the tumor cell as a mechanical load at different temperatures in such a way that the tumor cell is deformed. The elongation of the tumor cell is determined at the time of exercise; ii) The elongation of the substance contacted tumor cells at different temperatures is compared with reference data of the analysis of the elongation of similar untreated tumor cells at different temperatures.

The substance is then classified as a potential active substance for oncology if the values of the activation energy E _A and / or the critical temperature T _C RIT of the substance contacted tumor cells compared to the untreated tumor cells closer to the values of the activation energy E _A and / or the critical temperature T _C RIT of cells of the same tissue as the tumor, which are, however, healthy.

The substance is preferably classified as a potential active substance for oncology if the value of the activation energy E _A of the substance cells contacted with the tumor cells is lower in comparison to the untreated tumor cells.

Substances that are analyzed in a screening method of the invention include molecules that are of natural or synthetic origin. Preferred substances which are analyzed in a screening method according to the invention are selected from natural molecules, in particular peptides, proteins, nucleic acids, in particular siRNA, synthetic organic molecules, in particular monomers, polymers, synthetic inorganic molecules and small molecules.

In a screening method according to the invention tumor cells are examined, which either originate from a tissue sample of a patient or originate from a cell line. Preferably, tumor cells from cell lines are used in a screening method according to the invention because of their ease of culturing and availability.

The reference data used here is the analysis of the elongation of similar cells which were not contacted with the substance but were otherwise handled identically ("untreated" cells also here.) Therefore, when using cell lines, the analysis of the elongation of untreated tumor cells of the respective cell line is used for different cell lines Temperatures as a reference These data are compared to the elongation of cells from the same cell line that were contacted with the substance When using cells from tissue samples, preferably tumor tissue samples, untreated cells from the same tissue sample are analyzed as reference.

Particularly preferably, the data are also compared with normal reference data. Non-tumor cells of the same type serve for the collection of the data of the normal reference, for which the elongation at different temperatures is determined according to the same principle as for the tumor cells. If the screening method according to the invention with Tumor cell lines performed, are used as a normal reference cell lines that do not contain tumor cells. If the method according to the invention is carried out with tumor cells from tissue samples, it is preferable to use cells of the same tissue as the normal reference, but these cells are not degenerated into tumor cells, or alternatively cells of a healthy individual from the same tissue (for example, the screening method is performed on breast cancer cells) as normal reference either non-tumor cells of the breast tissue of the same patient or cells from breast tissue of a healthy individual are used). If the mechanical properties of the tumor cells are changed by contacting with the substance in the direction of the properties of the normal reference, this too is an indication that the substance is a potential active substance for oncology.

In a particular embodiment of a screening method according to the invention, the elongation at different temperatures under mechanical stress is determined by the same tumor cell. The values of the activation energy E _A and / or the critical temperature T _C RIT are particularly preferably determined for the tumor cell. Initially, the elongation of the untreated tumor cell at different temperatures under load is determined. For this, a method must be used in which the tumor cell is vital even after the determination of the elongation at different temperatures. For this purpose, the device according to the invention is preferably suitable. Subsequently, the tumor cell is contacted with the substance and then the elongation of the substance contacted with the tumor cell at different temperatures determined. In this embodiment, the influence of the substance on the mechanical properties of the tumor cells can be determined particularly well, since identical cells are analyzed before and after contact with the substance.

The method according to the invention can also be used for the thermal activation of tissue, for the characterization of soft matter and for proteomics on the basis of the use of the substance described here for the screening of substances as potential medicinal substances. In the following the invention will be explained in more detail by means of an exemplary embodiment and with reference to figures, in which:

1 shows the basic structure of the device according to the invention, with a microfluidic channel (5) with optical fibers (1 - 3) positioned perpendicular thereto, the middle fiber pair (2) being a stretch laser of a two-beam laser trap and the peripheral fibers (1 and 3 ) can be used as a heating laser;

FIG. 2: deformation data obtained according to the method according to the invention at different sample temperatures according to (1), (A) for healthy breast epithelial cells MCF-10A, for cancerous cells MDA-MB 231, (B) for embryo-based mouse fibroblasts Balb 3T3, for Chinese hamster ovary cells CHO, for cancerous breast epithelial cells MCF 7; unscaled (left column) and scaled by time shift factor a _T according to (2) (right column);

3 shows the time shift factors a _T according to (3) used for scaling the deformation data for (A) the cell strains MCF10A and MDA-MB 231 and (B) for the cell strains Balb 3T3, CHO and MCF 7;

4: the scaling factors (A) time shift factor a _T and (B) modulus shift factor bj as a function of the effective sample temperature.

Construction of the device

To investigate the mechanical response of living, suspended MCF10A, MDA-MB 231 breast epithelial cells, BALB 3T3 embryonic mouse fibroblasts, CHO Chinese hamster ovary cells (Cricetulus griseus) and MCF 7 cancerous breast epithelial cells to optically induced forces used a device according to the invention. The device in this case comprises a microfluidic optical Strecker structure (μ05), which has been modified according to the invention so that the temperature of the cells, on short time scales, in particular within milliseconds, is arbitrarily adjustable. For this purpose, according to the invention, the previously known from the prior art in the Optical Strecker, inherent coupling of heating and deformation is at least partially canceled. Due to the integration of laser heating into the optical stretcher according to the invention, cells held therein can be heated within milliseconds so that they are not exposed to direct irradiation by laser light. At the same time, the optically induced in the cells, mechanical stresses whose strength is determined solely by the performance of the stretch laser, kept constant.

The structure of the invention comprises a straight glass capillary with an inner diameter of δθμηη and a wall thickness of 40 μηη (ST8508, VitroCom, USA). This capillary was placed perpendicular to one through two laser beam trap. These comprises two monomode fibers arranged perpendicular to the capillary and aligned with each other at a distance of 200 μm, the measuring region being located between the fibers for the investigations of the mechanical properties of the cells (No. 4 in FIG. 2). All components of the assembly were embedded in a matching refractive index gel to prevent unwanted reflection and refraction at the capillary surfaces.

In order to control the temperature of the structure, the capillary was mounted on an aluminum sample holder, which was rinsed continuously with tempered water. The water was tempered by a circulating thermostat (F10, Julabo, Germany). By means of an external temperature sensor, which was installed in the vicinity of the measuring region, and a feedback control method, the temperature of the structure as a whole could be kept constant during the measurements and temperature changes of the structure as a whole within about 20min.

In order to realize temperature jumps of the cells within milliseconds, two additional optical fibers were also positioned perpendicular to the capillary and at a distance of 1 10 μηη to the measuring region. By emitting laser light having a wavelength of 1064 nm, the nutrient medium surrounding the sample in the measurement region was rapidly heated without exposing the cell to direct irradiation by the laser. Since the temperature rise expected in the measurement region depends strongly on the distance of the fibers of the heating laser, they were placed as close to the sample position as possible by etching standard HI-1064 monomode fiber down to a diameter of 80 μm using hydrofluoric acid.

Temperature calibration

To determine the heating power of the lasers used, a temperature calibration was carried out with the temperature-sensitive fluorescent dye Rhodamine B (Sigma-Aldrich, St. Louis, Missouri). This dye has a high temperature sensitivity in the range of 0 to 120 ° C. To record the temperature distribution in the capillary as far as possible, an Axioobserver Z1 in combination with a 10X objective (Carl Zeiss, Jena, Germany) was used. The dye solution was prepared from Rhodamine B (0.1 mM) and carbonate buffer (20 mM) and filled into the capillary together with the suspension containing the cells. The dye was calibrated by measuring the fluorescence intensity at various temperatures of the setup. To determine the temperature increase inside the glass capillary, the mean was determined from 6 independent measurements at temperatures between 15 and 40 ° C. This allowed the influence of the temperature of the sample holder or of Nonlinearities of Rhodamine B are minimized. As a result of the heating power, a temperature increase in the measuring region of 7 ° C / watt was determined by the laser and 26 ° C / watt by the laser.

cell preparation

MCF-10A cells (CRL-10317), a non-tumorigenic cell line, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in a 1: 1 mix of Dulbecco's modified Eagles's medium (DMEM) and Harns F12 medium. The mix was supplemented with 5% horse serum, 20 ng / ml epidermal growth factor, 10 μg ml insulin, 100 ng / ml cholera toxin, 500 ng / ml hydrocortisone and 100 U / ml penicillin / streptomycin. Prior to measurements, cells cultured in a 25 cm ^A 2 culture flask (TPP, Trasadingen, Switzerland) were washed twice with phosphate buffered saline and detached by the addition of 1 ml of 0.025% trypsin-EDTA solution. The detached cells were then suspended in 3 ml of culture medium and centrifuged at 100 g for 4 min. Finally, single cells at a concentration of about 5 ^× 10 5 cells / ml were re-suspended in culture medium and added to the device.

MDA-MB-231 cells (HTB-26), a tumor-like breast epithelial cell line, was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 100 μg / ml penicillin / streptomycin. Prior to measurements, cells cultured in a 25 cm ^A 2 flask (TPP, Trasadingen, Switzerland) were washed twice with phosphate buffered saline and detached by the addition of 1 ml of 0.025% trypsin-EDTA solution. The detached cells were then suspended in 3 ml of culture medium and centrifuged at 100 g for 4 min. Finally, single cells at a concentration of about 5 ^× 10 5 cells / ml were re-suspended in culture medium and added to the device.

Balb3T3 cells (CCL-163) are non-tumorigenic mouse fibroblasts obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% calf serum (BCS). Prior to measurements, cells cultured in a 25 cm ^A 2 flask (TPP, Trasadingen, Switzerland) were washed twice with phosphate buffered saline and detached by the addition of 1 ml of 0.025% trypsin-EDTA solution. The detached cells were then suspended in 3 ml of culture medium and centrifuged at 100 g for 4 min. Finally, single cells at a concentration of about 5 ^× 10 5 cells / ml were re-suspended in culture medium and added to the device. CHO cells (CCL-61) are non-tumor cells obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured with 10% fetal calf serum (FCS) -enriched Ham'sF-12. Prior to measurements, cells cultured in a 25 cm ^A 2 flask (TPP, Trasadingen, Switzerland) were washed twice with phosphate buffered saline and detached by the addition of 1 ml of 0.025% trypsin-EDTA solution. The detached cells were then suspended in 3 ml of culture medium and centrifuged at 100 g for 4 min. Finally, single cells at a concentration of about 5 ^× 10 5 cells / ml were re-suspended in culture medium and added to the device.

MCF7 cells (HTB-22) are tumor-like human mammary epithelial cells obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in 88% Eagle's minimal essential medium (PAA E15-825 supplemented with L-glutamine, 10 μg ml insulin (Sigma-Aldrich, I6634), 1 mM = 1 10 μg ml sodium pyruvate (Sigma-Aldrich, P5280) , 10% Fetal Calf Serum (FCS), 1% 100 g ml Penicillin / Streptomycin, and 1% Non Essential Amino Acids (PAA M1 1 -003). Prior to measurements, the cells resuspended in a 25 cm ^A 2 flask (TPP Trasadingen, Switzerland), washed twice with phosphate buffered saline, and detached by the addition of 1 ml 0.025% trypsin-EDTA solution The resuspended cells were then suspended in 3 ml culture medium and centrifuged at 100 g for 4 min individual cells with a concentration of about 5x10 ^Λ 5 cells / ml in culture medium and added to the device.

measurements

Over a period of 5 hours, the response of 685 MCF-10A cells to deformation was detected by the stretch laser at a power of P _str, etc _h ⁼ 700mW. One second before the deformation began, the heating lasers were activated to randomly emit a beam of power between 0 and 1700 mW. This caused a sudden increase in temperature in the measuring region. Since the temperature of the device as a whole is constantly adjustable and thus known by the circulating thermostat at any temperature, the effective temperature of the sample can be determined as follows

Teff ⁼ Tsetup + Pstretch + Pneiz ^ H (5)

The temperature increase is taken into account by the heating laser with a power P _He i _Z. According to the previously performed temperature calibration, the local temperature increase in the measuring region by the heating laser is ΔΤ _Η «7 ° C / watt. The measurements on the MDA-MB-231, BALB 3T3, CHO and MCF 7 cells were carried out analogously.

data analysis

The extraction of quantitative data from the experiments carried out on individual cells initially requires the digital processing of image stacks taken at 30 fps using video-phase-contrast microscopy. By means of an implemented edge detection algorithm, the perimeter in each cross section of each individual cell was determined with subpixel accuracy. In a second step, the orientation of the envelope of the cell was tracked through the captured image sequence so as to correct for measurement errors resulting from small cell cell rotations during the measurements. Data from cells showing large rotations or rotations about different axes during the measurements were excluded from further evaluation. The deformation of the remaining cells was determined as relative deformation ε (£) according to

, , (L _u (t) -L "(-ls <t <0s))

^W (Ln (-ls <t <0s)) ^{ ', where L (t) is the length of the cells measured parallel to the axis of the stretch laser.

Implementation of the TTS and determination of the activation energy

The creep behavior of the cells derived from the relative deformation measured at different temperatures and the power of the stretch lasers according to (1) derivable curves J (t) were overlapped by means of a self-constructed algorithm into a master curve. In doing so, after determining a reference temperature, all these curves were scaled according to (2), whereby the scaling factor aj was systematically varied to minimize the overlap error.

activation energy

Using the time shift factors aj determined in this way, the measured curves could be overlapped with respect to the creep behavior J (t) (compare Fig. 2 (A), (B)). According to the Arrhenius equation (3), the activation energy can be determined from the scaling factors aj. The measured shift factors appear linear, the slope being proportional to the activation energy for the viscous flow of the cells. This gives values for the activation energy of ~ 80 kJ / mol for MCF-10A cells, -170 kJ / mol for MDA-MB-231 cells (compare Fig. 3 (A)), ~ 75 kJ / mol for BALB 3T3 Cells, ~ 47 kJ / mol for CHO cells and ~ 141 kJ / mol for MCF 7 cells (FIG. 3 (B)). Diagnosis of cancers

From the deformation data shown in Figure 2 for MCF-10A, BALB 3T3 and CHO (healthy) and MDA-MB 231 and MCF 7 (cancerous) at different temperatures, it can be clearly seen that the response of the healthier cells is different from the cancerous ones ,

By determining the shift factors required for the overlap of the curves, in particular the time shift factor a _T , the healthy cells can be reliably distinguished from the cancerous cells (see Fig. 3 (A), (B)).

Critical temperature

In the above-mentioned operation of the cell characterizing method of the present invention, the temperature T of the structure as a whole was kept constant while the influence of a sudden increase in the sample temperature was determined. To determine how cells respond to changes in temperature over a longer period, the creep behavior of the cells at different temperatures of the structure was determined as a whole T _se t _U p. The cells had a few minutes to hours to adapt to the changing temperatures. At the beginning, the device was tempered as a whole to 37 ° C and about 200 cells measured within about 3 h. Subsequently, the sample holder and capillary were first cooled down to 25 ° C, then down to 15 ° C, and the measurements repeated at approximately the same number of cells and in the same range of laser power repeatedly Pstretch (chosen randomly between 500 and 1100 mW). With each cool down, it took about 20 to 25 minutes for the entire setup to be thermally equilibrated. Cells measured during the temperature transition were excluded from further evaluation of the data because the true temperature in the measuring chamber could not be accurately determined.

The thus determined creep behavior curves J (t) were superimposed according to (4) using a time shift factor aj and a modulus shift factor br. For the curves determined at 15 ° C, the value for bj is constant for all values of the laser power Pstretch. The curves determined at 25 ° C, however, can be overlapped only with different values for b _T. The same applies to measurements taken at 37 ° C. These measurements show that all cells exhibit thermorheologically complex behavior for a temperature T _e ff> T _C RiT, characterized by the necessity of a non-constant modulus shift factor b _T (see Fig. 4B). For the MCF-10A cells, the critical temperature was determined to be T _CRIT "52 ° C. The temperature T _C RIT is a value conserved for cells of the same type and therefore suitable for the characterization and differentiation of cells on the basis of their cell type. In addition one can from the Data conclude that a b _{T is} always necessary even when the cells are exposed to a characteristically long period of time at a new setup temperature.

Claims

claims

1) Apparatus for characterizing cells, tissue samples or particles, comprising a) a measuring chamber for receiving a sample contained in a liquid, b) a tempering device for adjusting the temperature of the measuring chamber and the liquid, c) a micro-rheological device for generating specific stress states in d) at least one heating laser with a beam path spaced from the sample, e) at least one detector for determining the geometric shape and / or the mechanical properties of the sample, and f) at least one detector for determining the temperature of the sample.

2) Device according to claim 1, characterized in that

the micro-rheological device comprises an optical tweezer, an optical straightener, an AFM force microscope, magnetic tweezers, a micropipette, an ultrasound generator, a microfluidic flow obstruction, and / or a device for generating electrophoresis.

3) Device according to claim 1, characterized in that a) the measuring chamber is formed as a microfluidic channel, b) the measuring chamber is continuously rinsed with tempered water, c) the mikrorheologische device by two, directed via photoconductive fibers in the microfluidic channel, d) the at least one heating laser is conducted via a separate light-conducting fiber into the microfluidic channel and e) at least one detector for determining the geometric shape and temperature of the sample in or relative to the microfluidic channel is arranged. I

Apparatus according to claim 3, characterized in that the light-conducting fibers of stretch laser and heating laser are arranged so that their beam paths do not overlap within the measuring chamber and / or the distance of the heating laser to the means of the stretch laser positioned sample between 1 μηη and 1000th μηη is.

Apparatus according to claim 3 or 4, characterized in that

the wavelength of the stretch laser is between 300 nm and 2000 nm and the

Wavelength of the heating laser is between 900 nm and 3000 nm.

Device according to one of claims 3 to 5, characterized in that a light microscope structure is arranged relative to the microfluidic channel and images of sample and channel to a suitable for the detection of optical information in a spectral range from 300 nm to 800 nm detector are transferable.

A method for characterizing cells, tissue samples or particles, wherein a sample to be examined is brought together with a liquid in a measuring chamber to a certain temperature, in the sample by means of a micro-rheological device targeted stress conditions are generated and thereby deformed, wherein previously, simultaneously or during deformation, but causally independently of this heating of the sample by up to 100 K in a time of 1 ms to 1000 ms, the deformation of the sample, depending on the forces generated by the micro-rheological device, the sample temperature and the Time is detected and derived from it biomechanical and thermodynamic parameters. A method according to claim 7, characterized in that a) a sample to be examined is brought together with a liquid in a microfluidic channel to a certain temperature, b) the sample is deformed by at least one divergent stretch laser, c) before, simultaneously or during the deformation is heated by means of at least one heating laser, a volume of liquid spaced from the sample by up to 100 K in a time of 1 ms to 1000 ms and thereby the sample is heated, d) the deformation of the sample depending on the performance of the stretch laser, the sample temperature and the time is detected by means of an optical structure.

9) Method according to claim 7 or 8, characterized in that

the wavelength of the heating laser is selected as a function of the wavelength-dependent absorption coefficient of the liquid used.

10) Method according to one of claims 7 to 9, characterized in that

the sample temperature is determined by the temperature-dependent intensity of the measured fluorescence radiation of a fluorescent dye in the liquid.

1 1) Method according to one of claims 7 to 10, characterized in that a) for a plurality of similar samples whose deformation, in dependence of the force exerted on them and the temperature T is determined, b) the data thus obtained in accordance with TTS means time shift factors aj -, scaled, c1) and from the time shift factors aj -, the cell-specific activation energy EA is determined and / or c2) the dependence of the time shift factors aj - on the temperature is qualitatively evaluated. 12) Method according to claim 1 1, characterized in that a) the data obtained according to TTS by means of a time shift factors

and a modulus shift factor are scaled byj b1) and the cell-specific critical temperature TCRIT is determined from the modulus shift factors byj and / or b2) the dependence of the modulus shift factors bj, i on the temperature is qualitatively evaluated.

13) Use of a device according to one of claims 1 to 6 for carrying out a method according to one of claims 7 to 12.

14) Use of a method according to any one of claims 7 to 12 or a device according to one of claims 1 to 6 for the prognosis and diagnosis of cancer.

15) Use of a method according to any one of claims 7 to 12 or a device according to any one of claims 1 to 6 for the screening of substances as potential medicinal agents.