DE102021113471A1 - Method and device for determining the state of suspended moving particles - Google Patents

Abstract

The invention relates to a method and a device 100 for determining the state of suspended moving particles using a radiation-activatable substance which, in the non-activated state, can penetrate into the interior of the particles or binds to their surfaces without influencing the state of the particles to be determined , and which, when activated, causes structures or components, e.g. proteins, to network in the particles to be examined.

Description

The present invention relates to the measurement of suspended particles moving either with or in the suspension. In particular, the present invention deals with the measurement of the properties of moving suspended particles, which are influenced by the movement of the particles.

Specifically, the subject matter of the present invention is a method for determining the state of suspended moving particles and a device for determining the state of suspended moving particles. In particular, the subject matter of the present invention is a device that is set up to carry out the method according to the invention.

The dynamics of flowing particles such as cells, microbes, protozoa and vesicles under microfluidic conditions is the subject of many research projects, which result in a whole range of possible applications. Relevant studies examine the interaction between the flow and the particles. Experimental approaches as well as numerical models are developed to describe the interaction dynamics in the micrometer or even submicrometer range and to improve their understanding.

An important question is the influence of external forces on the particles, which can be, for example, forces from the surrounding liquid (local or global pressure or flow changes), light-induced forces (e.g. optical tweezers) or forces due to static or dynamic electrical and /or magnetic fields. If such an external force acts on a particle, the shape of non-solid particles is usually affected. The resulting shape is often characteristic of the particular particle type or subtype.

So e.g. B. in the flow of red blood cells in microcapillary blood vessels, their shape is of great importance for the flow behavior and thus their transport, e.g. their speed. For a given mechanical constriction of a given size and cross-section, at relatively low flow velocities, the so-called “parachute” shape of the cells is induced, which has a large cross-sectional area. It ensures that the red blood cells are transported at a sufficient speed to enable sufficient gas exchange with the surrounding tissue. At relatively high flow velocities, however, the red blood cells tend to adopt the so-called "slipper" shape, an asymmetric shape that has a reduced effective cross-sectional area. This reduces the relative transport speed compared to the surrounding medium, as from the publication Kihm A, Kaestner L, Wagner C, Quint S, Classification of red blood cell shapes in flow using outlier tolerant machine learning, PLoS Comput Biol. 2018 Jun 15;14 (6):el006278. dot 10.1371/journal.pcbl.1006278. PMID: 29906283; PMCID: PMC6021115 is known. This feedback mechanism, based on the external shape of individual cells, is a small but important part of a higher-level control mechanism that regulates gas exchange (oxygen/carbon dioxide) throughout the organism. Varying the flow velocity, a different cell shape is dominant for each velocity, and a transition from “parachutes” to “slippers” can be observed. The underlying statistical distribution is called a "phase diagram".

Studies have shown that this cell-based feedback mechanism can be disturbed by inherited or acquired diseases, parasites (malaria) or medication. It was also found that the age of cells and the storage time, e.g. of blood transfusions, can influence this mechanism.

These studies were performed using 2D brightfield microscopy, which shows the projection of cells in flow under microfluidic conditions. This type of study provides enough information to motivate more detailed investigations in 3D using more sophisticated techniques such as 3D confocal microscopy, as evidenced by the publication Quint S, Christ AF, Guckenberger A, Himbert S, Kaestner L, Gekle S, Wagner C 3D Tomography of cells in microchannels, Appl. Phys. Latvia 2017; 111(10) results. First experiments with 3D confocal microscopy brought promising, highly detailed results for a better understanding of cell dynamics. However, due to the bandwidth limitations of these microscopes, high throughput cannot be expected, which poses a problem for statistical analysis.

Because of this limitation, alternative approaches that allow 3D imaging of cells in flow have been explored. However, all existing technologies (e.g. phase contrast microscopy) have the same limitations and require highly sensitive, large and expensive experimental setups.

This is where the present invention comes in, which has set itself the task of improving this situation.

The object of the present invention is therefore to specify a method which allows the state of suspended moving particles to be determined, it being possible to carry out the method with a significantly reduced outlay on equipment. Furthermore, it is the object of the present invention to specify a device that allows the state of suspended moving particles to be determined, with the structure of this device being significantly simplified compared to the devices previously known from the prior art.

These tasks are solved by a method according to claim 100 and by a device according to claim 17.

The dependent claims following these independent claims represent advantageous refinements and developments of the method according to the invention and the device according to the invention. The features of the subclaims can be combined with the features of the main claims both individually and in combination with the features of other subclaims, even if this is not explicitly stated. This also applies beyond the limits of the method and device claim categories.

The method according to the invention is intended to determine the state of suspended moving particles. The term “particle” is to be interpreted broadly. The term "particle" is intended to include objects whose condition can depend on movement in a suspension and whose size is so small that determining the condition to be determined on the individual particle requires more equipment. The typical size of such particles is below 100 micrometers, and in the case of biological particles also below 15 micrometers, the size specifications being to be understood as examples and not intended to limit the invention. In the context of the present invention, the term “particle” includes both animate and inanimate matter, human, animal or plant cells, viruses, vesicles, micelles, microbes and protozoa being mentioned here by way of example.

The method according to the invention comprises the use of a radiation-activatable substance which, in the non-activated state, can penetrate into the interior of the particles or binds to their surfaces without influencing the state of the particles to be determined. It goes without saying for the person skilled in the art that a certain influencing of the state of the particle to be determined by the radiation-activatable substance cannot be ruled out or is even unavoidable in many cases. What is meant is that the state to be determined can be detected with a good degree of certainty, even under the influence of the radiation-activatable substance, and in particular can be distinguished from other possible states of the particle with a good degree of certainty.

The radiation-activatable substance also has an activated state in which the substance causes crosslinking of structures or components, e.g. proteins, in the particle to be examined. In this way, the state of the particle to be determined is practically frozen and can be experimentally examined at a later point in time, in particular in a different environment.

In the context of the present invention, radiation-activatable means that the substance changes from the non-activated state to the activated state when it is exposed to suitable radiation, which will be discussed in more detail below.

1. a. Bereitstellen einer Suspension mit einer Vielzahl zu untersuchender Partikel, wobei die Suspension neben den zu untersuchenden Partikeln den strahlungsaktivierbaren Stoff im nicht aktivierten Zustand enthält,
2. b. Induzieren einer Bewegung der suspendierten Partikel im umgebenden Medium in einer Strömungskammer, und
3. c. Beaufschlagen zumindest eines Teilvolumens der Strömungskammer mit Strahlung, die dazu geeignet ist, den strahlungsaktivierbaren Stoff vom nicht aktivierten Zustand in den aktivierten Zustand zu versetzen, um den in den Partikeln enthaltenen oder anhaftenden strahlungsaktivierbaren Stoff zu aktivieren.

The method according to the invention has the following method steps:

1. a. Providing a suspension with a large number of particles to be examined, the suspension containing the radiation-activatable substance in the non-activated state in addition to the particles to be examined,
2. b. inducing movement of the suspended particles in the surrounding medium in a flow chamber, and
3. c. Subjecting at least one partial volume of the flow chamber to radiation which is suitable for changing the radiation-activatable substance from the non-activated state to the activated state in order to activate the radiation-activatable substance contained or adhering to the particles.

The suspension can include, for example, water as the liquid medium in which the particles to be examined are suspended. Of course, other liquid media are also possible. Furthermore, the suspension can contain further auxiliary substances such as salts, acids, bases, emulsifiers, stabilizers or nano-particles in order to set and/or stabilize certain states of the particles to be examined or to influence the density of the liquid medium.

The term flow chamber is also to be interpreted broadly in the context of the present invention. The term flow chamber encompasses all containers that can hold a minimum volume of the suspension and in which a movement of the particles according to the invention is induced in this way it can be that the liquid medium flows around the particles or the particles flow with the liquid medium or a combination of both. This also defines the meaning of the term movement of the particles in the context of the present invention.

For example, the flow chamber can be designed as a capillary, as a sample volume of a rheometer, or simply as a container that holds the suspension to be examined. The flow chamber is preferably designed in such a way that flow conditions of the suspension can be set in it, under which the state of the particles to be determined is set.

A movement according to the invention of the suspended particles to be examined can be caused, for example, by moving the suspension through the flow chamber, e.g. by pumping the suspension through the flow chamber. In the sample volume of a rheometer, the suspension is subjected to controlled shear, causing the particles to move relative to the surrounding liquid medium. Finally, it is also conceivable to indicate a movement of the particles to be examined in the suspension by subjecting the particles to an external force, which can originate, for example, from an (inhomogeneous) electric or magnetic field or from a light field (“optical tweezers”) . All of these examples are to be understood as examples and do not restrict the subject matter of the invention.

The method according to the invention allows the particles to be examined to be fixed or “frozen” in a moving state, i.e. the state of the particles no longer changes, even if the state of motion of the particles or the flow conditions to which the suspended particles are exposed change . With a suitable choice of the radiation-activatable substance, other parameters can also change, such as the medium in which the particles are suspended, without the state of the particles changing significantly as a result. This makes it possible to examine the particles fixed in the state to be determined in a subsequent process step under changed conditions, which opens up completely new experimental possibilities. For example, investigation methods can be used to determine the state of the particles that require stationary particles or would be very difficult or impossible to use as long as the particles are in the flow chamber, as is the case with many common microscopy methods, for example.

The state of the particles to be determined is advantageously their external shape or their internal structure. The three-dimensional character of the external shape or the internal structure of the particles or a two-dimensional projection of the three-dimensional character is particularly preferred.

The state of the particles to be determined is advantageously determined in a subsequent method step by means of an optical or scanning microscopic method. In the case of the optical methods, optical microscopy, light scattering, light diffraction, light absorption, phase modulation, fluorescence detection, or temporal or spatial modulation methods derived therefrom have proven to be particularly suitable.

In an advantageous development of the method according to the invention, the movement parameters of the particles are kept constant over a defined range of movement that extends through the partial volume. The state of the particles can advantageously be fixed by activating the radiation-activatable substance while the particles are in the aforementioned movement range. In this case, the said range of movement can be spatially limited in particular.

For this purpose, in an advantageous embodiment of the method according to the invention, it is provided that the partial volume of the flow chamber is only exposed to radiation if constant movement parameters of the particles are present in the defined movement range.

In the context of the method according to the invention, it has proven to be advantageous if the radiation-activatable substance is irradiated with electromagnetic radiation, in particular in the infrared, in the visible or in the ultraviolet spectral range or in the spectral range of X-rays or gamma radiation, or by means of particle radiation such as alpha or beta radiation can be switched to the activated state. However, the ability to be activated by means of sound waves, in particular by means of ultrasonic waves, has also proven to be advantageous and is intended to be encompassed by the general term radiation/irradiation in the context of the present invention.

Accordingly, electromagnetic radiation, particularly in the infrared, visible or ultraviolet spectral range or in the spectral range of X-rays or gamma radiation, or particle radiation such as alpha or beta radiation, is advantageously used in the method according to the invention for activating the radiation-activatable substance. But also the uses tion of sound waves, in particular ultrasonic waves, has proven to be advantageous.

Within the scope of the method according to the invention, preference is given to using a radiation-activatable substance which, in the activated state, is suitable for crosslinking proteins which are present in the particles to be examined. In particular, crosslinking of the amines of lysine or arginine side chains of proteins of the particles to be examined can be considered and has proven to be particularly advantageous.

It has also been found that when using a radiation-activatable substance that contains either an aryl azide, a diazirine or a diazirine-containing analog of the amino acids leucine (photo-leucine), methionine and p-benzoyl-phenylalanine or is selected from the Group consisting of aryl azides, diazirines and diazirine-containing analogues of the amino acids leucine (photo-leucine), methionine and p-benzoyl-phenylalanine can realize particular advantages.

It has proven to be particularly advantageous if the radiation-activatable substance has a modified glutaraldehyde, formaldehyde or paraformaldehyde, or if the substance is modified glutaraldehyde, formaldehyde or paraformaldehyde or a mixture thereof.

In an advantageous development of the method according to the invention, it is provided that the suspension is collected in batches after it has been exposed to radiation in the flow chamber. In particular, the suspension collected in batches can then be examined in turn, preferably in batches, in a subsequent method step in order to determine the state of the fixed particles in the suspension to be determined.

In a further advantageous development of the method according to the invention, it is provided that the constant movement parameters of the suspended particles in the defined movement range are changed after a predetermined measurement time has elapsed. This change preferably takes place in such a way that, after the change, constant movement parameters of the suspended particles are again present in the defined movement area, which, however, are different from the previous movement parameters.

Particular preference is given to combining particles which, at the time they were fixed, were exposed to the same movement parameters as a result of activation of the radiation-activatable substance, i.e. collected in one or more batches. No particles are added to these batches that were exposed to different movement parameters at the time of their fixation due to the activation of the radiation-activatable substance. In other words, in this advantageous development, a batch essentially only contains particles that were exposed to the same movement parameters at the time of their fixation by activation of the radiation-activatable substance.

This procedure provides batches that contain a large number of particles that were exposed to the same movement parameters at the time of their fixation by activation of the radiation-activatable substance. If the particles have essentially the same properties, this allows the use of statistical investigation methods for determining the state of the particles to be determined, which are based on the parallel investigation of a large number of particles, dynamic light scattering being mentioned here as an example. This can be the basis for a quick and reliable determination of the condition to be determined that requires little equipment.

If the aforementioned conditions are met, it has proven to be possible in an advantageous development of the method according to the invention to determine the state of more than 10,000 particles per hour, preferably more than 100,000 particles per hour and particularly preferably more than 100,000,000 particles per hour, which is the subject of particularly preferred embodiments of such a training.

Similarly high test numbers of 10,000 particles per hour and more can also be achieved with tests on individual particles. For this purpose, the use of methods that allow the state to be determined to be determined on individual particles, but at the same time to analyze a plurality of particles in parallel, has proven to be particularly advantageous. Three-dimensionally resolving optical microscopy methods are mentioned here by way of example, which allow a parallel determination of the state to be determined of a large number of particles that are simultaneously in the field of view of the relevant microscope.

In this context, it is pointed out that the method according to the invention can also be applied to a single suspended particle, which should also be covered by the wording of the independent method claim. As a rule, however, the suspension will contain a large number of suspended particles.

It is pointed out that it is also possible to feed the particles continuously to a suitable examination device after they have been fixed, instead of collecting the fixed particles in batches.

Finally, the subject of a further advantageous development of the method according to the invention is a method which provides a further method step in which the signals obtained with the optical or scanning microscopic method are evaluated by means of machine learning.

In a further advantageous development of the method according to the invention, the statistical distribution of the moving particles is determined at the time of their fixation.

A phase diagram of the moving suspended particles is particularly preferably determined.

The features, properties and advantages of a device according to the invention and of preferred configurations or advantageous developments are presented below, whereby to avoid repetition reference is made to the above statements on the features, properties and advantages of a method according to the invention, which mutatis mutandis to the device according to the invention and their preferred configurations or advantageous developments can be transferred, as is obvious to the person skilled in the art.

A device according to the invention is provided for determining the state of at least one, but preferably a plurality of moving particles. The device is based on the use of a radiation-activatable substance which, in the non-activated state, can penetrate into the interior of the particles or bind to their surface without significantly influencing the state of the particles to be determined, and which, in the activated state, enables crosslinking of structures or components, e.g. proteins, in the particles to be examined.

• a. eine Strömungskammer zur Aufnahme einer Suspension, in der die zu untersuchenden Partikel suspendiert sind,
• b. eine Bewegungseinrichtung, die dazu eingerichtet ist, eine Bewegung der suspendierten Partikel in der Strömungskammer zu induzieren, und
• c. eine Strahlungsquelle, die dazu eingerichtet ist, zumindest ein Teilvolumen der Strömungskammer mit Strahlung zu beaufschlagen, die dazu geeignet ist, den strahlungsaktivierbaren Stoff vom nicht aktivierten Zustand in den aktivierten Zustand zu versetzen.

The device has at least the following features:

• a. a flow chamber for receiving a suspension in which the particles to be examined are suspended,
• b. an agitator configured to induce movement of the suspended particles in the flow chamber, and
• c. a radiation source that is set up to expose at least a partial volume of the flow chamber to radiation that is suitable for changing the radiation-activatable substance from the non-activated state to the activated state.

In this connection it is pointed out that the device according to the invention is intended for determining the state of an individual suspended particle. As a rule, however, a suspension supplied to the device will contain a large number of suspended particles, which should also be covered by the wording of the independent device claim.

In an advantageous embodiment of the device according to the invention, the moving device is designed as a hose, piston, diaphragm or pressure-driven pump. The use of a pressure-driven pump as a movement device has proven to be particularly advantageous, since this type of pump allows a particularly precise control of the flow parameters in the flow chamber, in particular if the latter is of microfluidic design.

A pressure-driven pump is based on a pressure controller with a piezo-ceramic drive, which builds up pressure on a reservoir containing the suspension to be "pumped". The suspension then rises up a riser tube and then enters the flow chamber. Corresponding systems are offered, for example, by the company Elvesys—Microfluidics Innovation Center, Paris, France.

In an advantageous development of the device according to the invention, the radiation source is designed to activate radiation-activatable substance contained in or adhering to particles by irradiation if these particles are located in the partial volume.

In an advantageous embodiment of the device according to the invention, the movement device is set up to move the particles in the flow chamber by conveying the suspension through the flow chamber, by shearing the suspension in the flow chamber, by inducing a movement of the suspended particles in the suspension, or by a to induce any combination of two or more of the above measures.

In an advantageous embodiment of the device according to the invention, the device also has a movement device controller which is set up to control the movement device.

In a further advantageous development of this configuration of the device according to the invention, the movement device control is set up to control the movement device in such a way that the movement parameters of the particles are constant over a defined movement range that extends through the partial volume; in particular, a constant movement of the particles can occur set in the range of motion.

In an advantageous embodiment of the device according to the invention, the device also has a radiation source controller which is set up to control the radiation source.

In a further advantageous development of these refinements of the device according to the invention, the aforementioned controls are set up to activate the radiation source if constant movement parameters of the particles are present in the defined movement range.

It is obvious that the aforementioned controls do not have to be designed separately, but can also be combined in a common control.

In particular, one or both of the aforementioned controls can be implemented in software.

In a preferred embodiment of the device according to the invention, the radiation source provided according to the invention is set up to emit electromagnetic radiation, in particular in the infrared, in the visible or in the ultraviolet spectral range or of X-rays or gamma radiation, or of particle radiation. However, a radiation source that is set up to emit sound waves, in particular ultrasound, is also the subject of an advantageous embodiment of the device according to the invention.

In an advantageous development of the device according to the invention, the device also has an examination device which is set up to determine the state of the particles contained in the suspension.

The examination device is advantageously set up to determine the external shape or the internal structure of the particles. The examination device is particularly preferably set up to determine the three-dimensional character or a two-dimensional projection of the three-dimensional character of the external shape or the internal structure of the particles.

In a particularly preferred embodiment, the examination device is based on an optical or a scanning microscopic method.

It has proven to be particularly advantageous if the optical method involves optical microscopy, light scattering, light diffraction, light absorption, phase modulation, fluorescence detection, or temporal or spatial modulation methods derived therefrom.

In a particularly preferred embodiment, the examination device is set up to determine the state of more than 10,000 particles per hour, preferably more than 100,000 particles and particularly preferably more than 1,000,000 particles.

In a further advantageous development, the device according to the invention also has a collecting device which is set up for batchwise collecting of the suspension subjected to radiation.

The collecting device advantageously has a collecting device controller which is set up to control the collecting device. The collection device control also does not have to be designed separately, but can be combined in a joint control with the radiation source control and/or the movement device control. The collection device control can also be designed to be software-implemented.

In a further particularly advantageous development, the examination device has a processing unit that is set up to use machine learning to evaluate the signals obtained with the optical or scanning microscopic method, which correlate with the state of the particles to be determined.

The radiation source is advantageously set up to emit radiation, by means of which the radiation-activatable substance can be switched to the activated state.

It is advantageously provided in this device that the substance in the activated state is suitable for crosslinking proteins of the particles to be examined, in particular the amines of lysine or arginine side chains of proteins of the particles to be examined.

More advantageously, the substance may contain or be an aryl azide, a diazirine or a diazirine-containing analogue of the amino acids leucine (photo-leucine), methionine and p-benzoyl-phenylalanine be selected from the group consisting of aryl azides, diazirines, and diazirine-containing analogs of the amino acids leucine (photo-leucine), methionine, and p-benzoyl-phenylalanine.

Further advantageously, the substance can have a modified glutaraldehyde, formaldehyde or paraformaldehyde, or the substance can be modified glutaraldehyde, formaldehyde or paraformaldehyde or a mixture thereof.

Furthermore, the subject of the present invention is a device according to the invention, possibly also with the features of one or more advantageous configurations or developments, the device also comprising particles suspended in a suspension, the state of which is to be determined, the suspension also containing a radiation-activatable substance, which in the non-activated state can penetrate into the interior of the particles or bind to their surfaces without significantly influencing the state of the particles to be determined, and which in the activated state enables crosslinking of structures or components, e.g. proteins, in the to causes examined particles.

An exemplary embodiment of the invention is explained below with reference to the attached drawing. The exemplary embodiment is used by those skilled in the art to explain the invention in more detail, but does not restrict its scope. Nevertheless, features of the exemplary embodiment can be used individually or jointly to develop the subject matter of the claims, insofar as this is technically possible and advantageous for the person skilled in the art.

In the drawing shows 1 schematically an embodiment of a device 100 according to the invention.

The device 100 has a storage chamber 10 in which a suspension is stored, in which the particles to be examined are suspended. The particles are human red blood cells 1 suspended in an aqueous solution. A concentration of about 50 million cells per milliliter of suspension has proven itself, which corresponds to about 10 microliters of whole blood to 1 milliliter of suspension.

A movement device 20 designed as a peristaltic pump, piston, membrane or pressure-driven pump conveys the suspension via a hose system from the storage chamber 10 to a flow chamber 30. This flow chamber 30 is designed as a microcapillary with a rectangular or round cross-section, the cross-section being close to the typical cross-section adapted from human microcapillary blood vessels.

The pump also induces a movement of the suspended red blood cells 1 in the flow chamber 30. The operating parameters of the pump are selected in such a way that flow conditions are formed in the microcapillary in the suspension flowing through which are adapted to the flow conditions found in human capillary blood vessels will.

Furthermore, the device 100 has a radiation source 40 which is set up to generate UV radiation. This UV radiation is suitable for changing a radiation-activatable substance from the non-activated state to the activated state. The radiation source 40 is arranged in such a way that it applies UV radiation to a partial volume of the flow chamber 20 . The radiation source 40 is preferably an LED-based light source or laser, but alternatively a conventional mercury vapor lamp, halogen lamp or, in general, a gas discharge lamp can also be used.

The flow chamber 30 is made of glass or a polymer plastic, with both materials being made transparent or slightly attenuating for the UV radiation of the radiation source 30 .

The suspension which is supplied to the flow chamber 30 contains, in addition to the red blood cells 1 to be examined, a modified glutaraldehyde as a radiation-activatable substance, as is known from [reference 3].

Glutaraldehyde is widely used to fix cells and microorganisms in order to maintain their shape and possibly their metabolic state, preserving them for transport or long-term storage. When glutaraldehyde is added to a sample, the fixation process typically occurs within milliseconds, instantly “freezing” the shape of the cells. The fixation process is based on a chemical protein crosslinking in the cell and the cell membrane.

In the publication Schelkle KM, Schmid C, Yserentant K, Bender M, Wacker I, Petzoldt M, Hamburger M, Herten DP, Wombacher R, Schröder RR, Bunz UH, Cell Fixation by Light-Triggered Release of Glutaraldehyde, AngewChem Int Ed Engl .2017 Apr 18;56(17):4724-4728. dol: 10.1002/anle.201612112. Epub 2017 Mar22. PMID: 28328078 a chemically modified version of glutaraldehyde is proposed which has an additional employs a chemical functional group that masks the chemical reactivity of the substance in such a way that it becomes inactive under normal conditions. If the chemically modified glutaraldehyde is added to a sample, it can enter the cells via an attached ester group. However, the substance remains inactive in the cell until it is activated by the application of UV light. Upon application of the radiation, the masking group is detached from the main molecule and protein cross-linking begins within the cells. At this point, the dominant cell shape is fixed and conserved. This property makes the so-called “caged glutaraldehyde” an example of an ideal additive for studying cell shape dynamics in flow.

The modified glutaraldehyde thus has an activated state in which it causes crosslinking of proteins in the red blood cells 1 to be examined. In the non-activated state, however, this cross-linking property is masked. If this modified glutaraldehyde is irradiated with suitable UV radiation in the non-activated state, the masking is removed and it changes to an activated state in which it has the crosslinking properties mentioned.

The glutaraldehyde contained in the suspension which is fed to the flow chamber 30 is in the non-activated state.

The device 100 also has a movement device control 25, which is set up to control the pump in such a way that the movement parameters of the red blood cells 1 over a defined movement range, which extends through the partial volume of the flow chamber 30 to which UV radiation is applied by the radiation source 40 extends, are constant.

Furthermore, the device 100 has a radiation source controller 45 which is set up to control the radiation source 40, i.e. to control the exposure of the partial volume of the flow chamber 30 to UV radiation.

The aforementioned controls 25, 45 are integrated in a software-implemented common control 50 of the device 100. This is set up to activate the radiation source 40 when constant movement parameters of the red blood cells 1 are present in the defined movement range.

Downstream of the flow chamber 30 in the flow direction is an examination device 60 which is set up to determine the state of the red blood cells 1 contained in the suspension. In the exemplary embodiment shown, the state mentioned is the three-dimensional external shape of the red blood cells 1.

The examination device 60 has a scanning optical microscope, to which the red blood cells 1 fixed by UV radiation are fed in batches on a slide 2 after they have left the flow chamber 30 .

In the exemplary embodiment shown, the device 100 has a collection device 70 for this purpose, which is set up for collecting the suspension subjected to radiation in batches in separate collection containers 75 .

The collecting device 70 advantageously has a collecting device controller 71 which is implemented in software and is set up to control the collecting device 70 . The collection device control 71 is also integrated into the common control 50 in which it is combined with the radiation source control 45 and the movement device control 25 .

The scanning optical microscope creates layered images for each field of view perpendicular to the plane of the object slide 2, whereby a series of typically 50 to 60 consecutive tomographic images of the red blood cells 2 located in the field of view of the microscope, resolved in the plane of the object slide 2, is generated. The three-dimensional external shape of the red blood cells 1 located in the field of view can be determined from this series by means of a suitable image processing device 65 .

For this purpose, the examination device 60 has a processing unit 80 which is set up to use machine learning to evaluate the images obtained with the scanning optical microscope, which correlate with the state of the particles 1 to be determined.

In the exemplary embodiment shown, the examination device 100 is capable of determining the three-dimensional external shape of more than 10,000 red blood cells per hour.

• a. Bereitstellen einer Suspension mit einer Vielzahl zu untersuchender roter Blutkörperchen 1 in der Vorratskammer 10, wobei die Suspension neben den zu untersuchenden rote Blutkörperchen 1 modifiziertes Glutaraldehyd als strahlungsaktivierbaren Stoff im nicht aktivierten Zustand enthält, und Zuführen der Suspension zu der Strömungskammer 30 mittels der Pumpe,
• b. Induzieren einer Bewegung der suspendierten rote Blutkörperchen 1 im umgebenden Medium in der Strömungskammer 30 mittels der Pumpe, und
• c. Beaufschlagen eines Teilvolumens der Strömungskammer 30 mit UV-Strahlung, die dazu geeignet ist, den strahlungsaktivierbaren Stoff vom nicht aktivierten Zustand in den aktivierten Zustand zu versetzen, um das in den roten Blutkörperchen 1 enthaltene modifizierte Glutaraldehyd zu aktivieren und damit die äußere Form der strömenden roten Blutkörperchen 1 zu fixieren.

The device 100 shown above can be used, for example, to carry out a method which has the following method steps:

• a. Providing a suspension with a large number of red blood cells 1 to be examined in the storage chamber 10, the suspension containing modified glutaraldehyde as well as the red blood cells 1 to be examined contains lung-activatable substance in the non-activated state, and supplying the suspension to the flow chamber 30 by means of the pump,
• b. inducing movement of the suspended red blood cells 1 in the surrounding medium in the flow chamber 30 by means of the pump, and
• c. Applying UV radiation to a partial volume of the flow chamber 30, which is suitable for switching the radiation-activatable substance from the non-activated state to the activated state, in order to activate the modified glutaraldehyde contained in the red blood cells 1 and thus the external form of the flowing red blood cells 1 to fix.

The movement parameters of the red blood cells 11 in the flow chamber 30 are kept constant by means of the movement device control 25 over a defined movement range, which extends through the partial volume to which the radiation source 40 can be exposed to UV radiation.

The partial volume of the flow chamber 30 is exposed to UV radiation from the radiation source 40 when constant movement parameters of the red blood cells 1 are present in the defined range of movement. For this purpose, the radiation source 40 is controlled accordingly by the radiation source controller 45 . This activates the glutaraldehyde present in the red blood cells 1, thereby fixing the condition of the red blood cells 1.

In a subsequent method step, the fixed red blood cells 1 are collected in batches in separate collection containers 75 by means of the collection device 70 .

When the method is being carried out, it is provided that the constant movement parameters of the suspended red blood cells 1 in the defined movement range be changed after a predetermined measurement time has elapsed. This change takes place in such a way that, after the change, constant movement parameters of the suspended red blood cells 1 are again present in the defined range of movement, which, however, are different from the previous movement parameters.

Red blood cells 1 that were exposed to matching movement parameters at the time of their fixation by activation of the glutaraldehyde are combined into batches by means of the collection device 70, i.e. collected in one or more collection containers 75. No red blood cells 1 are added to these batches which were exposed to the different movement parameters at the time of their fixation due to the activation of the glutaraldehyde. This means that only red blood cells 1 are contained in a collection container 75 that were exposed to the movement parameters that matched at the time of their fixation. If the particles have essentially the same properties, this allows the use of statistical investigation methods for determining the state of the particles to be determined, which are based on the parallel investigation of a large number of particles, dynamic light scattering being mentioned here as an example. This can be the basis for a quick and reliable determination of the condition to be determined that requires little equipment.

Samples are taken from the collection containers 75, transferred to the slide 2 and fed to the scanning optical microscope. This uses the image processing device 65 to generate three-dimensional representations of the three-dimensional external shape of the red blood cells 1 in the field of view of the microscope, which can be displayed, for example, as a two-dimensional representation 3 on a connected display device 90 .

In a further method step, the images of the red blood cells 1 generated with the microscope are evaluated using machine learning.

In the exemplary embodiment, the basic idea of the invention is embodied in such a way that the shape of flowing red blood cells, which are suspended in a suspension containing modified glutaraldehyde, are fixed in a small spatial area by the application of UV light and their 3D shape is fixed when the red Blood cells reach a steady state within a precisely controlled and well-defined flow. Therefore, the subsequent imaging study of the red blood cells need not be performed at the time when the cells are in flux. Rather, cells can be separately, e.g. B. under different flow conditions, collected and mapped at a later time. The recordings can then be evaluated using artificial intelligence in order to obtain undistorted and fast results with high statistical relevance.

In summary, this approach allows the uptake of particles in the flow in three dimensions, independent of the prevailing flow regime and flow velocity. Other advantages are that the image exercise does not have to be carried out on site. Rather, the examinations can be carried out remotely and complex, vulnerable and expensive optical systems such as microscopes do not have to be provided on site. There are also no special regulations regarding the optical quality of the microfluidic system, which makes it possible, for example, to use standard glass capillaries as flow chambers. If no imaging components are involved at the time the cells flow through, no planar structures for the lowest light refraction such as e.g. B. rectangular channels can be used. Rather, channels with a round cross-section can also be used, which imitate the flow conditions more physiologically.

Reference List

1

Red blood cells

2

slide

3

Illustration of red blood cells

10

pantry

20

moving device

25

movement device control

30

flow chamber

40

radiation source

45

radiation source control

50

common control

60

examination device

65

image processing device

70

collection facility

71

collection facility control

75

collection container

80

processing unit

90

display device

100

contraption

Claims (35)

Method for determining the state of suspended moving particles using a radiation-activatable substance which, in the non-activated state, can penetrate into the interior of the particles or binds to their surfaces without influencing the state of the particles to be determined, and which crosslinks in the activated state of structures or components, e.g. proteins, in the particles to be examined, having the following process steps: a. Providing a suspension with a large number of particles to be examined, the suspension containing the radiation-activatable substance in the non-activated state in addition to the particles to be examined, b. inducing movement of the suspended particles in a flow chamber (30), and c. Subjecting at least a partial volume of the flow chamber (30) to radiation which is suitable for changing the radiation-activatable substance from the non-activated state to the activated state in order to activate the radiation-activatable substance contained or adhering to the particles. procedure according to claim 1 , characterized in that the movement of the particles in the flow chamber (30) by means of conveying the suspension through the flow chamber (30) or by inducing a movement of the suspended particles in the suspension or by inducing a shearing of the suspension in the flow chamber (30) or is induced by a combination of the above measures. procedure according to claim 1 , characterized in that the movement parameters of the particles are kept constant over a defined range of movement that extends through the partial volume. procedure according to claim 3 , characterized in that the partial volume of the flow chamber (30) is exposed to radiation when there are constant movement parameters of the particles in the defined movement range. procedure according to claim 1 , characterized in that the suspension is collected in batches after flowing through the flow chamber (30). procedure according to claim 1 , characterized in that after flowing through the flow chamber (30), the state of the particles is determined. procedure according to claim 3 , characterized in that the constant movement parameters of the suspended particles in the defined movement range are changed to deviating constant movement parameters after a predetermined measurement time has elapsed. procedure according to claim 1 , characterized in that the radiation is electromagnetic radiation, in particular in the infrared, in the visible or in the ultraviolet spectral range or from X-rays or gamma radiation, or to particle radiation or to Sound waves, in particular ultrasonic waves, are involved. procedure according to claim 1 , characterized in that the substance in the activated state is suitable for crosslinking proteins of the particles to be examined, in particular the amines of lysine or arginine side chains of proteins of the particles to be examined. procedure according to claim 9 , characterized in that the substance contains an aryl azide, a diazirine or a diazirine-containing analogue of the amino acids leucine (photo-leucine), methionine and p-benzoyl-phenylalanine or is selected from aryl azides, diazirines and diazirine-containing analogues of the amino acids leucine (photo-leucine), methionine and p-benzoyl-phenylalanine. procedure according to claim 1 , characterized in that the substance has a modified glutaraldehyde, formaldehyde or paraformaldehyde or the substance is modified glutaraldehyde, formaldehyde or paraformaldehyde or a mixture thereof. procedure according to claim 1 , characterized in that the state of the particles to be determined is their external shape or their internal structure, preferably their three-dimensional character or a two-dimensional projection of the three-dimensional character. procedure according to claim 12 , characterized in that the state of more than 10,000 particles per hour is determined. procedure according to claim 12 , characterized in that the outer shape or inner structure is determined by means of an optical or scanning microscopic method. procedure according to Claim 14 that the optical method is optical microscopy, light scattering, light diffraction, light absorption, phase modulation, fluorescence detection, or temporal or spatial modulation methods derived therefrom. procedure according to claim 12 that the signals obtained with the optical or scanning microscopic method are evaluated using machine learning. Device (100) for determining the state of a moving particle using a radiation-activatable substance which, in the non-activated state, can penetrate into the interior of the particles or binds to their surfaces without influencing the state of the particles to be determined, and in the activated State causes a crosslinking of structures or components, e.g. proteins, in the particles to be examined, having the following characteristics: a. a flow chamber (30) for receiving a suspension in which the particles to be examined are suspended, b. an agitator (20) arranged to induce movement of the suspended particles in the flow chamber (30), and c. a radiation source (45) which is set up to expose at least a partial volume of the flow chamber (30) to radiation which is suitable for switching the radiation-activatable substance from the non-activated state to the activated state. Device (100) according to Claim 17 , characterized in that the radiation source (45) is designed to activate radiation-activatable substance contained in particles or adhering to them by irradiation when these particles are in the sub-volume. Device (100) according to Claim 17 , characterized in that the moving device (20) is set up to move the particles in the flow chamber (30) by conveying the suspension through the flow chamber (30), by shearing the suspension in the flow chamber (30), by inducing a movement of the suspended particles in the suspension, or by any combination of the foregoing Device (100) according to Claim 17 , characterized in that the apparatus (100) further comprises a mover controller (25) adapted to control the mover (20). Device (100) according to claim 20 , characterized in that the movement device controller (25) is set up to control the movement device (20) in such a way that the movement parameters of the particles are constant over a defined movement range which extends through the partial volume. Device (100) according to Claim 17 , characterized in that the device (100) further comprises a radiation source controller (45) which is adapted to control the radiation source (45). Device (100) according to Claim 21 and 22 , characterized in that the controls are arranged to activate the radiation source (45) when in the defined movement constant movement parameters of the particles are present in the movement area. Device (100) according to Claim 17 , characterized in that the device (100) further comprises an examination device (60) which is set up to determine the state of particles contained in the suspension. Device (100) according to Claim 17 , characterized in that the device (100) further comprises a collecting device (70) which is set up for batchwise collecting of the suspension subjected to radiation. Device (100) according to Claim 17 , characterized in that the radiation source (45) is set up to emit electromagnetic radiation, in particular in the infrared, in the visible or in the ultraviolet spectral range or of X-rays or gamma radiation, or of particle radiation or of sound waves, in particular of ultrasound. Device (100) according to Claim 21 , characterized in that the state of the particles to be determined by the examination device (60) is their external shape or their internal structure, preferably their three-dimensional character or a two-dimensional projection of the three-dimensional character. Device (100) according to Claim 27 , characterized in that the examination device (60) allows the determination of the state of more than 10,000 particles per hour, preferably more than 100,000 particles per hour and in particular more than 1,000,000 particles per hour. Device (100) according to Claim 27 that the examination device (60) is based on an optical or scanning microscopic method. Device (100) according to claim 29 that the optical method is optical microscopy, light scattering, light diffraction, light absorption, phase modulation, fluorescence detection, or temporal or spatial modulation methods derived therefrom. Device (100) according to claim 29 , characterized in that the examination device (60) has a processing unit (80) which is set up to evaluate the signals obtained with the optical or scanning microscopic method by means of machine learning. Device (100) according to one or more of claims 17 until 31 , characterized in that the device (100) further comprises particles suspended in a suspension, the suspension containing a radiation-activatable substance which, in the non-activated state, can penetrate into the interior of the particles or bind to their surfaces without affecting the state to be determined of the particles, and which in the activated state causes a crosslinking of structures or components, eg proteins, in the particles to be examined. Device (100) according to Claim 32 , characterized in that the substance in the activated state is suitable for crosslinking proteins of the particles to be examined, in particular the amines of lysine or arginine side chains of proteins of the particles to be examined. Device (100) according to Claim 32 , characterized in that the substance contains an aryl azide, a diazirine or a diazirine-containing analogue of the amino acids leucine (photo-leucine), methionine and p-benzoyl-phenylalanine or is selected from aryl azides, diazirines and diazirine-containing analogues of the amino acids leucine (photo-leucine), methionine and p-benzoyl-phenylalanine. Device (100) according to Claim 32 , characterized in that the substance has a modified glutaraldehyde, formaldehyde or paraformaldehyde or the substance is modified glutaraldehyde, formaldehyde or paraformaldehyde or a mixture thereof.

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