WO2017013198A1 - Device for monitoring the three-dimensional movement of objects located in a liquid - Google Patents

Abstract

The invention relates to a device for monitoring the three-dimensional movement of objects (2), in particular biological objects, which are being held in a liquid and the behavior, in particular the movement, of which depends on the local properties in the liquid, in particular the temperature, the chemical composition, or the light conditions. The device comprises the following: - a monitoring chamber (5) for receiving the liquid provided with the objects (2), - a first lighting device (6) with a light source (7), said lighting device being equipped to locally change the properties in the liquid, and - an optical capturing unit (12) which is equipped to optically detect the objects (2) in the liquid, wherein the first lighting device (6) is equipped to introduce a light quantity into the monitoring chamber (5) in a desired three-dimensional distribution in a controlled manner, and the optical capturing unit is equipped to spatially detect the movement of the objects (2) and convert same into corresponding electronic signals.

Description

 Device for observing the three-dimensional movement of objects that are in a liquid

The invention relates to a method and a device for observing the three-dimensional movement of, in particular, living objects in a liquid.

Liquids and organisms have many opportunities to perceive chemical and / or physical properties in the environment and to navigate their distribution, especially along their gradients. Thus, for example, a chemical attractant diffuses out of an egg cell of the sea urchin Arbacia punctulata, so that over time a three-dimensional gradient of this attractant forms around the egg cell. Sperm can perceive this chemical attractant and orient themselves on their way to its rising gradient. This allows them to navigate directly towards the egg cell. This process is called chemotaxis.

Until now, the chemical gradient could only be produced in two dimensions under laboratory conditions, so that only a single observation of the two-dimensional movement of sperm along a two-dimensional gradient was possible. For example, a method is described in the article "Ca ²⁺ spikes in the flagellum control chemotactic behavior of sperm" (M. Böhmer et al., In the EMBO Journal Vol 24, NO 15, 2005, pp. 2741-2752) The sperm movement can be detected in two dimensions, whereby two-dimensional gradients of the attractant concentration are selectively generated and their influence on sperm movement in one plane is investigated the narrow confines of the observation chamber and hydrodynamic effects are suppressed, so that the underlying biochemical processes can only be insufficiently investigated.

The same is known from the article by Jansen in "Controlling fertilization and cAMP signaling in sperm by optogenetics" from eLife, 2015 4, where the sperm are collected by dark field microscopy Sample scattered light taken with a camera. For the lighting is not coherent , .

Defocusing can spatially capture an object.

In addition to the widely discussed example of sperm chemotaxis, knowledge of underlying biochemical processes is important in understanding a variety of cells, organisms, and particles. Examples include bacteria, algae and synthetic micro-swimmers.

The object of the present invention is now to provide a device and a method with which the said biochemical movement processes can be investigated in three dimensions.

The object underlying the invention is achieved by a device according to claim 1 and a method according to claim 7; preferred embodiments will become apparent from the dependent claims and the following description.

According to the invention, an apparatus and a method are provided for observing the three-dimensional movement of objects which are located in a liquid and whose behavior depends on the local distributions of special parameters prevailing in the liquid. In particular, the movement of the objects in the device should be observed. Relevant parameters are in particular the temperature and / or the chemical composition of the liquid and / or the distribution of the amount of light per volume element and in particular their gradients understood. The apparatus comprises a viewing chamber for receiving the liquid, a first exposure device for locally changing the properties of the liquid, and an optical recording unit for optically detecting the behavior of the particles in the liquid.

The device according to the invention is characterized in that the first exposure device is set up in such a way that a specific amount of light can be introduced into the observation chamber in a targeted manner and in a desired three-dimensional distribution in order to influence the spatial distribution of the parameters within the fluid. In addition, a second light source, in particular a laser, is provided, which radiates coherent light into the observation chamber. With a holographic recording unit then the three-dimensional movement of the objects is detected and converted into electronic signals that can be displayed on a screen. .

the spatial information of the object can be detected directly. Thus, the volume can be detected without time offset between the different focal planes. By using coherent light, the disadvantageous temporal offset between different focal planes can be avoided and the movement of the objects spatially detected.

The inventive method is intended for observation of the movement of particular biological living objects, which are in a liquid and their behavior, in particular their movement, is dependent on the prevailing in the liquid distribution of certain parameters, in particular the temperature, the chemical composition or the lighting conditions. The method comprises the steps: the defined local change of the parameters within the liquid by targeted introduction of first light rays into the observation chamber and the optical detection of the objects in the liquid. The method is characterized in that based on the first light beams a defined amount of light is introduced in a desired three-dimensional distribution in the observation chamber that second light rays of coherent light are irradiated into the observation chamber and that the movement of the objects of a holographic recording unit spatially detected and in corresponding electronic signals that can be displayed on a screen is converted.

In the context of the present invention, the term "objects" or "particles" is especially taken to mean cells, organisms and biological or chemical particles, such as latex beats or synthetic micro-floats.

The essence of the invention is now in particular that in the device specifically the properties of the liquid can be changed individually spatially. The generated effects of the targeted adjustable spatial distribution of the properties on the objects are also spatially detectable and can be subsequently analyzed.

Preferably, the liquid contains deliberately introduced components that change their properties when exposed to optical light rays at a certain wavelength. The first exposure device and the liquid are preferably matched to one another in such a way that the chemical composition can be selectively changed at individual locations of the observation chamber by the action of the first exposure device. In particular, so-called caged connections (English, cage-compound) are used. These are chemical compounds that release a certain substance when irradiated with light of certain wavelengths. This released substance then generates a desired reaction. By controlling the amount of light introduced to a location, the desired reaction can thus be specifically initiated at this location.

The main application of caged compounds is biochemical and cell biological research. Biologically active compounds are equipped with a photolabile protecting group, the "cage" and thus temporarily lose their biological function. By means of light irradiation, the photolabile protecting group is irreversibly cleaved from the biologically active compound and the previously inactive compound again has a biological activity.

The amount of light is the radiant energy that is calculated from the integral of the luminous flux during a certain time. By incident on a place amount of light in a certain frequency of light, for example, at this location a corresponding concentration of attractant can be generated. Through the targeted use of lenses, the amount of light can be increased or decreased at a desired location. The closer a location is arranged at the focal point of the lens, the greater is the amount of light incident on that location or in this volume element per unit of time. When a focused light beam is applied to different locations for the same time, the areas located closer to the focal point are exposed more intensely than other locations. Consequently, there forms a greater concentration of attractants.

Preferably, the optical pickup unit comprises a holographic camera. In this way, objects can not only be detected optically, but also the distance of the objects from the optical recording unit can be detected. This can be used to create 3D films of objects that can be evaluated by image processing.

The observation chamber is in particular a chamber in which the liquid is kept static during the observation, that is to say it is not subject to flow during the observation. However, it is not possible to replace the fluid before or after observation or to create a flow if necessary. e. ! it e e s e e e s e se eewa

Composition (in mM): 9 KCl, 423 NaCl, 9.27 CaCl 2, 22.94 MgCl 2, 25.5 MgSO 4, 0.1 EDTA, and 10 HEPES, with a pH of 7.8 adjusted with NaOH. It can be a predetermined number of objects, such as 10 ⁶ sea urchin sperm contained.

For generating a chemical gradient, the liquid may contain a chemical compound, for example, a "caged compound." Preferably, the chemical compound contains an attractant specific for the object In the chemical compound, preferably only a small portion of the specific attractant is freely available and therewith Preferably, the proportion of the free specific attractant is <0.1%, more preferably <0.05% and most preferably <0.01% of the chemical compound. Preferably, the chemical compound itself is not detectable by the object.

The liquid together with the objects is preferably introduced into the observation chamber via a perfusion system. The observation chamber preferably comprises an inlet for the sample into the observation chamber and an outlet from the observation chamber.

The first exposure device is provided for introducing a specific amount of light having a specific wavelength. The wavelength is preferably in the visible range, or in the UV range, preferably in the wavelength range of 300-400 nm. More preferably, the first exposure device comprises a light emitting diode (LED) as the first light source, but alternatively a corresponding laser with a corresponding wavelength may be used become. Preferably, the first light beams generated in the first light source are coupled into a light guide. The optical waveguide is preferably positioned so that the radiated first light beams can be introduced into the observation chamber along the optical axis of the objective via a dichoric mirror located below an objective. Preferably, the light intensity of the first light source is linearly adjustable. In addition to the dichoric mirror, an optical filter may be used so that the wavelength range can be further narrowed. Preferably, a system of lenses and / or a "pinhole" can be provided between the output of the light guide and the dichoric mirror.

The device according to the invention preferably comprises an optical recording unit, which comprises at least one objective and at least one camera. Preferably, the camera is a "high speed" camera which can take pictures at a rate of at least 200 pictures per second, preferably the lens used en. , orrg er eev, so too

Camera and the lens can be inserted. Preferably, a mechanical support for the introduction of additional optical components is provided.

Preferably, the device according to the invention comprises a second exposure device which illuminates the objects located in the observation chamber with coherent light of a predetermined wavelength range. The preferred coherent light source used is a laser having a wavelength range in the preferably visible range, more preferably in the wavelength range from 550 to 700 nm. Here it is expressly pointed out that the partial wavelength ranges can be taken out of this wavelength range. For example, a wavelength range of 675 - 685 nm can be taken out. As an alternative to the use of a laser, it is also possible to use an LED with the corresponding preferred wavelength in combination with a "pinhole." The light from the light source is preferably coupled into an optical waveguide The main propagation direction is defined by a vector pointing away from the optical waveguide on the center of the optical waveguide, the main propagation direction of the light bundle coming from the optical waveguide being positioned on the optical axis of the objective.

According to the invention, the wavelength of the light introduced by the first exposure device differs from the wavelength of the light introduced by the second light source, so that the light of the first exposure device can be used for physical and / or chemical gradients via, for example, "caged-compounds" For example, the light introduced by the first exposure means can change the chemical compounds in the sample or the temperature, or, trivially, the light intensity in a given wavelength range, without the light of the first exposure device interfering with the measurement of the holograms By changing the chemical compound in the sample, for example, the PH value, for example, the C0 ₂ content or, for example, a hole concentration, for example, the concentration of free m Resact or another attractant such as speractum, progesterone or the like. The concentration of free attractant is preferably increased by the light introduced by the first exposure device. The change preferably corresponds, for example, to the concentration of the free attractant or, for example, the pH or, for example, the CO ₂ content of one

v _{n r} o _n rnci _arl. "". , ™ = r +

 ucöiii i ii i iiBi i amount of light so that the change can be quantified t \ <ai in. the spatial distribution of the introduced Uchtes and the actual amount of light in the observation chamber is known, so that taking into account the diffusion of the chemical compound and the diffusion of the free attractant and taking into account the photolysis rate, the spatial distribution of the free attractant concentration can be determined in the observation chamber.

Preferably, each individual hologram picked up by the optical pickup unit can be used to numerically reconstruct the shape and orientation of the object. More specifically, by using a propagator such as the Rayleigh Sommerfeld propagator, a light field analogous to the light irradiated by the second exposure apparatus is numerically calculated. The numerically calculated light field can be calculated along the optical axis, also referred to below as the z axis. By mathematically folding each individual numerically calculated light field along the optical axis with the recorded hologram, the object along the optical axis can be reconstructed from a single hologram. In the thus numerically reconstructed volume, the position of the object on the z axis or the position of subareas of the object on the z axis can then be determined. For example, the position on the z-axis can be determined using the "Gouy phase anomaly." The "Gouy phase anomaly" is a contrast inversion, for example, from black to white, of the object at the respective focal position of the object.

Preferably, in a first step, the movement of the objects is detected spatially, without previously the properties in the liquid were defined by the first light beams influenced. In a second step, the properties in the liquid are then influenced in a defined manner by the first light beams. In a third step, the movement of the objects is again spatially detected. This approach can be used to immediately detect and analyze the effect of property changes on objects.

Preferably, the observation chamber comprises a perfusion device, which is electronically connected to the recording unit, the first and second light source, and is controlled in such a way that after successful completion of an observation the liquid in the observation chamber is automatically exchanged. Through an interaction of the perfusion device, the light sources and the recording unit, an optimal test procedure can be generated. In particular, the processes can be automated. First, the liquid in the observation chamber becomes automatic _au,,,

Take up. After a predetermined time, an automatic exchange of the liquid and a new observation takes place again. This process is advantageous because, in principle, objects tend to adhere to a wall of the observation chamber by accidental movement, but no longer detach from it. Over time, especially a few minutes, the liquid becomes so unusable for further observations. In this respect, automatic renewal of the liquid within predetermined time intervals makes sense.

The invention will be explained in more detail below with reference to the figures. Herein shows

FIG. 1 shows schematically the movement profile of a sperm cell on the way to the egg cell;

 FIG. 2 schematically shows a perspective view of an observation chamber in which a Gaussian light distribution is generated by means of an exposure device;

 FIG. 3 schematically shows the structure of a device according to the invention;

 Figure 4, the observation chamber of the device of Figure 3 with connected

 Perfusion.

FIG. 1 shows a sperm cell 2 in search of an ovum 1. The egg 1 exudes an attractant to attract the sperm cell 2. The three-dimensional movement of the sperm cell 2 is dependent on the three-dimensional concentration in particular of the gradient of attractant, which is to be examined. The egg 1 and the sperm cell 2 are in a liquid. The dashed lines indicate areas of each constant concentration of the attractant. On a spherical surface on which the point A is located, there is a concentration C (A) which is greater than the concentration C (B) in a spherical surface on which the point B is at a greater distance from the ovum 1 as the point A has. With the device according to the invention, the movement of sperm as a function of the concentration gradient of the attractant within the fluid can be examined.

FIG. 2 now shows a three-dimensionally extended observation chamber 5, which is filled with the liquid and in which a multiplicity of sperm cells 2 can move freely. The liquid is enriched with so-called caged compounds, which are sensitive to light of a specific wavelength or a specific wavelength range. If appropriate light hits the caged connection, this caged connection is split and an initially trapped substance is released. For this approved substance , , , v, rzu, to attract the sperm cells 1 is used. With a light input at the appropriate wavelength, the local release of the attractant can be controlled in three dimensions within the observation chamber.

For this purpose, a first exposure device 6 is provided which has a first light source 7 which emits first light beams 9 in the specific wavelength. Via a lens 8, the first light beams 9 are focused and focused on a focal point F. In the focal point F, the greatest light energy is introduced over a certain time. The caged compounds are thus most heavily exposed to light of the particular wavelength at focus F, thus most attractant from the caged compounds is released there. With the focal point F, the presence of an egg 1 is simulated. In this or a similar way, a large number of different light quantity distributions can be generated.

In the other areas outside the focal point F, although light energy is introduced; depending on the distance from the focal point, this amount of light per unit volume is much lower. According to the Gaussian light distribution, this results in a profile of the amount of light introduced per unit volume in the individual regions of the observation chamber, which has a direct influence on the three-dimensional concentration of released attractant in the observation chamber 5. In FIG. 2, by way of example, two planes are selected that intersect the focal point around which circles are indicated. The locations on the circular lines each have a concentration C (B |) and C (B _N ), which corresponds to the concentration C (B) of Figure 1. The concentration C (B,) or C (Bn) at the points Bl and BN is significantly lower than the concentration C (F) at the focal point.

By deliberately defocusing or using additional exposure devices, different three-dimensional light profiles can be introduced to different locations of the observation chamber 5. Consequently, far more complex three-dimensional gradients of attractant concentration in the observation chamber can be generated. Thus, galvanometric mirrors or acousto-optical deflectors can be used to deflect the light in different directions, or semitransparent masks.

FIG. 3 shows a schematic representation of an entire device according to the invention, which uses the exposure device according to FIG. Evident is the first light source 7 of the first exposure device 6, which emits the first light beams 9. about ,.

the first light beams 9 are directed in the direction of the observation chamber 5. Between the mirror 14 and the observation chamber 5, an objective 15 is arranged.

With the illumination device, a three-dimensional distribution of the attractant concentration is generated in the observation chamber 5, as explained above with reference to FIG. In addition, a second light source 10 is directed to the observation chamber 5, which illuminates the observation chamber 5 for observing the movement of the objects therein. The second light source 10 emits second coherent light beams 11 in a wavelength which have no influence on the caged connections. The sperm cells moving in the observation chamber are illuminated by the second coherent light beams 11. With a holographic camera 12, the processes in the observation chamber can then be observed. The holographic camera can now determine the positions of individual sperm cells in the observation chamber 5 with temporal and three-dimensional spatial resolution and convert it into electrical data. The movement profile of the individual investigating sperm cells can thus be processed and visualized on a computer 13. By means of the known light entry into the observation chamber, it is thus possible to generate links between the spatial concentration distribution of the attractant and the resulting spatial movement profile of individual sperm cells.

The observation chamber 5 has - as shown in Figure 4 - an inlet 19 and a drain 20. The inlet 19 is connected via a hose with a controllable pump 16. This pump 16 is connected to the computer 13 or other control unit, which in turn is connected to the optical pickup unit 12, to the first exposure means and to the second exposure means and to a control unit. Thus, the liquid can be selectively introduced from the sample container 17 into the observation chamber 5 with the sample without the observation chamber 5 itself has to be moved or replaced. In addition, the recording of the objects in the sample can be automated, this ensures a standardized and automated test procedure. LIST OF REFERENCE NUMBERS

1 egg

 2 sperm cell

 3 direction of movement

4 liquid

 5 observation chamber

6 exposure device

7 first light source

8 lens

 9 first rays of light

10 second light source

1 1 second light rays

12 holographic camera

13 computers

 14 dichromatic mirror

15 lens

 16 pump

 17 storage container

18 perfusion device

19 inflow

 20 outflow

F focus

Claims

claims 1. Device for monitoring the three-dimensional movement of in particular biological objects (2) in a liquid whose movement is dependent on gradients of certain parameters within the liquid, in particular of gradients of temperature, of certain chemical constituents and / or of light,  the device comprising:  an observation chamber (5) for receiving the liquid in which the objects (2) move, the observation chamber being dimensioned so that the objects can move freely in three dimensions,  a first exposure device (6) having a light source (7) adapted to change the local properties in the liquid,  - An optical recording unit (12), adapted for the optical detection of the objects (2) in the liquid, characterized in that the first exposure means (6) is adapted to selectively introduce a quantity of light in a desired three-dimensional distribution in the observation chamber (5), to influence the spatial distribution of parameters within the fluid, and  that a second light source (10) is provided which radiates coherent light (1 1) in the observation chamber (5), and  in that the optical recording unit is set up as a holographic recording unit in order to detect the three-dimensional movement of the objects (2) and to convert them into corresponding electronic signals. 2. Device according to the preceding claim  characterized, that the liquid contains components that change their properties when exposed to optical light rays at a certain wavelength. 2 and v0 u1i7i / i0w13 HU19I 8 i, "na" c "he _™ ine _Qr m _r) dj _o e _r r _w v" o _r rhe _a r _{r i} i _n g _Q e _r n, A4 "ncsnprr, ü" c _h he ₀ , PCT / EP2016 / 067379 characterized the first exposure device (6) and the composition of the liquid are coordinated with one another in such a way that the chemical composition can be selectively changed at individual locations of the observation chamber (5) by the action of the first exposure device (6). Device according to one of the preceding claims, characterized _» in that the optical recording unit is set up to numerically reconstruct from a single hologram the shape and the orientation of the object Device according to one of the preceding claims, characterized, in that the observation chamber (5) is connected to a perfusion device (18), which is electronically connected to the recording unit (12), the first and second light sources (7, 10), and is controlled in such a way that after successful completion of an observation, the liquid is automatically evacuated in the observation chamber (5), Device according to one of the preceding claims, characterized, in that the observation chamber (5) is arranged between an objective (15) of the recording unit (12) and the second exposure device (10) such that the first light beams (9) are transmitted from the first light source (7) via a semitransparent mirror (14) and then spatially localized by the lens (15) are introduced into the observation chamber (5). Method for monitoring the movement of, in particular, biological objects which are located in a liquid and whose movement is dependent on gradients of specific parameters within the liquid, in particular gradients of the temperature, of certain chemical constituents and / or of the density of light, comprising the following method steps:  - Defined local change of the parameters within the liquid by targeted introduction of first light beams (9) in the observation chamber (5), - optical detection of the objects (2) in the liquid, characterized in that on the basis of the first light beams (9) a defined amount of light in a desired three-dimensional distribution is deliberately introduced into the observation chamber (5),  that second light rays of coherent light (11) are irradiated into the observation chamber (5) and  the movement of the objects (2) is detected spatially with a holographic recording unit and converted into corresponding electronic signals. 8. Method according to the preceding claim,  characterized,  that, in a first step, the movement of the objects (2) is detected spatially, without the parameters within the liquid having been influenced in a defined manner by the first light beams (9),  that in a second step the parameters within the liquid are influenced in a defined manner by the first light beams (9),  that in a third step, the movement of the objects (2) is again detected spatially.