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WO2017013198A1 - Dispositif pour observer la migration tridimensionnelle d'objets qui se trouvent dans un liquide - Google Patents

Dispositif pour observer la migration tridimensionnelle d'objets qui se trouvent dans un liquide Download PDF

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Publication number
WO2017013198A1
WO2017013198A1 PCT/EP2016/067379 EP2016067379W WO2017013198A1 WO 2017013198 A1 WO2017013198 A1 WO 2017013198A1 EP 2016067379 W EP2016067379 W EP 2016067379W WO 2017013198 A1 WO2017013198 A1 WO 2017013198A1
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WO
WIPO (PCT)
Prior art keywords
liquid
light
objects
observation chamber
movement
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2016/067379
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German (de)
English (en)
Inventor
Ulrich Benjamin Kaupp
René Pascal
Luis Alvarez
Jan Jikeli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Stiftung Caesar Center of Advanced European Studies and Research
Original Assignee
Stiftung Caesar Center of Advanced European Studies and Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stiftung Caesar Center of Advanced European Studies and Research filed Critical Stiftung Caesar Center of Advanced European Studies and Research
Publication of WO2017013198A1 publication Critical patent/WO2017013198A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/144—Imaging characterised by its optical setup
    • G01N2015/1445—Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography

Definitions

  • 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.
  • a chemical attractant diffuses out of an egg cell of the sea urchin Arbacia franata, 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.
  • Defocusing can spatially capture an object.
  • 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.
  • 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.
  • 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.
  • a second light source in particular a laser, is provided, which radiates coherent light into the observation chamber.
  • a holographic recording unit 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.
  • the volume can be detected without time offset between the different focal planes.
  • 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.
  • 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.
  • 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.
  • 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.
  • caged compounds 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.
  • 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.
  • the optical pickup unit comprises a holographic camera.
  • 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.
  • the liquid may contain a chemical compound, for example, a "caged compound.”
  • 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.
  • 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.
  • 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.
  • 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.
  • the light intensity of the first light source is linearly adjustable.
  • an optical filter may be used so that the wavelength range can be further narrowed.
  • 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.
  • 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.
  • a mechanical support for the introduction of additional optical components is provided.
  • 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.
  • 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.
  • 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.
  • 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"
  • 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
  • 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
  • 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.
  • 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.
  • the object along the optical axis can be reconstructed from a single hologram.
  • 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.
  • 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.
  • the movement of the objects is detected spatially, without previously the properties in the liquid were defined by the first light beams influenced.
  • the properties in the liquid are then influenced in a defined manner by the first light beams.
  • 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.
  • 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.
  • 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.
  • an optimal test procedure can be generated.
  • the processes can be automated. First, the liquid in the observation chamber becomes automatic au,,,
  • 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
  • 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.
  • 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.
  • a first exposure device 6 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.
  • 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.
  • an objective 15 is arranged between the mirror 14 and the observation chamber 5.
  • a three-dimensional distribution of the attractant concentration is generated in the observation chamber 5, as explained above with reference to FIG.
  • 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.
  • the 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.
  • 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.
  • 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.
  • the recording of the objects in the sample can be automated, this ensures a standardized and automated test procedure. LIST OF REFERENCE NUMBERS

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  • Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un dispositif pour observer la migration tridimensionnelle d'objets (2), en particulier d'objets biologiques, qui sont contenus dans un liquide ainsi que leur comportement, en particulier leur déplacement, en fonction des conditions régnant localement dans le liquide, en particulier la température, la composition chimique ou les conditions d'éclairage. Ce dispositif comprend : - une chambre d'observation (5) destinée à recevoir le liquide contenant les objets (2), - un premier système d'éclairage (6) avec une source de lumière (7) conçu pour modifier localement les propriétés du liquide, - une unité d'acquisition optique (12) conçue pour l'acquisition optique des objets (2) dans le liquide, le premier système d'éclairage (6) étant conçu pour projeter de manière ciblée une certaine quantité de lumière selon une répartition tridimensionnelle souhaitée dans la chambre d'observation (5) et l'unité d'acquisition optique étant conçue pour enregistrer dans l'espace le déplacement des objets (2) et le convertir en signaux électroniques correspondants.
PCT/EP2016/067379 2015-07-21 2016-07-21 Dispositif pour observer la migration tridimensionnelle d'objets qui se trouvent dans un liquide Ceased WO2017013198A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102015111817.3A DE102015111817B4 (de) 2015-07-21 2015-07-21 Vorrichtung zur Beobachtung der dreidimensionalen Bewegung von Objekten, die in einer Flüssigkeit gehalten sind
DE102015111817.3 2015-07-21

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WO2017013198A1 true WO2017013198A1 (fr) 2017-01-26

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WO (1) WO2017013198A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014012031A1 (fr) * 2012-07-13 2014-01-16 The Regents Of The University Of California Suivi de sperme tridimensionnel (3d) exempt de lentille à haut débit

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8867803B2 (en) * 2010-04-20 2014-10-21 Eric J. Seibel Optical projection tomography microscopy (OPTM) for large specimen sizes
US9767341B2 (en) * 2010-12-14 2017-09-19 The Regents Of The University Of California Method and device for holographic opto-fluidic microscopy
US20150004637A1 (en) * 2011-11-23 2015-01-01 President And Fellows Of Harvard College Systems and methods for imaging at high spatial and/or temporal precision
US20160216192A1 (en) * 2013-09-05 2016-07-28 Empire Technology Development Llc Cell culturing and tracking with oled arrays

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014012031A1 (fr) * 2012-07-13 2014-01-16 The Regents Of The University Of California Suivi de sperme tridimensionnel (3d) exempt de lentille à haut débit

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BJÖRN KEMPER ET AL: "Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy", JOURNAL OF BIOPHOTONICS, vol. 3, no. 7, 1 July 2010 (2010-07-01), pages 425 - 431, XP055006175, ISSN: 1864-063X, DOI: 10.1002/jbio.201000035 *
JAN F. JIKELI ET AL: "Sperm navigation along helical paths in 3D chemoattractant landscapes", NATURE COMMUNICATIONS, vol. 6, 17 August 2015 (2015-08-17), pages 7985, XP055314638, DOI: 10.1038/ncomms8985 *
KAUPP U B ET AL: "THE SIGNAL FLOW AND MOTOR RESPONSE CONTROLLING CHEMOTAXIS OF SEA URCHIN SPERM", NATURE CELL BIOLOGY, MACMILLAN MAGAZINES LTD, GB, vol. 5, no. 2, 1 February 2003 (2003-02-01), pages 109, XP001181602, ISSN: 1465-7392, DOI: 10.1038/NCB915 *

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DE102015111817B4 (de) 2019-11-07
DE102015111817A1 (de) 2017-01-26

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