WO2019125195A1 - Chambre de mesure compacte compatible avec un système de pince optique dans des conditions de niveau d'oxygène contrôlées - Google Patents

Chambre de mesure compacte compatible avec un système de pince optique dans des conditions de niveau d'oxygène contrôlées Download PDF

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Publication number
WO2019125195A1
WO2019125195A1 PCT/PL2018/050069 PL2018050069W WO2019125195A1 WO 2019125195 A1 WO2019125195 A1 WO 2019125195A1 PL 2018050069 W PL2018050069 W PL 2018050069W WO 2019125195 A1 WO2019125195 A1 WO 2019125195A1
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Prior art keywords
chamber
measuring chamber
module
openings
compact
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PCT/PL2018/050069
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English (en)
Inventor
Marta WOŹNIAK
Marcin BACIA
Kamila Duś-Szachniewicz
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Uniwersytet Medyczny Im. Piastów Śląskich We Wrocławiu
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Publication of WO2019125195A1 publication Critical patent/WO2019125195A1/fr

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/26—Stages; Adjusting means therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/32—Micromanipulators structurally combined with microscopes

Definitions

  • the invention relates to a chamber for investigation of biomechanical properties of cells, particularly cancer cells, in the optical tweezers, in an atmosphere controlled for the concentration of oxygen.
  • Optical tweezers also called optical pliers, are a tool, which generates optical forces capable of manipulating objects of a size ranging from 0.4 pm to 20.0 pm in a microscope sample.
  • a proper shape of a laser beam having a wavelength in the infrared or visible light range allows investigating of mechanical properties of cells, determining cell membrane elasticity, intercellular interactions in cocultures of normal and cancer cells, biological materials, DNA strands.
  • the optical trap generated by the laser beam forming system originating from a highly focused laser beam, captures the objects present in the microscope sample in the focal plane of the microscope objective.
  • the optical tweezers system allows to directly or indirectly (using specialised dielectric nanospheres) measure displacements, forces acting on the biological structures, as well as the adhesive properties of the trapped cells.
  • Measuring chambers for microscopic observations are known in the art, see e.g. application US4974952A or US3726597A. Chambers enabling microscopic observations of living cells under controlled conditions are available on the market. Modern chambers are configured for specific types of microscopes, require partial casing of the microscope, which makes it impossible to move or carry them in a convenient manner. Additionally, they are characterised by impractical, considerable sizes.
  • a chamber for a microscope is known, allowing observation of living cells under the microscope with temperature control and under constant oxygen and carbon dioxide pressure (Environmental Chamber, Heat Controller, CO2 and O2 Controller by World Precision Instruments (175 Sarasota Center Blvd. Sarasota, FL 34240). However, this chamber has a considerable size, requires casing parts of the microscope and is unsuitable for moving.
  • chambers of small sizes are known in the art, but they are intended only for observations of cellular samples prepared on microscope slides or in cell cultures located in a Petri dish.
  • the construction of small measuring chambers is compatible with most of microscope tables, but not with the optical tweezers system that would allow manipulating objects of very small size.
  • One example may be a chamber for a microscope allowing observation of living cells located on a 35 mm diameter Petri dish. It is a small, mobile chamber having a cylindrical shape, with temperature control capability (https://www.microscopyu.com/applications/live-cell-imaging/live-cell-imaging-culture-chambers).
  • Another small chamber available on the market is a chamber in a temperature maintaining system (http://animalab.eu/pl/ etc./uniwersalny-system-ibidi-do-podtrzymywania-temperatury). It is a chamber of a small size, comparable to a typical microscope table, compatible with tables of all inverted microscopes and providing observations under precisely determined environmental conditions, including anaerobic conditions.
  • the ibidi system being applied allows maintaining a constant temperature and is intended for all kinds of cell observations using a high resolution microscope, TIRF, confocal microscope.
  • each of the known solutions is suitable only for a microscope and none of them can be implemented in the optical tweezers system in a direct and straightforward manner.
  • cancer cells metabolism based on glycolysis, which is also carried out in the presence of oxygen (Albert, Ina, Martin Hefti, and Vera Luginbuehl. "Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapy in vitro.” Neurological research 36.11 (2014): 1001 -1010). Because the microenvironment of cancer cells present within a tumour is characterised by a specific gas composition, that determines the metabolism of these cells, during the microscopic study of such samples, it is especially important to maintain the state of hypoxia, i.e. low oxygen concentration. All manipulations carried out, until now, on living cells in the optical tweezers were conducted under conditions of atmospheric oxygen level.
  • the object of the invention is therefore disigning a measuring chamber that provides maintaining of the specified oxygen conditions, including conditions of hypoxia, and that is compatible with the optical tweezers system.
  • a sealed measuring chamber having a small size, and maintaining an atmosphere of the desired composition, in medicine and biomedical engineering.
  • the object of the invention is a compact measuring chamber, characterised in that it comprises: a hermetic working chamber confined by walls:
  • a side right, two-part wall composed of a side wall (5) and a side wall (7) divided by a main panel (1 ),
  • the side wall (5) comprises an opening (A) for the secondary door (6)
  • the side wall (8) comprises gas inlet (B) and outlet (C) openings to/from the chamber
  • the front wall (3) comprises an opening (D) for the main door (4)
  • top (9a) and bottom (9b) walls comprise openings (E, F), located oppositely to each other, for microscope objectives;
  • the main panel (1 ) of the chamber is located, equipped with mounting openings (G), opening (F) for inserting the microscope objective, gas inlet openings (H), openings (I) for guides for elements supporting the measuring module, and, on the main panel (1 ), a measuring module (16) for samples is placed,
  • the said measuring chamber is compatible with optical tweezers system and allows performing measurements under controlled oxygen level conditions.
  • gaskets (10), preferably made of silicone, and rings (1 1 ) are placed on openings (E, F) for microscope objectives in the top (9a) and bottom (9b) walls.
  • the secondary door (6) comprises hinges (12).
  • the measuring chamber main panel comprises 4 mounting openings (G) fastening the structure to the microscope table.
  • the measuring chamber main panel comprises elements (13 a, b) supporting and reinforcing the structure.
  • the front wall (3) comprises an element (13c) supporting and reinforcing the structure.
  • the opening (F) for inserting the microscope objective is protected with gaskets, preferably made of silicone.
  • the measuring module (16) for samples is constituted by a dish for observation of microscopic cells, particularly cancer cells.
  • the compact measuring chamber according to the invention is connected to an oxygen level meter (17) placed locally on the gas outlet (C) of the chamber.
  • Another object of the invention is a system including the compact measuring chamber according to the invention connected to a module (18) supplying the specified atmosphere composition.
  • the chamber is connected to the module (18) supplying the specified atmosphere composition by a flexible tube.
  • the module (18) supplying the specified atmosphere composition comprises a gas source (19), a reducer (20), gas valves (21a, b), specifically ball valves, a gas pumping device (22), a mixing chamber (23), a gas outlet opening B to the measuring chamber.
  • the module (18) supplying the specified atmosphere composition comprises quick connectors (24) to flexible hoses.
  • the module (18) supplying the specified atmosphere composition contains gas, constituted by atmospheric air and nitrogen.
  • the specified atmosphere composition comprises 0.5-2% oxygen, preferably 0.5 to 19% oxygen.
  • the chamber according to the invention is compatible with an optical tweezers system including a camera system, dichroic mirrors, a single-mode optical fibre, a 980 nm laser diode, a collimator, a liquid crystal modulator, a microscope objective with large numerical aperture; a lens and Galvano-mirrors.
  • an optical tweezers system including a camera system, dichroic mirrors, a single-mode optical fibre, a 980 nm laser diode, a collimator, a liquid crystal modulator, a microscope objective with large numerical aperture; a lens and Galvano-mirrors.
  • the disclosed hermetic chamber according to the invention allows obtaining controlled oxygen conditions.
  • the chamber allows conducting studies of biomechanical properties of cancer cells in the optical tweezers under physiological oxygen conditions present inside the body (3-10% oxygen (23-70 mmHg)).
  • the chamber according to the invention also allows obtaining conditions of hypoxia present inside the tumour mass (0,5-2%).
  • the disclosed solution eliminates the necessity of preforming analyses under the atmospheric oxygen conditions, which do not reflect the actual concentration of gases inside the living organism.
  • a stable atmosphere composition in the chamber was achieved, characterised by an oxygen content of 5%. Additionally, the conditions were consistent during the subsequent initialisations of the measuring setup (with maintained settings of the control valves). The oxygen level fluctuations during prolonged measurement were equal to ⁇ 0.1 %, which coincides with the resolution of the meter being used. After opening the door for inserting the cell material (Fig. 2a, no. 6) for 10 s, the oxygen level in the chamber increased to the maximum of 6.5% (depending on the duration of opening) and returned to the level of 5% after 17.3 ⁇ 1.1 s.
  • the chamber according to the invention allows conducting such measurements under various aerobic conditions. See, example 2 below.
  • the optical tweezers system for measurement of physical properties of cells in a precisely controlled atmosphere allows performing optical manipulations under oxygen conditions resembling those in which the cells inside a living organism function (so called physioxia conditions).
  • the claimed solution allows isolating the measuring station from the outside atmosphere and removing the oxygen by pushing it out by a neutral gas (nitrogen).
  • a hermetic chamber for manipulations on living cells in the optical tweezers was designed, which constitutes a novel, additional element of a holographic optical tweezers system.
  • the chamber is intended for evaluating biomechanical properties of cells, including elasticity, resilience and adhesive properties under conditions of constant control of the oxygen concentration. It is located between two elements of the optical tweezers system: directly adjacent to the objective, from the bottom, and to the illuminator, from the top.
  • the chamber according to the invention is sealed and compatible with the optical tweezers system. Small dimensions of the chamber enable maintaining the air tightness of the chamber and maintaining the specified gas atmosphere, as well as providing its mobility.
  • the solution according to the invention may supplement the range of measuring chambers for cell observation under given environmental conditions, available on the market.
  • the claimed structure allows application for an entirely novel purpose - for observation of living cells, particularly cancer cells, in an optical tweezers system.
  • the invention allows analysing the biomechanical properties of cells under conditions of changing oxygen concentration in real time.
  • An additional advantage of the chamber being a part of an optical tweezers system according to the invention is that its structure and small size do not limit modulation of the optical tweezers in any way.
  • the optical setup of tweezers may be modified freely, while ensuring that the chamber will still be compatible with it.
  • Small dimensions of the chamber according to the invention ensure that, during inserting the sample, as well as applying the cells of the second cell line to the test dish, the time after which the expected atmosphere will be established in the chamber will be as short as possible. Due to small dimensions of the chamber, the time of returning to the set conditions is about 5 s. Applying a strictly controlled atmosphere also impacts the precision of conducted measurements.
  • optical tweezers system is not a closed system, it is working in the atmosphere present in the room and can be expanded further, depending on the requirements of the performed studies.
  • Figure 1 illustrates an optical tweezers system according to one embodiment of the invention.
  • the system comprises: SLM - a spatial light modulator, G - Galvano-mirrors, K1 - a standard camera, K2
  • DM1 - a dichroic mirror DM2 - a dichroic mirror
  • U - a measuring chamber handle LED - a sample illuminator, T1 - a l1 laser beam, T2 - a L2 laser beam, T3 - an observation path, T4 - an illuminator light beam.
  • Figure 2 illustrates the structure of the compact measuring chamber
  • 2a visualisation of parts constituting the chamber: 1 - a chamber main panel, 2 - a back wall, 3 - a front wall with a main door opening, 4 - a main door, 5 - a side wall with a secondary door opening, 6
  • a door for applying additional solutions to the measuring dish 7 - a bottom part of the side wall, 8 - a side wall with gas inlet (B) and outlet (C) openings to/from the chamber, 9a - a top wall with an opening (E) for the illuminator system objective, 9b - a top wall with an opening (E) for the microscope objective, 10 - silicone gaskets sealing the space around the microscope objectives, 11 - rings tightening the gaskets, 12 - door hinges, 13 a, b, c - additional elements supporting and reinforcing the entire structure; 14 a, b, c - elements supporting the measuring module, 15 - guides for the elements supporting the measuring module.
  • 2b visualisation of the finished chamber. 1 - a main panel of the chamber, 4 - a main door, 5 - a side wall with a secondary door opening (A), 6 - a door for adding additional solutions to the measuring dish, 7 - a bottom part of the side wall, 9a - a top wall with an opening (E) for the illuminator system objective, 10 - silicone gaskets sealing the space around microscope objectives, 1 1 - rings tightening the gaskets, 13 a, b, c - additional elements supporting and reinforcing the entire structure; 14 - a, c elements supporting the measuring module, 16 - a measuring module, 17 - an oxygen meter.
  • Figure 3 illustrates the structure of the main panel of the measuring chamber.
  • F an opening for inserting the microscope objective
  • G an opening fastening the structure to the microscope table
  • H gas outlet openings to the measuring chamber
  • I openings for guides of the elements supporting the measuring module.
  • Figure 4 illustrates an embodiment of the module (18) maintaining the specified atmosphere composition in the measuring chamber.
  • the module comprises a nitrogen cylinder (19), a reducer (20), a nitrogen valve (21a), an air valve (21 b), a fan (22) pumping the air, a mixing chamber (23) and a gas outlet opening (B) to the measuring chamber.
  • Figure 5 a, b, c presents photographs of an embodiment of the chamber mounted to the main panel of the microscope table of the optical tweezers system.
  • Figure 6 illustrates a Toledo line lymphoma cell in the optical trap, according to the description in Example 2.
  • Figure 7 illustrates a graph for the time of forming a stable connection between the lymphoma cells and HS-5 stromal cells under varying oxygen conditions. Student's t-test significance result, p ⁇ 0.0001.
  • a measuring chamber of size 130x58x60 mm was made of transparent acrylic cut using a laser plotter.
  • the structure of the compact measuring chamber is presented in Figs. 2a and 2b.
  • the main panel mounted to the optical tweezers system is the primary supporting element of the chamber (Fig. 3).
  • openings mounting the structure to the microscope table are present in the main panel, which enables moving the microscope table in three axes.
  • the main panel comprises openings, the largest of which is located in the central part of the panel and is intended for moving the icroscope objective towards the test sample.
  • the entire structure was closed with two walls parallel to the main panel, one under and one over the panel.
  • the top and bottom walls comprise openings, through which microscope objectives were inserted: the bottom one for sample observation and generating the optical traps, and the top one, coupled to the illuminator.
  • the chamber comprises two access openings closed by flaps.
  • the larger opening located on the longer side of the chamber, is intended for inserting the measuring module with the test sample.
  • the points of neutral gas inlet, present in the main panel, were designed near the access openings, which allows maintaining the desired atmosphere in the chamber.
  • Ready-made aquaristic couplings configured for working with pressured gases, were used as channel couplings.
  • the outlets of these couplings are located on the front side of the chamber.
  • the gas inlet was located on the bottom of the wall, whereas the outlet was located on the top part thereof.
  • a smaller chamber, constituting the measuring module with samples, is mounted to the main panel.
  • the chamber according to the invention is compatible with the measuring module, which is constituted by a 35 mm diameter dish, on which cells intended for microscopic observation are placed.
  • the dishes allow manipulating the cells in the optical tweezers system, due to the fact that the 170 pm thick specialised glass bottom enables the light beam to pass through.
  • the dish also provides sterile conditions for the conducted manipulations.
  • the measuring chamber is connected to the module maintaining the specified atmosphere composition by constantly supplying gases in the specified ratios, which provides desirable conditions of the flow system in the measuring chamber.
  • gases in the specified ratios
  • two components were mixed in the appropriate ratio: atmospheric air (drawn in real time from the ambient) and nitrogen from the high-pressure cylinder.
  • the nitrogen to air ratio allowing obtaining the oxygen concentration of 5%, was 4.2:1 , while the ratios were determined for the specified concentration.
  • nitrogen is drawn from a cylinder under a pressure of 200 bars.
  • the reducer mounted directly on the cylinder, reduces the working pressure of the gas to about 0.5 bar. Nitrogen is transported to the mixer module by a flexible tube.
  • a precise valve is located, enabling setting of the given gas flow to the mixing chamber.
  • the atmospheric air is pumped into the mixing chamber, using a radial fan.
  • an oxygen concentration of 0.5-2% is used.
  • the chamber allows conducting manipulations in an oxygen range of 0.5 to 19%.
  • a valve is located between the fan and the mixing chamber, by which air flow rate control is possible.
  • the nitrogen and air valves are connected to the mixing chamber by flexible tubes.
  • the structure of the module maintaining the specified atmosphere composition is presented in Fig. 4.
  • the above system was realised based on standard elements of hydraulic and gas installations.
  • the reducer was purchased as a ready-made part (Perun,2RBAz-0,3 model, cat. no. W242-7941 ).
  • As the valve a precise valve from an aquaristic CO2 installation (Eheim, cat. no. 4005510) was used. A three- way tube of size 1 ⁇ 2 was used as a mixing chamber.
  • the air valve is a hydraulic ball valve available on the market (valve GW 1/2" - GW 1/2" equation, cat. no. LM14523243).
  • the fan was fixed to the tubing element fitting to the air valve in use.
  • quick-couplings of flexible hoses from an aquaristic CO2 installation were used.
  • the flow of gases was calibrated before each use of the module, with an oxygen content meter placed in the measuring chamber (Greisinger GOX 100, cat. no. 600437). Additionally, oxygen was controlled during the measurements, in order to keep constant atmospheric conditions.
  • the presented module can be modified in order to add different gases to the atmosphere in the measuring chamber, by connecting another cylinder containing a desired gas, a reducer, a precise valve and a mixing chamber to the outlet of the mixing chamber.
  • a desired gas characteristic for a given tissue or organ
  • carbon dioxide concentration in the measuring chamber can be freely increased and controlled.
  • the entire chamber is moving independently from other elements of the optical tweezers system.
  • the air tightness around the objectives, which do not contact the chamber directly, was achieved by using silicone gaskets, deforming in the working range of the microscope table.
  • the gaskets were made by a mould cut out using the laser plotter.
  • the system of openings allows supplying nitrogen to the chamber by tubes and controlling the oxygen level in the chamber, while the concentration of gases is constantly controlled during observations.
  • Example 2 Using the chamber according to the invention for a comparative study of the adhesive properties of cells of the DLBCL lymphoma cell lines under normoxic (21 % O2) and physioxic (5% O2) conditions
  • Lymphoma cell lines (DB, Pfeiffer, Toledo, U2904, U2932, Ri-1 ) are cultured in a RPMI-1640 growth medium, recommended by ATCC and DSMZ banks (ATCC, cat. no. ATCC® 30-2001TM).
  • the growth medium was enriched with foetal bovine serum (ATCC, FBS, cat. no. ATCC® 30-2020TM).
  • ATCC, FBS foetal bovine serum
  • the cell cultivation was carried out in a humid atmosphere and in the presence of 5% CO2.
  • the cells intended for measurements under physiological oxygenconditions were cultured for 5 days in the presence of 5% O2 and 5% CO2 in an Eppendorf/New Brunswick Galaxy 48R incubator with oxygen level control in range of 1 -19%.
  • HS-5 line cell cultures ATCC, cat. no. ATCC® CRL-11882TM
  • ATCC American Type Culture Collection
  • cell line bank recommends the DMEM growth medium (ATCC, Dulbecco’s modified Eagle's medium, cat. no. ATCC® 30-2002TM).
  • the medium was additionally enriched with foetal bovine serum (ATCC, FBS, cat. no. ATCC® 30-2020TM).
  • ATCC foetal bovine serum
  • the cell cultivation was carried out in a humid atmosphere and in the presence of 5% CO2.
  • the cells intended for measurements under physiological oxygen level conditions were cultured in the presence of 5% O2 and 5% CO2 in an Eppendorf/New Brunswick Galaxy 48R incubator with oxygen level control in range of 1 -19%.
  • HS-5 cells after reaching 80% confluence in the culture flask were passaged, and their number and vitality was determined using trypan blue (Invitrogen, The CountessTM automated cell counter).
  • the cells in a number chosen experimentally 25,000 cells in 200 pi of DMEM medium
  • a sterile Petri dish having a glass bottom Glass bottom
  • 1 ml of the growth medium was added to the cells.
  • the confluence of morphologically mature cells in the dishes was equal to about 60%.
  • the system employed a microscope objective 100x NA 1.3, a holographic generation of optical traps using a LCoS Flamamatsu modulator with a resolution of 800x600, a laser beam control using Galvano- mirrors, an observation path for spectroscopic analysis, a video camera: CMOS max frame rate 10000 fps (frames per second).
  • a measuring dish with HS-5 fibroblasts was placed on the optical manipulator table inside the mounted measuring chamber.
  • the experiment under atmospheric oxygen conditions was carried out with the main door and the secondary door of the chamber being open (indicated in the chamber components diagram by numbers 4 and 6).
  • Physiological oxygen conditions (5%) in the chamber were achieved by pumping atmospheric oxygen out by neutral nitrogen gas.
  • lymphoma cell suspension 100 pi of lymphoma cell suspension was applied directly to the dish with previously prepared bone marrow stromal cells.
  • the secondary door opening was used (indicated in the chamber components diagram by number 5).
  • the time of cell settling to the bottom of the dish was about 5 minutes.
  • the cell was caught in the optical trap of 100 pN force and brought closer to the central part of the stromal cell in such a way, that the cells were kept in direct contact until they formed a stable co-culture (Fig. 6.).
  • the time rages of cell trapping determined experimentally were respectively: 5, 10, 20, 40, 60, 90, 120, 150, 180, 210, 240, 270, 300, 360, 420 s.
  • the experiment was repeated three times for the cells from 3.6 and 10 passages.
  • Table 3 The time required to form a stable connection between lymphoma cells of specified cell lines and the HS-5 stromal cells using optical tweezers under normoxia (21 % O2) and physioxia (5% O2) conditions. N - number of measured cells, Min - the shortest time of co-culture forming, Max - the longest time of co-culture forming, Ratio - a ratio of the time of co-culture forming under the 5% to 21 % oxygen level conditions.

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Abstract

L'invention concerne une chambre de mesure compacte pour l'étude des propriétés biomécaniques de cellules, en particulier des cellules cancéreuses, dans la pince optique, dans une atmosphère contrôlée au regard de la concentration en oxygène.
PCT/PL2018/050069 2017-12-21 2018-12-21 Chambre de mesure compacte compatible avec un système de pince optique dans des conditions de niveau d'oxygène contrôlées WO2019125195A1 (fr)

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PLP.424002 2017-12-21
PL424002A PL424002A1 (pl) 2017-12-21 2017-12-21 Kompaktowa komora pomiarowa kompatybilna z układem szczypiec optycznych w warunkach kontrolowanego stężenia tlenu

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