Microscope system and method for imaging sample

Abstract

To provide a microscope system that allows observing the reaction of a loosely mounted sample to a chemical stimulus in real time.SOLUTION: A microscope system (100) comprises a microscope stage (110) having a top surface (108) configured to have a sample carrier (106) arranged thereon. The sample carrier (106) is configured to receive at least one sample. The microscope system (100) further comprises an imaging system (112) configured to image the sample, and an injection device (116) configured to inject a predetermined amount of liquid into the sample carrier (106) by injecting multiple successive and temporally spaced jets of the liquid into the sample carrier (106). Each jet comprises a predetermined portion of the amount of liquid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microscope system. The present invention further relates to a method of imaging a sample using a microscope system. [Background technology]

[0002] Experiments in life sciences often require microscopic examination of samples that have been manipulated or treated with various chemicals, called reagents, to study and understand biological processes. These experiments typically require the injection of reagents into samples or their sample containers to examine the sample's response to chemical stimuli. Two broad categories of devices exist for performing injections. So-called microinjectors have extremely thin needles used to deliver small volumes of liquid, typically just a few nanoliters, through the cell wall and into cells. This injection is performed under microscope observation. This allows for real-time examination of the sample's response but requires human interaction. Microplate dispensers are specialized devices that can automatically and precisely deliver liquids and reagents into various microplates. However, microscopic observation of the prepared microplates is performed in a separate device, resulting in a time delay after the injection. Microscopic injection modules also exist that combine a microplate dispenser with a digital microscope for fluorescence, brightfield, color brightfield, and phase-contrast imaging of fixed samples placed in the wells of a microtiter plate.

[0003] One drawback of all known devices is that they are not suitable for high-speed volumetric imaging. In particular, known devices are not suitable for working with sensitive three-dimensional samples, such as organoids, or organisms, such as zebrafish or fruit flies, that cannot be easily mechanically fixed to the bottom of a sample carrier. These unfixed samples are easily displaced by turbulent flow during injection. Therefore, when examining biological samples, known devices are limited to adherent two-dimensional cell cultures or mechanically fixed samples, since such samples are less susceptible to the turbulence and flow caused by injection.

[0004] There are essentially two ways to address the fact that the sample's position changes during injection: one is to correct the sample's position relative to the observation volume in real time, and the other is to correct the position later. If the sample's response to a chemical stimulus is to be measured immediately, i.e., less than one second after injection, the correction must be performed in real time. Real-time correction requires a high-speed transmission illumination channel that rapidly acquires low-resolution images of the sample volume to measure any changes in the sample's position. The change in sample position is analyzed, and then a command is sent to the microscope stage, which corrects the position so that the sample remains centered in the field of view. Alternatively, if, for example, reagents are added to several wells of a plate and the sample's evolution is to be observed over several minutes or hours, images of the wells can be acquired in transmitted light mode, and each well of the plate can be scanned again after injection to identify the sample's new position in the well. However, with more than one sample per well, turbulence can cause the samples to mix, making subsequent assignment difficult or even impossible.

[0005] Both alternatives have their drawbacks. The first method requires hardware adaptation, high-speed image evaluation, and feedback to the stage controller. Even if technically feasible, this solution is very costly and requires significant development effort. The second method can significantly limit the feasibility of certain experiments, such as measuring the response during and immediately after injection. Furthermore, sample movement often also rotates the sample. It is unlikely that the same surface of the sample will still be facing the detection optics after movement. In this case, software-based tracking is extremely difficult and usually impossible. Summary of the Invention [Problem to be solved by the invention]

[0006] It is therefore a challenge to provide a microscope system that allows for the real-time observation of the response of a mounted, unfixed sample to a chemical stimulus. [Means for solving the problem]

[0007] The above-mentioned object is achieved by what is set forth in the independent claims. Advantageous embodiments are defined by the dependent claims and the following description.

[0008] The proposed microscope system comprises a microscope stage with an upper surface configured to have a sample carrier placed thereon, the sample carrier configured to accommodate at least one sample, an imaging system configured to image the sample, and an injection device configured to inject a predetermined amount of liquid into the sample carrier by injecting a plurality of successive, temporally spaced jets of liquid into the sample carrier, each jet comprising a predetermined partial volume of liquid.

[0009] Samples are typically contained in an embedding medium, such as water, an inorganic salt buffer, an aqueous, matrix-free growth medium, or another cell culture medium. A solution is added to the embedding medium to stimulate the sample. Adding a liquid to the embedding medium creates turbulence in the embedding medium. This turbulence can move the sample if it is not fixed to a sample carrier or cause shear stress in the case of a fixed sample. The amount of turbulence is determined by several factors, but one of the main factors is the amount of liquid injected. The more liquid injected, the more turbulence is created. Injecting liquid in sequential jets, each containing only a small amount of liquid, reduces the turbulence caused by each jet. Furthermore, because the jets are injected at intervals in time, the turbulence caused by each jet can dissipate. This also reduces the total amount of turbulence. Reducing turbulence also reduces sample movement and / or the shear stress experienced by the sample. Therefore, the proposed microscope allows for real-time observation of the response of samples, especially unfixed specimens, to reagents. Furthermore, because the sample is substantially stationary after injection, volumetric imaging techniques can be used to generate three-dimensional images of the sample during and immediately after the injection process, without the need for real-time repositioning of the sample relative to the observation volume.

[0010] The sample carrier may be a microplate, a Petri dish, a microscope slide, a chamber slide, or any other suitable sample carrier geometry. In particular, the needle has a gauge of 19-22. The point style may be, in particular, a type B or type AS.

[0011] In a preferred embodiment, the microscope system includes a control unit configured to predetermine at least one control parameter, in particular at least one of the portion size, the number of injections, and the interval between two immediately successive injections, and to control the injection of the injections based on the at least one control parameter. In this embodiment, the at least one control parameter can be controlled, for example, by user input. Alternatively or additionally, the at least one control parameter can be determined by a functional relationship or a look-up table correlating the at least one control parameter with a plurality of other parameters related to the sample carrier and / or the experiment of interest. This allows the injection of the injections to be customized to the individual sample carrier and the conditions of the experiment of interest. This allows a large number of sample carriers to be used for different experiments, making the microscope system more versatile.

[0012] In another preferred embodiment, the control unit includes a memory element. The memory element includes a lookup table correlating at least one sample carrier information item with at least one control parameter. The control unit is configured to predetermine the at least one control parameter based on user input of a value of the at least one sample carrier information item and based on the lookup table. In particular, the sample carrier information includes at least one of a sample carrier type, a sample carrier size, a sample carrier volume, a multiwell type, a number of wells, and a well volume. The lookup table may be predetermined by a manufacturer. Additionally or alternatively, a user may be able to modify the lookup table and customize it to suit their needs. In this embodiment, the at least one control parameter is set indirectly via the at least one sample carrier information item. Generally, the at least one sample carrier information item is more readily available to the user. This significantly improves the ease of use of the microscope system.

[0013] Preferably, the control unit is configured to predetermine at least one control parameter, so that the sample is not moved into or out of the field of view of the imaging system during injection of the jet. Whether the sample is moved out of the field of view during injection depends on the value of the at least one control parameter. Values ​​of the at least one control parameter for different values ​​of sample carrier information that do not move the sample can be determined experimentally and stored in a look-up table. Alternatively or additionally, the functional dependency can be determined in advance and stored in a memory element.

[0014] In another preferred embodiment, the imaging system is configured to capture at least one image of the sample during the injection of the liquid. This allows the sample's response to chemical stimuli to be examined without delay, which may be required in certain types of experiments, particularly in the study of neural tissue. This embodiment is therefore highly versatile.

[0015] In another preferred embodiment, each liquid aliquot has less than 10 μL, in particular less than 5 μL of liquid. By injecting liquid in aliquots of less than 10 μL, little or no turbulence occurs in most sample carriers.

[0016] In another preferred embodiment, the interval between two immediately successive injections is at least 0.5 seconds and at most 1.5 seconds. Turbulence dissipates over time. A lower limit of about 0.5 seconds is sufficient for turbulence to dissipate in most sample carriers. An upper limit of 1.5 seconds is still short enough to allow real-time observation of the sample's response.

[0017] In another preferred embodiment, the injection device has at least one needle with an angled tip. Preferably, the needle tip is bent at least 30° and at most 90°. Needles with angled tips can be better mounted in small mounting spaces, such as the sample chamber of a microscope system. Furthermore, the angled tip also reduces turbulence caused by the injection of the jet.

[0018] In another preferred embodiment, the tip of the needle and the upper surface of the microscope stage form an angle of at least 10° and at most 85°. In particular, the tip of the needle and the upper surface of the microscope stage form an angle of at least 10° and at most 50°. By injecting the liquid at an angle, i.e., rather than directly from above, the liquid injected into the sample carrier impinges on a larger cross-sectional area and travels a longer distance within the embedding medium. These two aspects allow turbulence caused by the injected liquid to dissipate more quickly, especially at shallow depths. This minimizes sample disturbance caused by the injected liquid. However, injecting the liquid at too shallow an angle can cause the jet to disperse, which can result in liquid loss and therefore an inaccurate injection volume.

[0019] In another preferred embodiment, the liquid flow rate is at least 5 μL / s and at most 250 μL / s. In particular, the liquid flow rate is at least 10 μL / s and at most 100 μL / s. The flow rate must be high enough to ensure clean droplet splitting at the end of the injection. In other words, the injected liquid must completely detach / detach from the tip, ensuring that the ejected liquid does not remain attached to the tip. However, the higher the flow rate, the greater the force exerted on the sample by the injected liquid. The above range represents a good compromise between having a flow rate low enough not to significantly displace the sample, while still allowing clean droplet splitting / detachment / detachment.

[0020] In another preferred embodiment, the injection device has a temperature control unit configured to control the temperature of the liquid. Injecting a liquid into the sample carrier not only changes the chemical environment of the sample, but also its temperature. In some experiments, especially those requiring careful incubation of the sample, rapid changes in temperature can cause undesirable side effects. Temperature control can be achieved by thermally coupling tubing between the pump and the injection tip to a temperature reservoir, for example, by running the tubing around / through a temperature-controlled metal block. In this embodiment, these side effects are avoided, thereby ensuring that the observed sample response to injection is a response to chemical stimulation alone.

[0021] In another preferred embodiment, the microscope system has a box microscope housing defining a sample chamber. The housing has a door for providing access to the sample chamber. The microscope stage is located at the bottom of the sample chamber. The sample carrier is housed within the sample chamber. The box microscope has a housing within which all microscope components are located. The housing typically has one or more openings for accessing the interior of the microscope. Due to the hermetically sealed or even sealed nature of the housing, box microscopes are particularly suitable for precisely controlling the environment of the sample, for example, by an atmospheric control unit.

[0022] The invention further relates to a method for imaging a sample with a microscope system, comprising the steps of placing the sample in a sample carrier, placing the sample carrier on a microscope stage of the microscope system, injecting a predetermined amount of liquid into the sample carrier by injecting a plurality of successive, temporally spaced jets of liquid into the sample carrier, each jet having a predetermined partial volume of liquid, and capturing at least one image of the sample with an imaging system of the microscope system during the injection of the liquid.

[0023] This method has the same advantages as the sample carrier and imaging system described above and can be supplemented using the features of the dependent claims directed to the sample carrier and imaging system.

[0024] In a preferred embodiment, the method comprises the additional steps of predetermining at least one control parameter, in particular at least one of the portion size, the number of injections and the interval between two immediately successive injections, and of injecting the injections based on the at least one control parameter.

[0025] In another preferred embodiment, the at least one control parameter is predetermined based on user input of a value of the at least one sample carrier information and based on a look-up table correlating the at least one sample carrier information with the at least one control parameter.

[0026] In another preferred embodiment, at least one control parameter is predetermined to prevent the sample from being moved outside the field of view of the imaging system during injection of the jet.

[0027] In another preferred embodiment, each aliquot of liquid has less than 10 μL of liquid.

[0028] In another preferred embodiment, the interval between two immediately successive injections is at least 0.5 seconds and at most 1.5 seconds.

[0029] In another preferred embodiment, the injection is performed by at least one needle with an angled tip.

[0030] In another preferred embodiment, the tip of the needle is bent at least 30° and at most 90°.

[0031] In another preferred embodiment, the jet is injected onto the sample carrier at an angle of at least 10° and at most 85° relative to the upper surface of the microscope stage, in particular at an angle of at least 10° and at most 50° relative to the upper surface of the microscope stage.

[0032] In another preferred embodiment, the liquid flow rate is at least 5 μL / s and at most 250 μL / s. In particular, the liquid flow rate is at least 10 μL / s and at most 100 μL / s.

[0033] In the following, specific embodiments will be described with reference to the drawings. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 is a schematic diagram of a microscope system according to one embodiment. [Figure 2] 2 is a schematic plan view of a sample chamber of the microscope system according to FIG. 1; [Figure 3] FIG. 3 is a schematic front view of a sample chamber of the microscope system according to FIGS. 1 and 2; [Figure 4] FIG. 4 is a schematic side view of a sample chamber of the microscope system according to FIGS. 1 to 3. [Figure 5] FIG. 5 is a schematic detailed view of a sample chamber of the microscope system according to FIGS. 1 to 4. [Figure 6] FIG. 10 is a schematic detailed view of a sample chamber of a microscope system according to another embodiment. [Figure 7] FIG. 10 is a schematic plan view of a sample chamber of a microscope system according to yet another embodiment. [Figure 8] FIG. 10 is a schematic plan view of a sample chamber of a microscope system according to yet another embodiment. [Figure 9] FIG. 10 is a schematic plan view of a sample chamber of a microscope system according to yet another embodiment. [Figure 10] FIG. 1 is a schematic diagram of a sample carrier and needle tip. [Figure 11] 1A and 1B are two schematic plan views of a sample carrier. [Figure 12] 1 is a graph showing the flow rate of the liquid exiting the tip over time. DETAILED DESCRIPTION OF THE INVENTION

[0035] FIG. 1 is a schematic diagram of a microscope system 100 according to one embodiment.

[0036] The microscope system 100 is illustratively formed as a box microscope having a microscope housing 102. The microscope housing 102 forms a sample chamber 104 configured to accommodate a sample carrier 106 in which one or more samples 502 (see FIG. 5 ) are disposed. Sealing the sample carrier 106 within the microscope housing 102 allows for precise control over the environment of the sample 502, for example, via an incubation control unit for controlling the temperature, humidity, and gas composition of the sample chamber 104. The sealed sample 502 is also shielded from the environment, thereby protecting both the sample 502 and the environment from accidental contamination. Furthermore, the sample chamber 104 can easily be converted into an incubation chamber and / or a sterile environment, as required by the experiment.

[0037] The sample carrier 106 is positioned on the upper surface 108 of a microscope stage 110, which is positioned below the sample chamber 104. The microscope stage 110 is movable along two orthogonal directions, i.e., the microscope stage 110 is a so-called XY table. By moving the microscope stage 110, it is possible, for example, to select individual cavities or wells of the sample carrier 106 or to select particular regions of interest of a single sample 502 for observation.

[0038] The microscope system 100 according to FIG. 1 is illustratively configured as a transmitted light microscope system. An imaging system 112 for imaging a sample is illustratively disposed in a component space below the microscope stage 110, and an illumination system 114 for illuminating the sample 502 is disposed above the microscope stage 110 in the sample chamber 104. The optical axes of the imaging system 112 and the illumination system 114 are aligned, such that illumination light emitted by the illumination system 114 passes through the sample 502 before entering the imaging system 112. In an alternative embodiment, the positions of the imaging system 112 and the illumination system 114 can be reversed. In another alternative embodiment, both the imaging system 112 and the illumination system 114 are disposed on the same side of the microscope stage 110. Additional components, such as a power supply or various filters, may be disposed in the component space below the sample chamber.

[0039] The microscope system 100 further includes an injection device 116 configured to inject a liquid, e.g., a reagent, into the sample carrier 106. The injection device 116 includes a needle 118 disposed within the sample chamber 104, the needle 118 having a curved tip 120 for injecting the liquid into the sample carrier 106. The injection device 116 also includes a liquid reservoir 122 disposed outside the sample chamber 104 for storing the liquid. The liquid reservoir 122 illustratively consists of a single container. However, the liquid reservoir 122 may include two or more containers, e.g., standard-sized disposable plastic tubing, each storing a different liquid. The liquid reservoir 122 is connected by a first fluid line 124 to a pump 126, illustratively formed as a syringe pump 126. Alternatively, the pump 126 may be formed as a peristaltic pump or any other suitable pump. The pump 126 includes a syringe 128 with a barrel and plunger, a motor-driven plunger driver, and a three-way valve 130. The motor-driven plunger driver is configured to move the plunger of the syringe 128, thereby drawing fluid into or expelling fluid from the barrel. In a first mode of operation, the three-way valve 130 is configured to allow the syringe 128 to draw fluid from the fluid reservoir 122 into the barrel. In a second mode of operation, the three-way valve 130 is configured to allow the syringe 128 to deliver fluid stored in its barrel to the needle 118 via a second fluid line 132. The shorter the second fluid line 132, the smaller the minimum volume of fluid that can be injected into the sample carrier 106. Another factor affecting the minimum volume of fluid that can be injected is the consistency of the fluid line. For lengths of less than 100 cm, a minimum volume of approximately 1 μL to 3 μL is achievable.

[0040] FIG. 2 is a schematic plan view of the sample chamber 104 of the microscope system 100 according to FIG.

[0041] The sample carrier 106 is centrally positioned above the microscope stage 110 and is illustratively formed as a multiwell plate having 24 wells 200. The wells 200 are arranged in four rows and six columns. To uniquely identify each well 200, the rows are labeled A through D and the columns are labeled 1 through 6. The wells 200 are filled with an embedding medium 504 (see FIG. 5 ) in which individual samples 502 are accommodated. A reagent in liquid form is added to the sample carrier 106, and a reaction from the sample 502 is observed.

[0042] The needle 118 is positioned above the sample carrier 106. To inject liquid into each well 200, the tip 120 can be positioned above each well 200 of the sample carrier 106 by moving either the sample carrier 106 or the needle 118. As can be seen in Figure 2, the angled tip 120 is slightly twisted to one side, i.e., towards the top of Figure 2. This ensures that the needle 118 is not in the beam path of the illumination light and does not cast a shadow on the sample 502.

[0043] FIG. 3 is a schematic front view of the sample chamber 104 of the microscope system 100 according to FIGS.

[0044] The needle 118 has a base 300 connected to a second fluid line 132 that is parallel to the top surface 108 of the microscope stage 110, and an angled tip 120. As can be seen in Figure 3, the angled tip 120 and the top surface 108 of the microscope stage 110 form an angle of approximately 40°, which means that the jet also strikes the surface of the embedding medium 504 at an angle of incidence of approximately 40°.

[0045] Figure 4 is a schematic side view of the sample chamber 104 of the microscope system 100 according to Figures 1 to 3. The view in Figure 4 shows the sample chamber 104 as seen from the right side of Figure 3.

[0046] The needle 118 is rotatably positioned about the longitudinal axis of its base 300. As can be seen in FIG. 4, the angled tip 120 does not point straight down, but is rotated approximately 30° to the right in FIG. 4. By allowing the needle 118 to rotate, the angle of incidence of the jet striking the surface of the embedding medium 504 can be precisely controlled. Typically, the needle 118 has a tip 120 bent by 45°, 60°, or 90°, corresponding to angles of incidence of 45°, 30°, or 0°, respectively. If a different angle of incidence is required, the tip 120 must be manually bent, which is both imprecise and time-consuming. By providing a means for rotating the needle 118, the angle of incidence can be precisely and automatically controlled. Furthermore, the tip 120 is not positioned in the beam path of either the imaging system 112 or the illumination system 114. Therefore, the tip 120 does not cast a shadow on the sample 502.

[0047] FIG. 5 is a schematic detailed view of the sample chamber 104 of the microscope system 100 according to FIGS.

[0048] One well 200 of the sample carrier 106 is positioned above the detection objective 500 of the imaging system 112. A sample 502 is contained in an embedding medium 504 in that well 200 and is observable through an optically transparent bottom window 506 of the well 200. The angled tip 120 is positioned directly above the well 200 currently being observed. However, the tip 120 does not reach the well 200, allowing the sample carrier 106 to move freely.

[0049] FIG. 6 is a detailed schematic diagram of the sample chamber 104 of a microscope system 600 according to another embodiment.

[0050] In this embodiment, the sample carrier 602 is illustratively shaped as a Petri dish with a transparent window portion 604 located at the bottom of the Petri dish. A large biological specimen, such as a zebrafish, nematode, or fruit fly specimen, is contained in a nutrient solution as the sample 502. The angled tip 120 is positioned above the sample 502 so that the jet strikes the surface of the nutrient solution 606 slightly to the side of the sample 502. The tip 120 does not reach the sample carrier 602, allowing the sample carrier 602 to move freely.

[0051] FIG. 7 is a schematic plan view of a sample chamber 104 of a microscope system 700 according to yet another embodiment.

[0052] The microscope system 700 according to Fig. 7 differs from the microscope system 100 according to Figs. 1 to 5 in that it includes a second needle 702. The second needle 702 is positioned next to the first needle 118, and their bases 300, 704 are parallel. The tips 120, 706 of both needles 118, 702 are positioned above one well 200, so that both needles 118, 702 can be used to inject liquid into that particular well 200. The two needles 118, 702 are connected to liquid reservoirs 122 by fluid lines 132, 708, respectively, and can therefore be used to inject two different liquids into the sample carrier 106, 602.

[0053] FIG. 8 is a schematic plan view of a sample chamber 104 of a microscope system 800 according to yet another embodiment.

[0054] The microscope system 800 according to Figure 8 is distinguished from the microscope system 700 according to Figure 7 in that a second needle 702 is positioned opposite the first needle 118. The two needles 118, 702 are positioned such that their bases 300, 704 lie on a common axis. The tips 120, 706 are positioned such that a gap 802 is formed between the two needles 118, 702 and above the well 200. This gap 802 allows for observation and illumination of the sample 502 placed in the well 200.

[0055] FIG. 9 is a schematic plan view of the sample chamber 104 of a microscope system 900 according to one embodiment.

[0056] The microscope system 900 according to Figure 9 is distinguished from the microscope system 700 according to Figure 7 in that the first needle 118 and the second needle 702 are angled toward each other. The bases 300 and 704 of the two needles 118, 702 enclose an angle of approximately 25°. Additionally, the tips 120, 706 are bent toward each other.

[0057] FIG. 10 is a schematic diagram of a sample carrier 1000 and the tip 120 of a needle 118.

[0058] Liquid ejected from tip 120 towards sample carrier 1000 forms a jet 1002. As can be seen in Figure 10, jet 1002 widens with distance from tip 120 due to diffusion, which can result in loss of liquid and therefore an inaccurate jet volume. It can therefore be advantageous to keep the distance between tip 120 and surface 1004 of embedding medium 504 as short as possible. As can also be seen in Figure 10, jet 1002 is slightly curved downwards due to gravity.

[0059] Two schematic plan views of the sample 504 are shown in FIG.

[0060] Two figures show the movement of the sample 502 due to the turbulence caused by the injection of the jet 1002. The initial positions 1100a, 1100b of the sample 502 are shown as solid black circles. The final positions 1102a, 1102b of the sample 502 are shown as white circles with solid black outlines. The field of view 1104 of the microscope system 100, 600, 700, 800, 900 is shown as a large circle with a dashed black outline. The first figure shows the semicircular movement caused by the injection of the jet 1002. The sample 502 is moved from the initial position 1100a to the final position 1102a along the semicircle, as indicated by the first arrow P1. The second figure shows the linear movement caused by the injection of the jet 1002. The sample 502 is moved from the initial position 1100b to the final position 1102b along the straight line, as indicated by the second arrow P2. The distance between the initial position and the final position is smaller than the diameter of the sample 502 and much smaller than the field of view 1104 of the microscope system 100 .

[0061] FIG. 12 is a graph showing the flow rate of the liquid exiting the tip 120 over time.

[0062] The horizontal axis represents time t in seconds, and the vertical axis represents flow rate f in μL per second. As can be seen in FIG. 12, the liquid injection is divided into five distinct portions 1200, 1202, 1204, 1206, and 1208 with different flow parameters. Most syringe pumps allow the user to set parameters for these five portions. The first portion 1200, referred to as the start flow, corresponds to an initial flow rate of 10 μL / s. The second portion 1202, referred to as the slope up, corresponds to an increase in the flow rate until a flow rate of 95 μL / s is reached. During the second portion 1202, the flow rate increases by 8 μL / s per second. The third portion 1204, referred to as the top flow, corresponds to a flow rate of 95 μL / s. During the fourth portion 1206, called the slope down, the flow rate is changed by 40 μL / s per second until a final flow rate of 150 μL / s is reached. The fifth portion 1208, called the end flow, corresponds to a final flow rate of 150 μL / s.

[0063] While the exact values ​​of the flow rates during the start flow, slope up, and end flow have very little effect, the values ​​of the flow rates during the top flow and slope down affect whether the sample 502 is transferred during injection and whether clean droplet splitting can be achieved. The flow rate during the top flow has the greatest effect on whether the sample 502 is transferred and should not be higher than 200 μL / s. The change in flow rate during the slope down can be as slow as 8 μL / s per second. Any slow change will only result in clean droplet splitting if combined with a higher flow rate during the end flow of about 350 μL / s.

[0064] The same reference numerals are used in all figures to refer to the same, similar or identically functioning elements. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0065] While some aspects have been described in the context of an apparatus, it will be apparent that these aspects also represent a description of a corresponding method, where a block or apparatus corresponds to a step or feature of a step, and similarly, aspects described in the context of a step also represent a description of a corresponding block or item or feature of a corresponding apparatus. [Explanation of symbols]

[0066] 100 Microscope System 102 Microscope Housing 104 Sample Chamber 106 Sample Carrier 108 Top surface 110 Microscope Stage 112 Imaging System 114 Lighting System 116 Injection device 118 needles 120 Tip 122 Fluid Reservoir 124 Fluid line 126 Pump 128 Syringe 130 valves 132 Fluid pipeline 200 wells 300 base 500 objective lens 502 samples 504 Embedding medium 506 Windows 600 Microscope System 602 Sample Carrier 604 Windows 606 Nutrient Solution 700 Microscope System 702 needle 704 Base 706 Tip 708 Fluid line 800 Microscope System 802 Gap 900 Microscope System 1000 sample carriers 1002 injection 1004 Surface 1100a,1100b initial position 1102a,1102b Final position 1104 Field of view 1200,1202,1204,1206,1208 part P1, P2 arrows

Claims

1. A microscope system (100, 600, 700, 800, 900), comprising: a microscope stage (110) having a top surface (108) configured to have a sample carrier (106, 602, 1000) placed thereon, the sample carrier (106, 602, 1000) configured to accommodate at least one sample (502); an imaging system (112) configured to image the sample (502); an injection device (116) configured to inject a predetermined volume of liquid into the sample carrier (106, 602, 1000) by injecting a plurality of successive, temporally spaced jets (1002) of liquid into the sample carrier (106, 602, 1000), each jet (1002) having a predetermined partial volume of the liquid; A microscope system (100, 600, 700, 800, 900) comprising:

2. the microscope system (100, 600, 700, 800, 900) has a control unit configured to predetermine at least one control parameter, in particular at least one of the amount of the partial dose, the number of injections (1002) and the interval between two immediately successive injections (1002), and to control the injection of the injections (1002) based on the at least one control parameter, 2. The microscope system (100, 600, 700, 800, 900) of claim 1.

3. the control unit has a memory element, the memory element having a look-up table correlating at least one sample carrier (106, 602, 1000) information with at least one of the control parameters, and the control unit is configured to predetermine at least one of the control parameters based on a user input of a value of at least one of the sample carrier (106, 602, 1000) information and based on the look-up table.

3. A microscope system (100, 600, 700, 800, 900) according to claim 2.

4. the control unit is configured to predetermine at least one of the control parameters so that the sample (502) is not moved into or out of a field of view (1104) of the imaging system (112) during the injection of the jet (1002).

3. A microscope system (100, 600, 700, 800, 900) according to claim 2.

5. the imaging system (112) is configured to capture at least one image of the sample (502) during the injection of the liquid.

2. The microscope system (100, 600, 700, 800, 900) of claim 1.

6. each aliquot of liquid having less than 10 μL of said liquid; 2. The microscope system (100, 600, 700, 800, 900) of claim 1.

7. the interval between two immediately successive injections (1002) is at least 0.5 seconds and at most 1.5 seconds; 2. The microscope system (100, 600, 700, 800, 900) of claim 1.

8. The injection device (116) has at least one needle (118) with an angled tip (120).

2. The microscope system (100, 600, 700, 800, 900) of claim 1.

9. The tip (120) of the needle (118) is bent at least 30° and less than 90°. A microscope system (100, 600, 700, 800, 900) according to claim 8.

10. The tip (120) of the needle (118) and the top surface (108) of the microscope stage (110) form an angle of at least 10° and at most 85°. A microscope system (100, 600, 700, 800, 900) according to claim 8.

11. The tip (120) of the needle (118) and the top surface (108) of the microscope stage (110) form an angle of at least 10° and at most 50°. A microscope system (100, 600, 700, 800, 900) according to claim 10.

12. The flow rate of the liquid is at least 5 μL / s and at most 250 μL / s; 2. The microscope system (100, 600, 700, 800, 900) of claim 1.

13. the flow rate of the liquid is at least 10 μL / s and at most 100 μL / s; A microscope system (100, 600, 700, 800, 900) according to claim 12.

14. the injection device (116) having a temperature control unit configured to control the temperature of the liquid; 2. The microscope system (100, 600, 700, 800, 900) of claim 1.

15. The microscope system (100, 600, 700, 800, 900) includes a box microscope housing (102) defining a sample chamber (104), the housing (102) having a door for providing access to the sample chamber (104), the microscope stage (110) being positioned below the sample chamber (104), and the sample carrier (106, 602, 1000) being housed within the sample chamber (104).

2. The microscope system (100, 600, 700, 800, 900) of claim 1.

16. 1. A method for imaging a sample (502) with a microscope system (100, 600, 700, 800, 900), the method comprising: placing the sample (502) in a sample carrier (106, 602, 1000); placing the sample carrier (106, 602, 1000) on a microscope stage (110) of the microscope system (100, 600, 700, 800, 900); injecting a predetermined volume of liquid into the sample carrier (106, 602, 1000) by injecting a plurality of successive, temporally spaced jets (1002) of liquid into the sample carrier (106, 602, 1000), each jet (1002) having a predetermined fractional volume of the liquid; capturing at least one image of the sample (502) by an imaging system (112) of the microscope system (100, 600, 700, 800, 900) during the injection of the liquid; A method having the following.

17. The method comprises: - determining at least one control parameter in advance, in particular at least one of the amount of the partial dose, the number of injections (1002) and the interval between two immediately successive injections (1002); performing said injection of said injection (1002) based on at least one of said control parameters; having 17. The method of claim 16.

18. the at least one control parameter is predetermined based on user input of a value of at least one sample carrier (106, 602, 1000) information and based on a look-up table correlating the at least one sample carrier (106, 602, 1000) information with the at least one control parameter.

18. The method of claim 17.

19. At least one of the control parameters is predetermined so that the sample (502) is not moved into or out of a field of view (1104) of the imaging system (112) during the injection of the jet (1002).

18. The method of claim 17.

20. each aliquot of liquid having less than 10 μL of said liquid; 17. The method of claim 16.

21. the interval between two immediately successive injections (1002) is at least 0.5 seconds and at most 1.5 seconds; 17. The method of claim 16.

22. said injection being performed by at least one needle (118) having an angled tip (120); 17. The method of claim 16.

23. The tip (120) of the needle (118) is bent at least 30° and less than 90°.

23. The method of claim 22.

24. the jet (1002) is injected into the sample carrier (106, 602, 1000) at an angle of at least 10° and at most 85° relative to the upper surface (108) of the microscope stage (110); 17. The method of claim 16.

25. the jet (1002) is injected into the sample carrier (106, 602, 1000) at an angle of at least 10° and at most 50° relative to the top surface (108) of the microscope stage (110); 25. The method of claim 24.

26. The flow rate of the liquid is at least 5 μL / s and at most 250 μL / s; 17. The method of claim 16.

27. the flow rate of the liquid is at least 10 μL / s and at most 100 μL / s; 27. The method of claim 26.

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