Automated, imaging-based and five-axis liquid handling platform

Abstract

The invention is related to an automated, imaging-based, five-axis liquid handling platform to automatically perform pipetting processes applied during experimental studies, especially microbiological studies. The platform in question consists of two main structures: liquid handling system and imaging system. Thanks to the liquid handling platform, cell seeding, medium change and system cleaning can be performed automatically. A software is used for the operation of the invention. In this way, an automated and fast system is obtained, and the pipetting processes applied in wide ranges of experimental studies, especially microbiological studies, can be performed automatically while operator-related errors may be prevented. Thanks to the liquid handling platform subjected to the invention, the biophysical properties of cells can be determined by monitoring confluency, and in-vitro studies such as cell-based drug discovery can be performed.

Description

[0001] AUTOMATED, IMAGING-BASED AND FIVE-AXIS LIQUID HANDLING PLATFORM

[0002] Technical Field

[0003] The invention relates to a five-axis automated liquid handling platform with an integrated imaging base. Through the liquid handling platform subject to this invention, microbiological studies such as cell seeding, media replacement, and system cleaning can be performed automatically. The operation of the mentioned system is managed by a dedicated software. As a result, an automatic and fast system is obtained, allowing pipetting processes commonly applied during experimental studies, particularly in microbiological research, to be automated, thus eliminating human-induced errors.

[0004] STATE OF THE ART

[0005] Micropipettes are commonly used laboratory tools designed to accurately and precisely transfer liquid volumes in the microliter (pL) range. They are available in both single-channel and multi-channel configurations. Single-channel micropipettes are frequently used in laboratories conducting research in fields such as molecular biology, microbiology, immunology, cell culture, analytical chemistry, biochemistry, and genetics. Manual micropipettes with varying volume capacities and precision levels are widely utilized worldwide. When using micropipettes, the pipette button is pressed to the first step to draw liquid into the tip. The button is then slowly released to aspirate the liquid, and upon pressing the button to the second stop, the liquid is dispensed. This process is referred to pipetting. Studies requiring pipetting, e.g., cell culture-based studies, drug development, serology, virology and microbiology, are long-term manual processes that require minimal errors. User- related errors and potential contamination that may occur in these studies can adversely impact both the process and reliability of the results. Automated pipetting systems are needed to minimize user-related errors and the risk of contamination. Traditional cell culture processes are manual processes that require long-term repetition, careful work, and minimal errors. Traditional cell cultures are manual processes that require long-term repetition, cautious work, and minimal errors. Solutions used in cell culture procedures must be pipetted precisely in the desired amount and the risk of contamination caused by the pipette tip must be minimized. With developing technology, automated cell culture systems are also frequently used to increase the consistency of cell culture processes and reduce the risk of contamination.

[0006] The current technique commonly employs manual micropipettes that operate using a traditional piston-driven air displacement mechanism. These micropipettes are capable of transferring liquid volumes across a wide range, typically from 0.1 pL to 1000 pL. Manual micropipettes do not have high precision for volumes below 1 pL. Although they can be used reliably for many applications above 1 pL, error rate is high due to the fact that the air displacement working principle is affected by parameters such as temperature, tip residues, surface contact and user experience [1], These errors negatively affect the reliability of the studies and increase in costs. The ergonomic structure of individuals is also at risk while constantly performing manual micropipetting, which causes hand-musculoskeletal disorders [2], Therefore, in the use of manual micropipettes, there are problems such as inconsistencies in the measurements, user-related errors, high costs due to errors, and hand-musculoskeletal disorders encountered by users performing manual measurements.

[0007] As another application of this technique, confluency calculation is performed in biological research, cell therapy development and production studies. Confluency is determined manually by observing cells through a microscope and counting them one by one in the classical method. However, in this method, changes in confluency are estimated by the operators, which creates variations between tests and negatively affects the consistency of the process and outputs. In this case, in the tests based on cell culture; the risk of inter-user variability cannot be prevented.

[0008] In another known state of the art, liquid handling in automated cell culture systems is based on the principle of changing the pipette tip. For high-volume liquid handling processes, pipette tips must be filled at certain intervals. Therefore, for high-volume liquid handling processes, the problem of constantly changing pipette tips cannot be prevented.

[0009] In another known state of the art, in cell culture; solutions and cells must be stored under certain conditions (such as 37.5°C temperature, 5% carbon dioxide rate, 95% humidity). However, there is no incubator environment that maintains these parameters for the solutions used in the automated liquid handling devices, and no system for the insulation of the tubings is used in liquid transfer. In this case, the problem of not being able to provide the proper storage conditions for the solutions cannot be prevented.

[0010] The limitations and shortcomings of current technical solutions, e.g., the frequent inability to obtain precise measurements with manual micropipettes, user-induced errors, high cost due to operator-dependent errors, variability in test results among users, and the risk of musculoskeletal disorders in users performing repetitive manual measurements, along with challenges like the constant need to change pipette tips for high-volume liquid handling operations in automated cell culture systems, have highlighted the need for advancements in this field.

[0011] Brief Description and Purposes of the Invention

[0012] The invention relates to a five-axis liquid handling platform with an automated imaging base. Thanks to the liquid handling of the invention, cell seeding, medium change and system cleaning in microbiological studies can be done automatically. The liquid handling platform of the invention enables the determination of biophysical properties of cells through confluency monitoring and facilitates in-vitro studies, including cell-based drug discovery.

[0013] One purpose of the invention is to achieve high-efficiency and high-resolution automation in live cell imaging. With the focus stacking and image stitching algorithms in the liquid handling platform of the invention, high-resolution images are produced with the camera and objective lens system integrated into the platform and the efficiency in cell imaging is increased by providing automation of live cell imaging.

[0014] Another purpose of the invention is to enhance the resolution of the imaging for the transparent samples in cell imaging studies. For this reason, phase-contrast microscopy is performed by integrating a phase-contrast objective and phase rings into the imaging mechanism used in the liquid handling platform..

[0015] Another purpose of the invention is to preserve cell phenotype and culture quality during cell tests in microbiological studies. The liquid handling platform of the invention allows for the determination of the optimal time required for cell passaging, thereby preserving cell phenotype and maintaining culture quality. Another purpose of the invention is to provide the users with precise micro-stepping and reliable flow rate control in microbiological studies. The high-resolution pump used in the liquid handling platform of the invention helps to perform precise micro stepping and reliable flow rate control in adjustable liquid volumes.

[0016] Another purpose of the invention is to provide control of cells subjected to liquid handling processes. In the liquid handling platform of the invention, cells subjected to liquid handling processes can be imaged thanks to the fact that liquid handling and imaging functionalities are integrated on the same platform.

[0017] Another purpose of the invention is not to damage cell samples during liquid withdrawal in microbiological studies. With the fourth axis movement capability of the liquid handling platform, the well plates are held in an angled position and cell seeding, medium dispensing or liquid aspiration processes can be performed from the corners of the wells without damaging the samples.

[0018] Another purpose of the invention is to accelerate the process and improve efficiency in confluency tests while eliminating user-dependent variability in test results. In the liquid handling platform, the automatization of confluency analysis enables users to make rapid decisions about cell culture and increases the efficiency of cell culture-based analyses, paving the way for the acceleration of the analyses where large numbers of tests are conducted.

[0019] Another purpose of the invention is to provide a controlled and contamination-free environment for cells and solutions by regulating parameters such as temperature, humidity and carbon dioxide. For this reason, the solutions to be used in liquid handling and the cells to be seeded in the wells are kept in an incubator consisting of two nested boxes within the system. In order to maintain these conditions during the liquid transfer to the wells, the tubings used in the system are insulated.

[0020] Thanks to the automated, imaging-based and five-axis liquid handling platform, it is aimed to automatically perform the pipetting processes during wide range of experimental studies to achieve precise measurements by increasing the efficiency of cell culture-based analyses, to prevent hand-musculoskeletal system disorders in user conducting manual measurements, to perform the liquid dispensing-aspiration processes without damaging the cell samples, to provide high efficiency and high resolution in live cell imaging, and to preserve cell phenotype and culture quality.

[0021] Figure Description

[0022] Figure 1: Schematic of the automated, imaging-based, five-axis liquid handling platform

[0023] Figure 2: Perspective view of tube change using the revolver (17) and the motor (15)

[0024] Figure 3: Perspective view of the objective lens change using the revolver (19) and the motor (30)

[0025] Figure 4: Sensor (98) integrated into the fourth axis to which the holder (32) is attached

[0026] Figure 5: Sensor (99) integrated into the fifth axis to which the tube holders (16, 58, 59) are attached

[0027] Figure 6: Sensors (100, 101, 102) integrated into the x, y, z axes of the three-axis Cartesian robot (65)

[0028] Definitions of the Elements / Parts of the Invention n = Number of Solutions

[0029] 1. Solution

[0030] 2. Tubing

[0031] 3. Low resolution pump

[0032] 4. Manifold

[0033] 5. Valve

[0034] 6. Tubing

[0035] 7. Flow Sensor

[0036] 8. Tubing 9. Solution

[0037] 10. Rotary Valve

[0038] 11. Tubing

[0039] 12. Waste bottle

[0040] 13. Low resolution pump

[0041] 14. Tubing

[0042] 15. Motor

[0043] 16. Tubing holder

[0044] 17. Revolver

[0045] 18. Achromatic objective lens

[0046] 19. Revolver

[0047] 20. Lens tube

[0048] 21. Tube Lens

[0049] 22. Lens tube

[0050] 23. Camera

[0051] 24. Motor

[0052] 25. Well plate

[0053] 26. Tubing

[0054] 27. High resolution pump

[0055] 28. Tubing

[0056] 29. Phase-contrast objective lens

[0057] 30. Motor

[0058] 31. Shaft 32. Holder

[0059] 33. Driver board

[0060] 34. Electrical connection

[0061] 35. Driver board

[0062] 36. Electrical connection

[0063] 37. Driver board

[0064] 38. Electrical connection

[0065] 39. Driver board

[0066] 40. Electrical connection

[0067] 41. Driver board

[0068] 42. Electrical connection

[0069] 43. Driver board

[0070] 44. Electrical connection

[0071] 45. Driver board

[0072] 46. Electrical connection

[0073] 47. Processor

[0074] 48. Driver board

[0075] 49. Electrical connection

[0076] 50. Electrical connection

[0077] 51. Electrical connection

[0078] 52. Electrical connection

[0079] 53. Electrical connection

[0080] 54. Electrical connection 55. Electrical connection

[0081] 56. Electrical connection

[0082] 57. Electrical connection

[0083] 58. Tubing Holder

[0084] 59. Tubing Holder

[0085] 60. Waste container

[0086] 61. Electrical connection

[0087] 62. Tubing

[0088] 63. Tubing

[0089] 64. Phase ring

[0090] 65. Three-axis Cartesian robot

[0091] 66. Motor

[0092] 67. Shaft

[0093] 68. Driver board

[0094] 69. Electrical connection

[0095] 70. Electrical connection

[0096] 71. LED array

[0097] 72. Diffuser

[0098] 73. Motorized switcher

[0099] 74. Incubator

[0100] 75. Heater

[0101] 76. Carbon dioxide source

[0102] 77. Valve 78. Carbon dioxide filter

[0103] 79. Humidity source

[0104] 80. Humidity sensor

[0105] 81. Temperature sensor

[0106] 82. Carbon dioxide sensor

[0107] 83. Driver board

[0108] 84. Electrical connection

[0109] 85. Electrical connection

[0110] 86. Electrical connection

[0111] 87. Electrical connection

[0112] 88. Electrical connection

[0113] 89. Electrical connection

[0114] 90. Fan

[0115] 91. Electrical connection

[0116] 92. Tubing

[0117] 93. Tubing

[0118] 94. Ultraviolet C (UVC) source

[0119] 95. Electrical connection

[0120] 96. Electrical connection

[0121] 97. Display

[0122] 98. Sensor for crash detection

[0123] 99. Sensor for crash detection

[0124] 100. Sensor for collision detection 101. Sensor for collision detection

[0125] 102. Sensor for collision detection

[0126] Detailed Description of the Invention

[0127] The invention relates to a five-axis automatic liquid handling platform with an imaging base. The liquid handling platform in the invention minimizes user-dependent pipetting errors and increases efficiency compared to manual pipetting by automatically performing liquid handling operations in a wide range of areas such as serology, virology, microbiology and drug discovery. The platform allows automated cell seeding and medium change on the well plate. The calculation of confluency with the imaging system integrated in the liquid handling platform prevents user-dependent errors by ensuring that the cell passage is timely performed. Thus, the invention ensures that the liquid handling processes are performed in a way to increase the reliability of the cellular information. Through the liquid handling platform described in the invention, confluency monitoring enables the determination of the biophysical properties of cells, making it possible to conduct in-vitro studies, e.g., cell-based drug discovery.

[0128] The automated, imaging-based, and five-axis liquid handling platform described in the invention, comprises:

[0129] (n = Number of Solutions)

[0130] • A well plate (25) on which cell seeding, medium change and system cleaning are performed in an automated manner,

[0131] • A manifold (4) with as many inlets as the number of solutions (n) intended to be used in the automation,

[0132] • n tubings (2) providing the fluidic connection between the manifold (4) and the solutions used in automation in the coarse adjustment system,

[0133] • n valves (5) for directing the solution (1) to the outlet, • A driver board (35) with n ports for opening and closing each valve (5),

[0134] • n electrical connections (36) between the valves (5) and the driver board (35),

[0135] • A low-resolution pump (3) that aspirates the solution (1),

[0136] • A tubing (26) providing the fluidic connection between the low-resolution pump (3) and the manifold (4),

[0137] • A driver board (33) for controlling the pump (3),

[0138] • An electrical connection (34) between the pump (3) and the driver board (33),

[0139] • A flow sensor (7) to keep the flow rate constant provided by the pump (3),

[0140] • A tubing (6) for the fluidic connection between the pump (3) and the flow sensor (7),

[0141] • A driver board (33) used for the pump (3),

[0142] • A processor (47) for the control of the flow sensor (7),

[0143] • Electrical connections between the driver board (33) and the flow sensor (7) and the processor (47),

[0144] • A rotary valve (10) with two more ports than the number of solutions (9) intended for automation in the fine-tuning system,

[0145] • A tubing (62) for the fluidic connection between the rotary valve (10) and the solutions (9),

[0146] • A driver board (39) for the control of the pump (27),

[0147] • An electrical connection (42) between the pump (27) and the driver board (39),

[0148] • A driver board (41) for the control of the rotary valve (10),

[0149] • An electrical connection (40) between the rotary valve (10) and the driver board (41),

[0150] • An incubator (74) consisting of two nested boxes in which solutions will be stored at certain parameters,

[0151] • A heater (75) located in the incubator and 2-port driver board (83) that controls it,

[0152] • A carbon dioxide source (76), carbon dioxide filter (78) and valve (77), • A tubing (92) providing fluidic connection between carbon dioxide source (76) and carbon dioxide filter (78),

[0153] • A tubing (93) providing the fluidic connection between the carbon dioxide filter (78) and the valve (77),

[0154] • A 2-port driver board (83) controlling the valve (77),

[0155] • An electrical connection (84) between the driver board (83) and the processor (47),

[0156] • A motorized switcher (73) providing automatic switching between the phase ring (64) and the diffuser (72),

[0157] • An electrical connection (91) between the fan (90) and the processor (47),

[0158] • An electrical connection (87) between the humidity source (79) and the processor (47),

[0159] • An electrical connection (88) between the humidity sensor (80) and the processor (47),

[0160] The coarse adjustment system of the automated, imaging-based and five-axis liquid handling platform described in the invention, comprises:

[0161] • Solution (1),

[0162] • A tubing (2) for fluidic connection between the solution and the manifold,

[0163] • An electrical connection (36) between the manifold (4), the valve (5), and the driver board (35),

[0164] • An electrical connection (51) between the processor (47) and the driver board,

[0165] • A low-resolution pump (3),

[0166] • A driver board (33),

[0167] • An electrical connection (34) between the low-resolution pump (3) and the driver,

[0168] • An electrical connection (50) between the driver board (33) and the processor (47),

[0169] • A tubing (26) for fluidic connection between the manifold (4) and the low-resolution pump (3), • A flow sensor (7),

[0170] • A tubing (6) for fluidic connection between the flow sensor (7) and the low-resolution pump (3),

[0171] • An electrical connection (61) between the flow sensor (7) and the processor (47),

[0172] • A tubing holder (16),

[0173] • A tubing (8) for the fluidic connection between the flow sensor (7) and the tubing holder (16)

[0174] The fine-tuning system of the automated, imaging-based and five-axis liquid handling platform described in the invention, comprises:

[0175] • Solution (9),

[0176] • Rotary valve (10),

[0177] • A tube (62) for fluidic connection between solution (9) and rotary valve (10),

[0178] • High resolution pump (27),

[0179] • Atubing (63) forfluidicconnection between rotary valve (10) and high-resolution pump (27),

[0180] • A driver board (39),

[0181] • An electrical connection (42) between driver board and high-resolution pump (27), an electrical connection (52) between driver board (39) and processor (47),

[0182] • A tubing holder (58),

[0183] • A tubing (11) for fluidic connection between rotary valve (10) and tubing holder (58),

[0184] The waste system of the automated, imaging-based and five-axis liquid handling platform described in the invention, comprises:

[0185] • A waste bottle (12),

[0186] • A low resolution pump (13), • A tubing (28) for fluidic connection between waste bottle (12) and low-resolution pump (13),

[0187] • A driver board (37),

[0188] • An electrical connection (38) between low resolution pump (13) and driver board (37),

[0189] • An electrical connection (54) between driver board (37) and processor (47),

[0190] • A tubing holder (59),

[0191] • A tubing (14) for fluidic connection between low resolution pump (13) and tube holder (59),

[0192] • A waste container (60),

[0193] The imaging system of the automated, imaging-based and five-axis liquid handling platform described in the invention, comprises:

[0194] • A camera (23),

[0195] • A lens tube (22),

[0196] • A tube lens (21),

[0197] • A lens tube (20),

[0198] • A revolver (19),

[0199] • A motor (30),

[0200] • A driver board (45),

[0201] • An electrical connection (46) between the motor (30) and the driver board (45),

[0202] • An electrical connection (56) between the driver board (45) and the processor (47),

[0203] • An achromatic objective lens (18) and phase-contrast objective lens (29),

[0204] • An LED array (71),

[0205] • A phase ring (64),

[0206] • A diffuser (72),

[0207] • A motorized switcher (73), Coarse Adjustment Operating Method of the Platform:

[0208] 1. Opening the valve (5) connected to the desired solution (1) for automation by the driver board (35) based on a command from the processor (47).

[0209] 2. Activating the low-resolution pump (3) by applying a specific voltage through the driver board (33), allowing the solution (1) to be aspirated into the manifold (4).

[0210] 3. Measuring the flow rate of the solution (1) aspirated by the low-resolution pump (3) using the flow rate sensor (7) and transmitting the data to the processor (47).

[0211] 4. Transferring the solution (1) from the flow sensor (7) to the tubing holder (16) through tubing (8).

[0212] 5. Using the data received by the processor (47) to stabilize the user-defined flow rate and controlling the voltage applied to the low-resolution pump (3) using a control algorithm to maintain the flow rate.

[0213] 6. Rotating the revolver (17) by the driver board (43) based on a command from the processor (47), aligning the tubing holder (16) connected to the tubing (8) in the coarse adjustment system with the well plate (25).

[0214] 7. Obtaining well coordinates from the processor (47) and adjusting the x, y, and z axes of the three-axis Cartesian robot (65) to these coordinates.

[0215] 8. Setting the holder (32) containing the well plate (25) at a specific angle by rotating the shaft (31) using the motor (24).

[0216] 9. Activating the motor (66) through the driver board (68), based on a command from the processor (47), rotating the shaft (67), and moving it into the well.

[0217] 10. Performing the liquid handling process on the well plate (25) using the tubing holder (16).

[0218] 11. After completing the process, activating the motor (66) through the driver board (68), based on a command from the processor (47), rotating the shaft (67), and moving it upward. The fine-tuning operating method of the platform includes the following process steps:

[0219] 1. Connection of the port on the rotary valve to which the solution to be handled is connected to the high-resolution pump by the driver board according to the command from the processor,

[0220] 2. High-resolution pump (27) aspirates the solution (9) at a certain flow rate and transmits it to the outlet port to which the tubing (11) in the fine-tuning is connected,

[0221] 3. Rotating the rotary tube (17) by the driver board (43) according to the command from the processor (47) and matching the tubing holder (58) to which the tubing (11) in the fine- tuning is connected on the well plate (25),

[0222] 4. Receiving the well coordinates by the processor (47) and bringing the x, y, z axes (65) to these coordinates,

[0223] 5. Bringing the holder (32) where the well plate (25) is located to a certain angle with the shaft (31) rotated by the motor (24),

[0224] 6. Operating the motor (66) by the driver board (68) according to the command from the processor (47) and rotating the shaft (67) ensuring its movement into the well,

[0225] 7. Performing the liquid handling process on the well plate (25) with the tubing holder (58),

[0226] 8. Operating the motor (66) by the driver board (68) according to the command from the processor (47) and ensuring the upward movement of the shaft (67) by rotating it.

[0227] Operating Method of the Platform's Waste System includes the following process steps:

[0228] 1. Rotating the revolver (17) by the driver board (43) according to the command from the processor (47) and matching the tubing holder (59) to which the tubing (14) in the waste system is connected, on the well plate (25),

[0229] 2. Taking the well coordinates by the processor (47) and bringing the x, y, z axes of the three- axis Cartesian robot (65) to these coordinates,

[0230] 3. Bringing the holder (32) where the well plate (25) is located to a certain angle with the shaft (31) rotated by the motor (24), 4. Operating the motor (66) by the driver board (68) according to the command from the processor (47) and ensuring the movement of the shaft (67) by rotating it into the well,

[0231] 5. Operating the low-resolution pump (13) at the highest flow rate by the driver board (37) according to the command from the processor (47),

[0232] 6. Transferring the waste withdrawn by the low-resolution pump (13) to the waste bottle (12) via the tubing (28) transfer,

[0233] 7. At the end of the process; the motor (66) is operated by the driver board (68) according to the command from the processor (47), and the shaft (67) is rotated and its upward movement is secured,

[0234] Operating Method of the Platform's Imaging System includes the following process steps:

[0235] 1. The motor connected to the revolver (19) is activated by the driver board (45) according to the command from the processor (47), aligning either the achromatic objective lens (18) or the phase-contrast objective lens (29) with the well plate (25).

[0236] 2. Taking the well plate coordinates by the processor and bringing the x,y,z axes to these coordinates.

[0237] 3. Placement of the diffuser (72) or phase ring (64) between the well plate (25) and the LED array (71) by a motorized switcher (73) .

[0238] 4. Taking images by camera (23).

[0239] With the automated imaging-based liquid handling platform, which is the subject of the invention, cell seeding, medium change and system cleaning are performed automatically on the well plate (25). With the imaging feature of the system, confluency is calculated via the system software. Thus, errors that may occur due to user intervention are minimized and the pipetting process becomes practical. Figure 1 shows the liquid handling platform that can perform imaging for well plates (25). The platform consists of two main structures: the liquid handling system and the imaging system. The liquid handling system has two settings: The coarse adjustment, which is achieved using a low-resolution pump, and the fine-tuning which is achieved using a high-resolution pump.

[0240] In the coarse adjustment system; a manifold (4) is used that has the number of inlets sufficient to handle the targeted solutions (1). For n solutions (1), the manifold (4) with n inlets is used. The fluidic connection between the solutions (1) and the manifold (4) is provided with n number of tubings (2). The manifold (4) has a single outlet, n number of valves (5) are used to direct the desired solution (1) to the outlet. A driver board (35) with n ports is used to open and close each valve (5). There are n number of electrical connections (36) between the valves (5) and the driver board (35). After the valve (5) is opened forthe desired solution (1), the low- resolution pump (3) is operated and the solution (1) is aspirated. The fluidic connection between the low-resolution pump (3) and the manifold (4) is provided with a tubing (26). The control of the low-resolution pump (3) is provided with a driver board (33). There is an electrical connection (34) between the low-resolution pump (3) and the driver board (33). In order to keep the flow rate provided by the low-resolution pump (3) constant, a flow sensor (7) is used. The fluidic connection between the low-resolution pump (3) and the flow sensor (7) is provided by a tubing (6). The driver board (33) and the flow sensor (7) used for the low- resolution pump (3) are controlled by a processor (47). There are electrical connections (50), (61) between the driver board (33), the flow sensor (7) and the processor (47), respectively. The processor (47); performs the flow control by the control loop method (P control, I control, D control, PD control, PI control, PID control, adaptive control, fuzzy logic control, neural network control, MPC and other control algorithms) and creates a feedback loop between the flow sensor (7) and the low-resolution pump (3).

[0241] In the fine-tuning system; a rotary valve (10) with two more ports than the number of solutions (9) is used. For example, for n solutions (9), a rotary valve (10) with n+2 ports is used. The fluidic connection between the rotary valve (10) and the solutions (9) is provided by a tubing (62). One of the ports of the rotary valve (10) is connected to the high-resolution pump (27) via a tubing (63). The control of the high-resolution pump (27) is provided by a driver board (39). There is an electrical connection (42) between the high-resolution pump (27) and the driver board (39). The control of the rotary valve (10) is provided by a driver board (41). There is an electrical connection (40) between the rotary valve (10) and the driver board (41). In addition to the coarse adjustment and fine-tuning systems, the liquid handling system also includes a waste system untulizing a low-resolution pump (13) to aspirate remaining solutions. The waste solutions dispensed by the low-resolution pump (13) are sent to a waste bottle (12). The fluidic connection between the low-resolution pump (13) and the waste bottle (12) is provided by a tubing (28). The control of the low-resolution pump (13) is provided by a driver board (37). There is an electrical connection (38) between the low-resolution pump (13) and the driver board (37).

[0242] The tubings coming out of the coarse adjustment system, fine-tuning system and waste system [respectively (8), (11), (14)] are positioned in a revolver (17) with the help of the tubing holders [respectively (16), (58), (59)]. For the desired liquid handling, the revolver (17) is rotated and positioned in the position where the tubing holder is located via a motor (15). Figure 2 shows how the changes between the tubings are made with the revolver (17). The control of the motor (15) is provided by a driver board (43). There is an electrical connection (44) between the motor (15) and the driver board (43).

[0243] The imaging system captures the images of the biological materials in the well plate (25) with brightfield microscopy and phase-contrast microscopy methods. There are two microscopy setups in the same optical compartment. In the brightfield microscopy setup; relative to the sample plane, there are an achromatic objective lens (18), a lens tube (20), a tube lens (21), a lens tube (22) and a camera (23), respectively. In the phase contrast microscopy setup; relative to the sample plane, there are a phase-contrast objective lens (29), a phase ring (64), diffuser (72), motorized switcher (73), a lens tube (20), a tube lens (21), a lens tube (22) and a camera (23), respectively. The two microscopy setups share the lens tube (20), tube lens (21), lens tube (22) and camera (23).

[0244] The achromatic objective lens (18) and the phase-contrast objective lens (29) are placed in a revolver (19). Regardless of the microscopy device to be used for imaging, the revolver (19) is rotated and positioned to the position where the lens of the invention is located via a motor (30). Figure 3 shows the mechanism for switching between the two objective lenses using the revolver (19). The control of the motor (30) is provided by a driver board (45). The tube lens (21) in the invention is used jointly for both objective lenses (achromatic objective lens (18) and phase-contrast objective lens (29)). There is an electrical connection (46) between the motor (30) and the driver board (45). In the liquid handling platform, which is the subject of the invention, there is a holder (32) where the well plate (25) and the diffuser (72) are placed. The diffuser (72) ensures that the illumination from the LED array (71) is distributed homogeneously over the sample, thus preventing shadow formation on the image. The tilting movements of the holder (32) will be provided by rotating a shaft (31) on which the plate holder is placed, by a motor (24). The control of the motor (24) is provided by a driver board (48). There is an electrical connection (49) between the motor (24) and the driver board (48).

[0245] The phase ring (64) can be attached / detached manually or by the motorized switcher (73) when phase-contrast imaging is to be performed. The holder (32) in which the well plate (25), phase ring (64) and LED array (71) are placed is fixed on the shaft (31). The shaft (31) is rotated by the motor (24); and the holder (32) is kept at a certain angle. Thus; the tubing (8, 11 or 14) that enters vertically into the well plate (25) standing at an angle can perform the liquid dispensing from the side wall of the well.

[0246] In liquid handling and imaging processes; the invention includes a three-axis Cartesian robot (65) that can be positioned over the desired well on the well plate (25).

[0247] To address contamination issues that may occur due to the dead volume in the manifold (4) and rotary valve (10) during the processes in the coarse and fine adjustment systems; the manifold (4) and rotary valve (10) are cleaned with cleaning buffers between different liquid handling processes. The cleaning buffers coming out of the manifold (4) and rotary valve (10) are emptied by positioning the relevant system tubing in a waste container (60) with the help of the three-axis Cartesian robot (65).

[0248] In order to prevent the imaging system from colliding with the well plate (25) during liquid handling operations on a plate positioned within the well plate (25), the imaging system is mounted on a shaft (67). By rotating this shaft with a motor (66), the tube holders (16), (59), (58) are lifted up. The control of the motor (66) is provided by a driver board (68). There is an electrical connection (69) between the motor (66) and the driver board (68).

[0249] There are independent electrical connections between the processor (47) and all driver boards (33, 35, 37, 39, 41, 43, 45, 48, 68) included in the invention. Electrical connection (51) for the driver board (35), electrical connection (52) for the driver board (39), electrical connection (53) for the driver board (41), electrical connection (54) for the driver board (37), electrical connection (55) for the driver board (43), electrical connection (56) for the driver board (45), electrical connection (57) for the driver board (48), electrical connection (70) for the driver board (68).

[0250] The images captured from the sample are transferred to a processor, e.g., computer, singleboard computer and all processors by the camera (23). In cases where the samples being imaged are optically thick, meaning they have different focal points, high depth of field and high-resolution images focused at each point can be obtained by combining the images taken from different sections with the focus stacking method. In well plates with large well areas e.g., 6, 12, 24, the bottom of the wells is scanned with a three-axis Cartesian robot (65). The images obtained with the camera (23) are obtained with a wide field of view and images in which the entire well is seen by using the image stitching method. When optically thick samples are desired to be imaged in well plates with large well areas (25); focus stacking and image stitching methods are used together to generate images with a wide field of view and high depth of field. The solutions (1) used in the coarse adjustment system and the solutions (9) used in the fine-tuning system are stored in an incubator (74) consisting of two nested boxes. The incubator (74) allows cells and microorganisms to grow and reproduce under optimal conditions by keeping the temperature, carbon dioxide and humidity parameters stable within certain ranges. There is a heater (75) and a temperature sensor (81) in the incubator. There is an electrical connection (86) between the heater (75) and the 2-port driver board (83). There is an electrical connection (84) between the processor (47) and the 2-port driver board (83). There is an electrical connection between the temperature sensor (81) and the processor (47). The heater (75) heats the incubator environment to a certain temperature value. The temperature sensor (81) constantly measures the ambient temperature and transmits it to the processor (47). The processor (47) controls the temperature value of the environment with a feedback algorithm. For example; when the ambient temperature is above the desired temperature value (such as 37.5°C), the processor (47) disables the heater (75) and ensures that the environment cools down and reaches the desired temperature value. Otherwise, when the ambient temperature is below the desired temperature value, the processor (47) sends a high current to the heater (75) and causes the heater to heat up more, thus increasing the ambient temperature and fixing it at the desired temperature value.

[0251] There is a carbon dioxide sensor (82) and a valve (77) inside the incubator. There is an electrical connection (95) between the carbon dioxide sensor (82) and the processor (47). There is an electrical connection (85) between the valve (77) and the 2-port driver board (83). The valve (77) ensures that the carbon dioxide source (76) located outside the incubator (74) is released into the incubator (74) in a controlled manner. The carbon dioxide gas to be given into the incubator is first passed through the carbon dioxide filter (78) via a tubing (92) from the carbon dioxide source (76). Then, the gas coming out of the carbon dioxide filter is transferred to the valve (77) via a tubing (93) and given to the incubator (74). The amount of carbon dioxide in the incubator (74) environment is controlled by the feedback algorithm. If the amount of carbon dioxide in the environment is insufficient, the control algorithm ensures that the valve (77) remains open for the calculated period of time, allowing carbon dioxide to enter the environment. Continuous measurement is taken with the carbon dioxide sensor (82) and when there is sufficient carbon dioxide in the environment, the valve (77) is closed by the driver board (83).

[0252] There is a humidity source (79) and a humidity sensor (80) inside the incubator. There is an electrical connection (87) between the humidity source (79) and the processor (47). There is an electrical connection (88) between the humidity sensor (80) and the processor (47). The humidity rate inside the incubator (74) is controlled by the feedback algorithm. The measurement data continuously received by the humidity sensor (80) is sent to the processor (47). In cases where the humidity rate is insufficient, the humidity source (79) is operated to increase the humidity of the environment and is turned off by the processor (47) when the desired value is reached.

[0253] The parameters of the incubator (74) are displayed to the user in real-time on the display (97).

[0254] There is a fan (90) inside the incubator and an electrical connection (91) between this fan (90) and the processor (47). The fan (90); distributes the heat, carbon dioxide gas and humidity of the environment homogeneously to the incubator environment.

[0255] There is an ultraviolet C (UV-C) source (94) in the incubator (74). There is an electrical connection (95) between the ultraviolet C source (94) and the processor (47). UV-C rays sterilize the incubator (74) environment by destroying the DNA of bacteria, viruses and other microorganisms. This feature reduces the risk of contamination of the cell culture and ensures reliable and reproducible results. When the three encoder motors inside the three-axis Cartesian robot (65) are removed from the system and replaced with motors without encoders, a new version of the system is created. Similarly, the encoder motors responsible for the up / down movements of the shaft (67), which holds the tubing holders, are replaced with non-encoder motors. Additionally, the encoder motor controlling the shaft (31), to which the holder (32) is connected, is also replaced. Together, these changes result in a new version of the system. In this case, new components have been added to prevent any collisions in the system. One sensor (98, 99, 100, 101, 102) has been added to the x, y, z axes of the three- axisCartesian robot (65), the fourth axis to which the holder (32) is connected and the fifth axis to which the tubing holders (16, 58, 59) are connected. Figure 4 shows the sensor added to the fourth axis to which the holder (32) is connected (98). Figure 5 shows the sensor (99) added to the fifth axis to which the tubing holders (16, 58, 59) are connected. Figure 6 shows the sensors (100, 101, 102) added to the x, y, z axes of the three-axis Cartesian robot (65). These sensors can be optical sensors, proximity sensors such as infrared sensors, motion sensors such as gyroscopes, pressure sensors, current sensors, acoustic sensors, touch sensors, cameras, and all sensors that can detect impact or provide current location information.

Claims

CLAIMS1. An automated, imaging-based, and five-axis liquid handling platform comprising:• A well plate (25) where automated processes such as cell seeding, medium change, and system cleaning are performed,• A manifold (4) with n inlets sufficient to handle the number of solutions intended for automation,• n tubings (2) providing fluidic connections between the solutions (1) and the manifold (4) in the coarse adjustment system,• n valves (5) for directing the solutions (1) toward the outlet,• A driver board (35) with n ports for opening and closing each valve (5),• n electrical connections (36) between the valves (5) and the driver board (35),• A low-resolution pump (3) that draws the solution (1),• A tubing (26) providing fluidic connection between the low-resolution pump (3) and the manifold (4),• A driver board (33) for controlling the pump (3),• An electrical connection (34) between the pump (3) and the driver board (33),• A flow sensor (7) to maintain constant flow rate provided by the pump (3),• A tubing (6) creating a fluidic connection between the pump (3) and the flow sensor (7),• A driver board (33) for controlling the pump (3),• A processor (47) for controlling the flow sensor (7),• Electrical connections (50) and (61) between the driver board (33), the flow sensor (7), and the processor (47),A rotary valve (10) with two more ports than the number of solutions (9) for handling in the fine-tuning system,• A tubing (62) establishing a fluidic connection between the rotary valve (10) and the solutions (9),• A driver board (39) controlling the pump (27),• An electrical connection (42) between the pump (27) and the driver board (39),• A driver board (41) controlling the rotary valve (10),• An electrical connection (40) between the rotary valve (10) and the driver board (41).• An incubator (74) consisting of two nested containers for maintaining solutions under specific conditions,• A heater (75) located inside the incubator and controlled by a two-port driver board (83),• A carbon dioxide source (76), carbon dioxide filter (78), and valve (77),• A tubing (92) providing a fluidic connection between the carbon dioxide source (76) and the carbon dioxide filter (78),• A tubing (93) ensuring a fluidic connection between the carbon dioxide filter (78) and the valve (77),• A two-port driver board (83) controlling the valve (77),• An electrical connection (84) between the driver board (83) and the processor (47),• A motorized switcher (73) enabling automated switching between the phase ring (64) and the diffuser (72),• An electrical connection (91) between the fan (90) and the processor (47),• An electrical connection (87) between the humidity source (79) and the processor (47),• An electrical connection (88) between the humidity sensor (80) and the processor (47),• An electrical connection (89) between the temperature sensor (81) and the processor (47),• An electrical connection (95) between the ultraviolet C (UVC) source (94) and the processor (47), and• An electrical connection between the carbon dioxide sensor (82) and the processor (47).

2. A platform according to Claim 1, characterized by comprising a liquid handling system that includes a coarse adjustment system, a fine-tuning system, and a waste system.

3. A coarse adjustment system of the platform according to Claim 1, comprising:• A solution (1),• A tubing (2) providing fluidic connection between the solution (1) and the manifold (4),• A manifold (4),• A valve (5),• A driver board (35),• An electrical connection (36) between the valve (5) and the driver board (35),• An electrical connection (51) between the processor (47) and the driver board (35),• A low-resolution pump (3),• A driver board (33) for controlling the pump,• An electrical connection (34) between the low-resolution pump (3) and the driver board (33),• An electrical connection (50) between the driver board (33) and the processor (47),• A tubing (26) providing a fluidic connection between the manifold (4) and the low- resolution pump (3),• A flow sensor (7),• A tubing (6) providing a fluidic connection between the flow sensor (7) and the low- resolution pump (3),• An electrical connection (61) between the flow sensor (7) and the processor (47),• A tubing holder (16),A tubing (8) providing a fluidic connection between the flow sensor (7) and the tubing holder (16).

4. A fine tuning system of the platform according to Claim 1, comprising:• A solution (9),• A rotary valve (10),• A tubing (62) providing a fluidic connection between the solution (9) and the rotary valve (10),• A high-resolution pump (27),• A tubing (63) providing a fluidic connection between the rotary valve (10) and the high- resolution pump (27),• A driver board (39),• An electrical connection (42) between the driver board (39) and the high-resolution pump (27),• An electrical connection (52) between the driver board (39) and the processor (47),• A tubing holder (58),• A tubing (11) provides a fluidic connection between the rotary valve (10) and the tubing holder (58).

5. A waste system of the platform according to Claim 1, characterized by comprising:• A waste bottle (12),• A low-resolution pump (13),• A tubing (28) providing a fluidic connection between the waste bottle (12) and the low- resolution pump (13),• A driver board (37),An electrical connection (38) between the low-resolution pump (13) and the driver board (37), 1• An electrical connection (54) between the driver board (37) and the processor (47),• A tubing holder (59),• A tubing (14) providing a fluidic connection between the low-resolution pump (13) and the tubing holder (59), and• A waste container (60)6. An imaging system of the platform according to Claim 1, comprising:• A camera (23),• A lens tube (22),• A tube lens (21),• A lens tube (20),• A revolver (19),• A motor (30),• A driver board (45),• An electrical connection (46) between the motor (30) and the driver board (45),• An electrical connection (56) between the driver board (45) and the processor (47),• An achromatic objective lens (18) and a phase-contrast objective lens (29),• An LED array (71),• A phase ring (64),• A diffuser (72), and• A motorized switcher (73).

7. A platform according to Claim 1, enabling automated processes such as cell seeding, medium change, and system cleaning on the well plate (25).

8. A platform accordingto Claim 1, where one of the ports of the rotary valve (10) is connected to a high-resolution pump (27) through a tubing (63).

9. A platform according to Claim 1, which includes a waste system with a low-resolution pump (13) for aspirating small volumes of solutions.

10. A platform according to Claim 1, which includes a waste bottle (12) to which waste solutions aspirated by the pump (13) are sent.

11. A platform according to Claim 1, which includes a tubing (28) providing a fluidic connection between the pump (13) and the waste bottle (12).

12. A platform according to Claim 1, which includes a driver board (37) for controlling the pump (13).

13. A platform according to Claim 1, which includes an electrical connection (38) between the pump (13) and the driver board (37).

14. A platform according to Claim 1, which includes tubing holders [(16), (58), (59)] positioned within the revolver (17), holding tubings [(8), (11), (14)] coming from the coarse adjustment system, fine-tuning system, and waste system, respectively.

15. A platform according to Claim 1, which includes a driver board (43) for controlling the motor (15).

16. A platform according to Claim 1, which includes an electrical connection (44) between the motor (15) and the driver board (43).

17. A platform according to Claim 1, which includes an imaging system for visualizing biological materials on the well plate (25) using brightfield and phase-contrast microscopy techniques.

18. A platform according to Claim 1, which includes the following components arranged sequentially and relative to the sample plane: a diffuser (72), an achromatic objective lens (18), a lens tube (20), a tube lens (21), another lens tube (22), and a camera (23) in the brightfield microscopy configuration.

19. A platform according to Claim 1, which includes, the following components sequentially arranged relative to the sample plane: a phase-contrast objective lens (29), a phase ring (64), a lens tube (20), a tube lens (21), another lens tube (22), and a camera (23) in the phasecontrast microscopy configuration.

20. A platform according to Claim 1, comprising both brightfield and phase-contrast microscopy configurations, sharing the following components: a lens tube (20), a tube lens (21), another lens tube (22), and a camera (23).

21. A platform according to Claim 1, which includes an achromatic objective lens (18) and a phase-contrast objective lens (29), both mounted inside a revolver (19).

22. A platform according to Claim 1, which includes a driver board (45) for controlling the motor (30).

23. A platform according to Claim 1, where the tube lens (21) is shared by both the achromatic objective lens (18) and the phase-contrast objective lens (29).

24. A platform according to Claim 1, which includes an electrical connection (46) between the motor (30) and the driver board (45).

25. A platform according to Claim 1, comprising a holder (32) where the well plate (25) is placed.

26. A platform according to Claim 1, where the tilting motions of the holder (32) are performed by rotating a shaft (31) to which the holder is attached, driven by a motor (24).

27. A platform according to Claim 1, which includes a driver board (48) for controlling the motor (24).

28. A platform according to Claim 1, comprising an electrical connection (49) between the motor (24) and the driver board (48).

29. A platform according to Claim 1, comprising a removable phase ring (64), which can be attached or detached by the operator when phase-contrast imaging is required.

30. A platform according to Claim 1, which includes a holder (32) fixed on the shaft (31), a well plate (25), a phase ring (64), a diffuser (72), a motorized switcher (73), and an LED array (71).

31. A platform according to Claim 1, comprising a three-axis Cartesian robot (65) to position the desired well on the well plate (25) during liquid handling and imaging processes.

32. A platform according to Claim 1, where the imaging system is mounted on a shaft (67) to prevent collisions with the well plate (25) during liquid handling processes.

33. A platform according to Claim 1, which includes a driver board (68) for controlling the motor (66).

34. A platform according to Claim 1, comprising an electrical connection (69) between the motor (66) and the driver board (68).

35. A platform according to Claim 1, with independent electrical connections between the processor (47) and all driver boards (33, 35, 37, 39, 41, 43, 45, 48, 68, 83).

36. A platform according to Claim 1, comprising the following electrical connections:Electrical connection (51) for the driver board (35),Electrical connection (52) for the driver board (39),Electrical connection (53) for the driver board (41),Electrical connection (54) for the driver board (37),Electrical connection (55) for the driver board (43),Electrical connection (56) for the driver board (45),Electrical connection (57) for the driver board (48),Electrical connection (70) for the driver board (68),Electrical connection (84) for the driver board (83).

37. A platform according to Claim 1, comprising an electrical connection (91) between the processor (47) and the fan (90).

38. A platform according to Claim 1, comprising an electrical connection (87) between the processor (47) and the humidity source (79).

39. A platform according to Claim 1, comprising an electrical connection (88) between the processor (47) and the humidity sensor (80).

40. A platform according to Claim 1, comprising an electrical connection (89) between the processor (47) and the temperature sensor (81).

41. A platform according to Claim 1, comprising an electrical connection (95) between the processor (47) and the ultraviolet C source (94).

42. A platform according to Claim 1, comprising an electrical connection (96) between the processor (47) and the carbon dioxide sensor (82).

43. A platform according to Claim 1, comprising an incubator (74) and a display (97).

44. A platform according to Claim 1, which includes insulated tubings (2, 26, 6, 8, 62, 11, 14, 28) for maintaining temperature stability.

45. A platform according to Claim 1, which includes motors with encoders (24, 73, 30, 15, 66).

46. A platform according to Claim 1, comprising three encoder-equipped motors within the three-axis Cartesian robot (65).

47. A platform according to Claim 1, comprising collision-prevention sensors (98, 99, 100, 101, 102).

48. A coarse adjustment operating the method of the platform according to Claim 1, comprising the following steps: i. Opening the valve (5) connected to the desired solution (1) for automation by the driver board (35) based on a command from the processor (47), ii. Activating the low-resolution pump (3) by applying a specific voltage through the driver board (33), allowing the solution (1) to be aspirated into the manifold (4), iii. Measuring the flow rate of the solution (1) aspirated by the low-resolution pump (3) using the flow sensor (7) and transmitting the data to the processor (47), iv. Transferring the solution (1) from the flow sensor (7) to the tubing holder (16) through tubing (8), v. Using the data received by the processor (47) to stabilize the user-defined flow rate and controlling the voltage applied to the low-resolution pump (3) with a control algorithm to maintain the flow rate, vi. Rotating the revolver (17) by the driver board (43) based on a command from the processor (47), aligning the tubing holder (16) connected to the tubing (8) in the coarse adjustment system with the well plate (25),vii. Obtaining well coordinates from the processor (47) and adjusting the x, y, and z axes of the three-axis Cartesian robot (65) to these coordinates, viii. Rotating the shaft (31) holding the well plate (25) using the motor (24) to set the well plate holder (32) at a specific angle, ix. Activating the motor (66) by the driver board (68) based on a command from the processor (47), rotating the shaft (67), and enabling its movement into the well, x. Performing liquid handling on the well plate (25) using the tubing holder (16), xi. Activating the motor (66) by the driver board (68) based on a command from the processor (47), rotating the shaft (67), and moving it upward.

49. A fine tuning operating method of the platform according to Claim 1, comprising the following process steps: i. Establishing a connection between the port on the rotary valve (10), to which the desired solution (9) is connected, and the high-resolution pump (27) through the driver board (41), based on a command from the processor (47), ii. Aspirating the solution (9) at a specified flow rate using the high-resolution pump (27) and transferring it to the outlet port connected to the tubing (11) within the fine- tuning system, iii. Rotating the revolver (17) using the driver board (43), based on a command from the processor (47), and aligning the tubing holder (58) connected to the tubing (11) over the well plate (25), iv. Obtaining the well coordinates from the processor (47) and adjusting the x, y, and z axes (65) accordingly, v. Setting the holder (32) containing the well plate (25) at a specific angle by rotating the shaft (31) with the motor (24), vi. Activating the motor (66) through the driver board (68), based on a command from the processor (47), rotating the shaft (67), and moving it into the well,vii. Performing the liquid handling process on the well plate (25) using the tubing holder (58), viii. Activating the motor (66) through the driver board (68), based on a command from the processor (47), rotating the shaft (67), and moving it upward.

50. A waste system operating method of the platform according to Claim 1, comprising the following steps: i. Rotating the revolver (17) through the driver board (43), based on a command from the processor (47), and aligning the tubing holder (59) connected to the tubing (14) within the waste system over the well plate (25), ii. Obtaining the well coordinates from the processor (47) and adjusting the x, y, and z axes of the three-axis Cartesian robot (65) accordingly, iii. Setting the holder (32) containing the well plate (25) at a specific angle by rotating the shaft (31) with the motor (24), iv. Activating the motor (66) through the driver board (68), based on a command from the processor (47), rotating the shaft (67), and moving it into the well, v. Operating the low-resolution pump (13) at maximum flow rate through the driver board (37), based on a command from the processor (47), vi. Transferring the waste drawn by the low-resolution pump (13) to the waste bottle (12) through tubing (28), vii. Activating the motor (66) through the driver board (68), based on a command from the processor (47), rotating the shaft (67), and moving it upward.

51. An operating method of the imaging system of the platform according to Claim 1, comprising the following steps: i. Activating the motor connected to the revolver (19) through the driver board (45), based on a command from the processor (47), aligning either the achromatic objective lens (18) or the phase-contrast objective lens (29) with the well plate (25),ii. Obtaining well coordinates from the processor (47) and adjusting the x, y, and z axes (65) accordingly, iii. Positioning the diffuser (72) or the phase ring (64) between the well plate (25) and the LED array (71) using the motorized switcher (73), iv. Capturing images with the camera (23).

Images