Devices, methods and systems for culture sample development monitoring

Abstract

The present invention relates to the field of testing and evaluating biological samples and provides an apparatus for incubating samples, comprising at least one independently accessible module adapted for incubating at least one of a plurality of samples, wherein the at least one module is in operative association with a light source and a movable optical detection member adapted for movement about an observation axis through the module to effect scanning of an observation area. The present invention also provides a method of evaluating viability of incubated samples, comprising the steps of placing biological samples in a substantially elliptical arrangement within an incubation chamber of an independently accessible module; imaging individual samples of the substantially elliptical arrangement with a driven optical detection member in an X-Y plane normal to an observation axis through the module to obtain time-lapse measurements of development of the individual samples.

Description

[0001] This application is a divisional application of Chinese invention patent application 201480024632.X, filed on March 3, 2014, entitled "Apparatus, method and system for monitoring the development of cultured samples".

[0002] Related applications

[0003] This application claims priority to Australian Provisional Patent Application No. 2013900700, filed March 1, 2013, entitled “Method and System for Monitoring the Development of Culture Samples”, and Australian Provisional Patent Application No. 2013903928, filed October 11, 2013, also entitled “Method and System for Monitoring the Development of Culture Samples”, the descriptions of which are incorporated herein by reference in their entirety and for all purposes. Invention Field

[0004] This invention relates to the field of testing and evaluating biological samples. The following description of the invention concerning the imaging and evaluation of biological samples will be convenient, particularly regarding the imaging and evaluation of zygotes, embryos, oocytes, and stem cells located in a culture space; however, it should be understood that the invention is not limited to this use. For example, the invention is also used to provide optimal and safe culture conditions for incubation during embryonic development. Background Technology

[0005] The singular form of the word "inventor" as used in this specification may refer to one (singular) inventor or more than one (multiple) inventors of the present invention.

[0006] It should be understood that any discussion of documents, devices, actions, or knowledge in this specification is included to explain the context of the invention. Furthermore, the discussion throughout this specification arises from the inventor's practice and / or the inventor's identification of certain related technical problems. In addition, any discussion of materials such as documents, devices, actions, or knowledge in this specification is included to explain the context of the invention in accordance with the inventor's knowledge and experience; therefore, any such discussion should not be considered as an admission that any such material forms part of the prior art or common general knowledge in the relevant field in Australia or elsewhere on or prior to the priority date of the disclosure and claims herein.

[0007] Assisted reproductive technology (ART) has become increasingly important as a means of assisted reproduction in developed countries. The background is that after its introduction to the United States in 1981, approximately 150,000 ART cycles were performed in the US in 2010, resulting in 47,090 safe births and 61,564 babies. While ART use remains relatively low compared to potential demand, its use has increased significantly over the past decade, leading to the assumption that 1% of all babies born annually in the US now utilize in-vitro fertilization (IVF), compared to 2-4% in other countries. This is also supported by a recent online article from the US Centers for Disease Control and Prevention (http: / / www.cdc.gov / art).

[0008] IVF involves hormonal stimulation of a woman's ovaries to mature multiple oocytes, which are then removed, fertilized in a laboratory, cultured for 2 to 6 days, and then transferred back to her uterus for pregnancy. Oocytes that have replicated their chromosomes and undergone cell lysis twice by day 2 and reach the 4-cell stage by day 3, and the 8-cell stage by day 3, are more likely to produce offspring compared to oocytes that have replicated their chromosomes and undergone only one cell lysis, reaching the 2-cell stage by day 2 and the 4-cell stage by day 3. The embryonic developmental pattern that demonstrates widely accepted embryo viability and leads to subsequent successful pregnancy outcomes (despite patient-specific factors) is appropriate and timely, i.e., cell division occurs in a normal manner and at the appropriate time.

[0009] Little is known about the fundamental pathways and events of early human embryonic development, including factors that may help predict developmental success or failure. Therefore, to increase the chances of pregnancy through IVF, multiple embryos are often transferred to the uterus, despite potentially leading to well-documented adverse outcomes (see, for example, Pinborg 2005). 1 ).

[0010] In response to this problem, many IVF protocols extend embryo culture to day 5 or 6 to transfer a single blastocyst. This practice has successfully reduced the risk of multiple pregnancies for women under 36 years of age, while producing higher implantation / pregnancy rates per transferred embryo. However, many patients' fertilized eggs do not form blastocysts in culture. Furthermore, well-studied mouse embryo models have shown that the rapid cleavage rate that occurs in vivo between the 4-cell and 16-cell stages cannot be reproduced in vitro under existing culture conditions. Because blastocyst formation begins at defined intervals after fertilization, independent of the number of cell divisions, mouse embryos developing in vivo have more than twice as many cells at the blastocyst stage compared to embryos developing in culture. If this is the case with human embryos, then extended culture may result in blastocysts with fewer cells available for fetal development—a possible explanation for some IVF babies reported to have low birth weight (Kiessling et al., 1991).

[0011] Oocytes required for IVF procedures are aspirated via a vaginal ultrasound-guided needle. One to over 40 oocytes can be retrieved, but typically 10 to 20 are used. The oocytes are then placed in a culture medium based on human fallopian tube fluid and cultured at 37°C. Then, typically 100,000 to 200,000 sperm are added to the oocytes in a droplet of medium, or a single sperm is injected directly into the oocyte using intracytoplasmic sperm injection (ICSI). Fertilization is recorded 12 to 20 hours later by the presence of pronuclei indicating that fertilization has occurred, derived from both the father (from sperm) and the mother (from the egg). Fertilization rates can vary between 0 and 100%, but an average of approximately 6-70% is considered normal. Embryos with the “best” morphological grade are then selected for transfer.

[0012] Many factors influence the in vitro development of mammalian preimplantation embryos. Besides proper temperature control and culture medium formulation, human embryos are generally susceptible to oxidative stress. Therefore, human embryos are typically cultured at low oxygen concentrations (approximately 2-7%), although some centers still utilize atmospheric oxygen concentrations (approximately 20%).

[0013] As IVF procedures take on increasing clinical significance, the morphological assessment of retrieved oocytes remains rather superficial (Rienzi et al., 2011). 2Typical studies of in vitro collected oocytes are limited to the assessment of the presence and general morphology of the cumulus using a stereomicroscope. Subsequently, a rapid assessment is also performed using an inverted microscope after ablation (removal of cumulus cells), including assessment of the cytoplasm, perivitelline space, and zona pellucida (Rienzi et al., 2011). This evaluation provides very superficial information about the developmental stage [Medium 1 (MI) or MII] and quality (by looking for signs of regression in the cytoplasm, polar bodies, or zona pellucida). Subsequently, ICSI (intracytoplasmic sperm injection) is performed on MII oocytes, from which point the developmental potential of the resulting embryo is estimated solely based on the morphology of the embryo body, regardless of the quality of the oocyte from which it is derived (Rienzi et al., 2011).

[0014] Once fertilized embryos are cultured, morphological evaluation becomes a crucial step. At predetermined checkpoints, typically daily or every other day for in vitro culture, routine inverted microscopic examinations are performed, and quantitative characterization is performed using internationally recognized standards, despite some concerns regarding the predictive values ​​of these parameters. (Cummins et al., 1986) 3 Emiliani et al., 2006 4 ).

[0015] Many different methods have been developed to identify embryos with high implantation potential. The most widely supported strategy for selecting viable embryos relies on the number of blastomeres at the time of embryo transfer and the embryo's morphological grade (Beuchat et al., 2008). 5 Embryo grading is defined as a grade assigned to an embryo based on one of a limited number of internationally recognized embryo grading standards. However, these morphological aspects are not sufficiently correlated with embryo viability to allow for definitive identification of the optimal embryos capable of producing a successful pregnancy. Several alternative strategies have been proposed to improve the prognostic accuracy of embryo viability estimates, including selecting embryos that undergo early division (Shoukir et al., 1997). 6 ), cultured to the blastocyst stage (Gardner et al., 1998) 7 ), and the scoring of pronuclear (PN) stage conjugates (Ebner et al., 2003). 8 The study analyzed the metabolic profile of embryos and examined their chromosomal composition after cell biopsy.

[0016] While the methods described above offer improvements, they remain inherently subjective measurements, and several algorithm-driven automated scoring systems have been designed to further improve the predictive accuracy of embryo scoring. These include pronuclear zygote scoring systems (Beuchat et al., 2008). More recently, time-lapse imaging techniques have been incorporated into some scoring algorithms, along with phenotypic measurements, including cleavage timing estimation (ARAV 2008). 9 ), blastocyst development rate (Cruz et al., 2011) 10 Phenotypic measurements include mitosis timing, cytokinesis, and zona pellucida thickness (Wong et al., 2010). 11 Regardless of the morphological scoring system used, time-lapse imaging techniques inherently increase the predictive accuracy of embryo scoring (Montag et al., 2011). 12 ).

[0017] International patent application number WO 2012 / 047678 (Auxogyn) discloses a system for automated imaging and evaluation of human embryos, oocytes, or pluripotent cells, describing automated culture dish detection and well occupancy determination. Additionally, multi-well culture dishes and illumination components for bimodal imaging are described. These devices are used to identify, or facilitate the identification of, in vitro embryos and oocytes that may be used to treat human infertility. The apparatus of WO 2012 / 047678 includes a standard incubator having one or more racks for supporting the imaging system. The imaging system has a loading platform and is placed within the incubator to image one or more embryos cultured in culture dishes mounted on their loading platforms. In other words, several complete imaging systems are placed in situ with the incubator for one or more embryos associated with the culture dishes mounted on each imaging system.

[0018] Generally, minimizing patient confusion or misidentification of biological samples is important. In current systems, including those with time-lapse imaging, it is often necessary to hand-label the lid of the culture dish containing the biological sample, or the sample may not be labeled in the same way on both the dish and the lid. Since the embryo is retained in the culture dish, it should be noted that the dish lid can be detached from the embryo. Furthermore, the culture dish can be removed and placed in different locations, causing the time-lapse imaging to no longer match the actual embryo.

[0019] Regarding embryo viability, current incubator systems can operate on a 'set and forget' basis. In other words, a single temperature is set for the entire instrument. Furthermore, embryo development may not be enhanced during the culture process.

[0020] Current systems may also introduce varying degrees of disruption to the culture environment of biological samples. For example, a non-time-lapse 'benchtop' incubator may require periodic removal of culture dishes from the controlled environment. Regarding the specific time-lapse system disclosed in WO 2012 / 047678 (Auxogyn), this system only provides time-lapse devices, with multiple devices placed in a large incubator; therefore, no control of the incubator environment is provided for any individual patient's biological sample. For example, Unisense FertiliTech AS's Embryoscope... TM Incubation systems, collectively known as time-lapse systems, may require all culture dishes to be placed in a shared environment. Therefore, removing a patient's culture dish could affect other patient samples. Furthermore, these systems involve a single camera and a shared environment. The result could be the destruction of patient samples because the continuous movement of samples within their environment, using an instrument with only one camera, can damage the samples. Summary of the Invention

[0021] One objective of the implementation schemes described herein is to overcome or mitigate at least one of the aforementioned disadvantages of the relevant technical systems, or at least to provide a useful alternative to existing technical systems.

[0022] In a first aspect of the embodiments described herein, an apparatus for automatically evaluating cultured samples is provided, comprising at least one independently accessible module adapted to incubate at least one of a plurality of samples, wherein the at least one module is operatively associated with a light source and a movable optical detection member adapted to move about an observation axis passing through the module to scan an observation area.

[0023] The movement of the movable optical detection component can be limited to one or a combination of the following: an XY plane perpendicular to the observation axis, and a Z direction including the observation axis. Preferably, the available movement of the movable optical detection component includes the ability of the optical detection component to translate freely in an XY plane perpendicular to the optical observation direction of the optical detection component, and to have further degrees of freedom of movement in the orthogonal Z direction including the optical observation direction. In specific embodiments, the movement of the movable optical detection component can be substantially eccentric or orbital.

[0024] Preferably, the movable optical detection component is adapted to be moved via an elliptical rotating objective system or, more generally, a rotating objective system. The at least one module may include a lid and latching mechanism for sealing a culture chamber within the module and having a controlled environment. The module may include components for controlling the gas composition and temperature within at least the culture chamber for holding cultured samples. Preferably, the at least one module further includes a balancing component. The optical detection component may include one or a combination of a camera and a microscope operatively connected to the elliptical rotating objective system. A preferred device may further include a culture dish comprising a plurality of spaced-apart micropores for receiving cultured samples, wherein the culture dish is adapted to be placed within the module. Further, the device may also include an alignment component for precisely positioning the culture dish relative to the optical detection component.

[0025] In another aspect of the embodiments described herein, a method for assessing the viability of cultured samples is provided, comprising the following steps:

[0026] Biological samples are placed in a generally elliptical arrangement within the culture chamber of an independently accessible module.

[0027] Imaging a substantially elliptical individual sample using an optical detection element in the XY plane perpendicular to the observation axis passing through the module, to obtain a time-lapse record or measurement of the individual sample's development.

[0028] The method described above may further include the step of sending images of individual samples to a data processing component to obtain time-lapse records and measurements of the individual sample's development. The method may also include the step of independently controlling one or a combination of temperature, gas supply, CO2 level, and humidity within an independent culture chamber. The step of imaging individual samples preferably includes using gamete ligation as a reference point for assessing subsequent developmental events in the sample during time-lapse measurements.

[0029] Other aspects and preferred forms disclosed in the specification and / or defined in the appended claims form part of the description of the invention.

[0030] In fact, the embodiments of the present invention originate from the following implementation, assuming that a stable environment with controlled conditions can be provided and maintained for the cultured sample, wherein the viability of the cultured sample can be observed and evaluated without interfering with the development of adjacent or neighboring samples using a movable detection component.

[0031] This invention provides a modular system for the maintenance and imaging of conjugates, embryos, oocytes, and pluripotent cells, enabling high-throughput culture of these cells in a highly controlled, optimal environment. The system includes a built-in optical inspection (microscope / camera) system with image acquisition and remote processing. The optical inspection system incorporates a unique elliptical rotating objective lens that enables multi-well scanning without disturbing the developing cultured sample (e.g., embryo).

[0032] The further applicability of embodiments of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art based on this detailed description. Brief description of the attached figures

[0033] Those skilled in the art will better understand the further disclosure, objects, advantages, and aspects of preferred and other embodiments of the invention by referring to the following description of embodiments in conjunction with the accompanying drawings, which are given by way of illustration only and therefore do not limit the disclosure herein, and wherein:

[0034] Figure 1 A biological sample culture system according to a preferred embodiment of the present invention is shown;

[0035] Figure 2 The time-shifting incubator module shown according to a preferred embodiment of the present invention is displayed as removed, as... Figure 1 The biological sample culture system shown;

[0036] Figure 3 The preferred embodiment of the invention is shown. Figure 2 A cross-sectional view of the time-lapse incubator module in the diagram;

[0037] Figure 4 A camera with a rotating lens assembly according to an embodiment of the present invention is shown;

[0038] Figure 4a A preferred system for environmental control of a culture chamber according to a preferred embodiment of the present invention is shown;

[0039] Figure 5 A camera with a rotating lens assembly that moves toward a plurality of culture dishes, according to a preferred embodiment of the invention;

[0040] Figure 6 A camera with a fixed lens assembly that moves toward a plurality of culture dishes along the x and y axes, according to a preferred embodiment of the invention, is shown.

[0041] Figure 7 A rotating lens, according to a preferred embodiment of the invention, is shown moving toward each embryo position; it includes markings for identifying individual samples.

[0042] Figure 8 The simplest form of a petri dish according to a preferred embodiment of the invention is shown; the petri dish is configured to identify individual samples with labels and includes a user gripping component;

[0043] Figure 9 A petri dish of a preferred embodiment of the present invention is shown in a close-up cross-section;

[0044] Figure 9 A close-up cross-sectional view shows an alternative petri dish according to an embodiment of the present invention.

[0045] Figure 10 An improved petri dish according to one embodiment of the present invention is shown;

[0046] Figure 11 The illustration shows a petri dish with an abutment as a positioning pin according to a preferred embodiment to ensure that the petri dish is repeatedly repositioned in the correct position;

[0047] Figure 12 The image quality achievable using embodiments of the present invention is shown, wherein, Figure 12 (a) shows a 2PN embryo. Figure 12 (b) shows a 2-cell embryo. Figure 12 (c) Showing a blastocyst in the process of hatching, and Figure 12 (d) Shows hatched and incubating embryos.

[0048] Figure 13 The image shown is captured using POC2 in a preferred embodiment of the invention, a) unmasked and b) having a circular dark-field mask;

[0049] Figure 14 An alternative biological sample culture system for embryos according to another preferred embodiment of the invention is shown;

[0050] Figure 15 The time-shifting incubator module shown in the alternative embodiment according to the invention is removed, as shown below. Figure 14 The embryo culture system shown;

[0051] Figure 16 Alternative preferred embodiments according to the invention are shown as follows: Figure 15 A cross-sectional view of the incubator module over time;

[0052] Figure 17A camera with a rotating lens assembly according to an alternative embodiment of the present invention is shown;

[0053] Figure 18 Another preferred system for environmental control of a culture chamber according to an alternative embodiment of the invention is shown. Detailed description

[0054] The following terminology definitions will apply in the context of this specification.

[0055] The term "embryo" is used both to refer to the zygote formed when two haploid gametes (e.g., an unfertilized oocyte and a sperm cell) unite to form a diploid totipotent cell, such as a zygote, and to refer to the embryo resulting from immediate subsequent cell division (i.e., embryonic cleavage), and then through the morula (i.e., the 16-cell stage) and blastocyst stages (with differentiated trophectoderm and inner cell mass).

[0056] Oocytes are used to refer to unfertilized female reproductive cells or gametes.

[0057] A zygote is a single cell formed when two haploid gamete cells (e.g., an unfertilized oocyte and a sperm cell) unite to form a diploid totipotent cell.

[0058] Pluripotent cells are used to refer to any cell that is capable of differentiating into multiple cell types in an organism. Examples of pluripotent cells include stem cell oocytes and 1-cell embryos (i.e., zygotes).

[0059] Stem cells are used to refer to (a) cells or groups of cells that are capable of self-renewal and (b) have the potential to produce differentiated cell types.

[0060] Mitosis, or the mitotic cell cycle, refers to the cellular events that lead to the replication of chromosomes and the division of those chromosomes and the cytoplasm of the cell into two daughter cells. The mitotic cell cycle consists of two phases: interphase and mitotic phase.

[0061] The first cleavage event is the first division, that is, the division of the oocyte into two daughter cells, which is cell cycle 1. When the first cleavage event is completed, the embryo consists of 2 cells.

[0062] The second cleavage event is the second set of divisions, where the primary daughter cell divides into two granddaughter cells. After the second cleavage, the embryo consists of four cells.

[0063] Cytokinesis / cell division is the mitotic phase in which a cell undergoes cell division, that is, the mitotic phase in which the cell's nuclear material and its cytoplasm are divided to produce two daughter cells.

[0064] The first cytokinesis is the first cell division event after fertilization, in which the fertilized oocyte divides to produce two daughter cells. The first cytokinesis usually occurs about one day after fertilization.

[0065] Second cytokinesis is the second cell division event observed in the embryo, namely, the division of the daughter cells of the fertilized oocyte into the first group of granddaughter cells.

[0066] Reference Figure 1 Embodiments of the present invention include a device 10, which is a modular system for culturing and continuously monitoring biological or cultured samples. This device is particularly suitable for the culture and imaging of zygotes, embryos, oocytes, and pluripotent cells.

[0067] The preferred device includes multiple incubator modules 20, such as Figure 1 The device shown has a cover 13 and an open latch 12, which can independently operate and control multiple incubator modules 20, each capable of temperature monitoring and control, gas monitoring and control, microscopic observation and image capture, time-lapse image processing, and connection to external data analysis equipment.

[0068] In such Figure 3 In further detail of a preferred form shown, each module 20 has a lid 33 operated by a lid latch 32, which seals the incubation chamber 36 to isolate it from the external environment and allows independent access to the chamber 36. In effect, this provides proper removal of cultured samples without any disturbance to adjacent modules 20. This offers a significant advantage over conventional modular incubators, where all cell cultures are exposed to altered atmospheric and temperature conditions when the door / lid 33 is opened to remove the cultures. Figure 1 An example of this device, denoted as 10, is generally shown. In practice, there is no limit to the number of modules 20 that may be incorporated into each device 10. Figure 3 As shown in the details, each module includes an individual culture chamber 36 for accommodating multiple time-lapse culture dishes 39 and equilibration dishes 31, and PCBs 37 and 46 for controlling the heating of the environment. Associated with the operation of module 20 are optical detection components, which include, for example, Figure 3 As shown, there is a camera 43, a moving mechanism 42 (preferably Z-stacking and focusing Y-axis movement control), a lens positioning motor 44, and a rotating lens 41 that works in combination with a light source 34.

[0069] Reference Figure 2 Each individual incubator module 20 can be removed from the device 10 independently of other modules 20, for example, for repair or operation. The removal of module 20 does not affect the functionality of other modules 20 in the device 10. Figure 3An embodiment of an incubator module 20 intended for use within device 10 is shown, which can be securely placed within said device 10. The internal ambient temperature of each incubation chamber 36 is controlled to a predetermined value using heaters 37, 46 and temperature sensors. In a preferred embodiment, two heaters are used to heat the chambers, one located on the lid 37 and the other on the stage 46. In a preferred embodiment, the temperature is set to 37°C. Each module 20 is provided with an inlet for a gas supply 38 and a valve for maintaining a predetermined gas flow rate. In a preferred embodiment, a premixed gas, typically composed of one or a combination of oxygen, carbon dioxide, and nitrogen, is supplied into the incubation chamber 36 via said inlet and valve.

[0070] In another embodiment, gases (typically oxygen, carbon dioxide, and nitrogen) are supplied to the device via separate inlets and mixed on a board before being supplied to the incubation chamber 36. In this embodiment, the mixing can provide an atmosphere consisting of approximately 5% oxygen, approximately 6% carbon dioxide, and approximately 89% nitrogen; in a further embodiment, the gases are mixed to provide an atmosphere consisting of approximately 20% oxygen, approximately 5% carbon dioxide, and approximately 75% nitrogen.

[0071] The gas can be humidified before being supplied to the incubation chamber 36, with the aim of maintaining a humid atmosphere in the chamber. Figure 4a In this process, gas flows through a tube into the aqueous solution in the vial. The moistened gas then flows upwards from the vial into a controlled incubation chamber. (Example:) Figure 4a As shown, humidification of the gas is achieved by supplying gas directly through a pipe into a small bottle containing a water-based solution. Figure 4a The portion of module 20 shown includes a vial 53 containing an aqueous solution. Moisturizing gas rises through the aqueous solution in vial 53 into an incubation chamber or individual culture chamber 52. In one embodiment, the aqueous solution consists only of water. In an alternative embodiment, the aqueous solution may contain water and an additive such as glycerol. An optical sensor 54 is attached to the end of the tube or located within vial 53 to detect the presence of air bubbles, thereby ensuring no gas blockage.

[0072] Each module 20 is equipped with an object holder in which the cell culture dish can remain substantially stationary during culture, allowing for consistent observation and imaging of cells or tissues. (See reference) Figure 11In a preferred embodiment, the precise positioning of the petri dish is achieved using an alignment member or abutment 111, for example, in the form of three locating pins and a movable latch. In other embodiments, the object holder may include any number of locating pins and / or latches 111. The object holder has an opening or window through which light can be transmitted to the microscope objective.

[0073] Each module 20 includes an area within the incubation chamber for additional culture dishes. This culture dish area does not allow microscopic observation of cell cultures that may be contained within the dishes, but allows the user to culture unmonitored samples or to equilibrate the culture medium before using cell cultures.

[0074] Each module is provided with an optical detection component, which may include one or a combination of a camera system or microscope for monitoring cultured samples, cells, or tissues. The microscope or camera system may be any suitable design known in the art. In a preferred embodiment, the microscope is a simple tube microscope. In alternative embodiments, the microscope design may be selected from any of the following: a simple tube microscope, a Hoffman modulation phase-contrast microscope, a differential interference contrast microscope, a dark-field microscope, or a phase-contrast microscope.

[0075] In examples of microscope use, monocular microscopes include a 10x objective lens, a spacer tube with a light aperture or hole, and a CMOS sensor for image capture. In one embodiment, a diffuser and a circular aperture are positioned between the light source and the sample to illuminate the sample with oblique light and provide increased contrast in the captured image. Additionally, extra filters or diffusion masks can be introduced into the light path as needed. In a preferred embodiment, a condenser lens system can be used to enhance the uniformity of light illuminating the sample. The optical design within the preferred embodiment provides sufficient contrast in the acquired images to identify features of the cultured sample, such as polar bodies, pronuclei, nucleoli, and inner cell mass (ICM), in addition to events such as cleavage, blastocyst expansion, and hatching. In an alternative embodiment, the optical detection components include an image sensor, such as a CCD camera.

[0076] Preferably, each microscope is equipped with an objective lens positioning motor for automatic and / or manual focusing.

[0077] In this embodiment, the microscope's illumination source is provided by a light-emitting diode (LED) with a wavelength of 550 nm and variable intensity. In other embodiments, the light source may have different wavelengths. As those skilled in the art will understand, the wavelength and power output of the illumination source can be selected to minimize phototoxic damage or stress to the cultured sample, cells, or tissue of interest. To further minimize illumination-related stress, it is preferable to turn on the illumination source only during observation or imaging during the culture process. Images captured by sensors of the optical detection components can be processed and analyzed by external data processing or computer systems, or by image processing components associated with the operation of the device, and in some embodiments by image processing components within the device itself.

[0078] A particularly advantageous feature of embodiments of the present invention is that it provides an elliptical rotating objective lens system as part of the optical inspection component of a microscope and / or camera, thereby providing eccentric movement of the optical inspection component capable of scanning the area of ​​observation. Figure 4 An exemplary drive mechanism for elliptical rotation is shown. The advantage of this innovation is that multiple embryos or biological samples can be imaged without moving the culture container. Figure 4 As shown, a camera 43 is housed within a camera holder 51 having a spacer tube 49 leading to a motor belt assembly, the motor belt assembly including a motor belt 48 driven by a lens positioning motor 44, which provides movement of the objective lens 47. Figure 4 The rotating lens assembly provides eccentric movement to achieve scanning of the imaging area. The ability to move the objective lens in this manner while maintaining good image quality depends on using a low-power objective lens and being assisted by a side illumination path. In a preferred embodiment, lens movement can be facilitated by a simple stepper motor.

[0079] Figure 5 Another embodiment of the invention is shown, wherein multiple culture containers or time-lapse dishes 57 are included within a modular device. In this embodiment, a microscope / ellipse drive mechanism with a rotating lens assembly 56 is moved along a guide mechanism to acquire images from multiple culture containers without disturbing the containers. Typically, such a drive mechanism enables movement in two directions (X & Y), thereby achieving fine-scale control over image positioning and quality, such as... Figure 6 As shown.

[0080] Figure 7 An exemplary movement of the rotating lens is shown, which enables the optical detection component to be positioned at each of multiple culture sample locations on a time-lapse culture dish. For example, this allows for the detection of several embryos in a conditioned environment. Figure 8An exemplary culture dish 90 is shown, which contains multiple culture sample wells 103 for time-lapse detection of culture sample dishes, and prep wells 94 provide flexibility for users to prepare media or embryos. Furthermore, Figure 9 Given Figure 8 A close-up of the disassembled culture dish shows the culture sample well 103, with a fluid control wall 91, divots 92 for positioning the cultured sample (e.g., an embryo), and markers 90 for identifying the individual samples.

[0081] Figure 9 A provides an exploded close-up of the improved culture sample wells, showing the fluid control wall 91, channel 93, and divot 92 for positioning the cultured sample (e.g., embryo).

[0082] Figure 10 and 11 An improved petri dish design is shown, in which a user gripping area 101 is further provided along the marked area 102. Furthermore, Figure 11 A preferred component is shown, by which embodiments of the invention can provide precise positioning and repositioning of the petri dish within device 10 for reliable optical inspection. For example... Figure 11 As shown, alignment member 111 or base is provided in the form of a locating pin to ensure that the dish can be repeatedly repositioned in the correct position. Alternative alignment members, such as stops, indentations or other equivalents, may be used within or operatively associated with the support base or wall of the chamber to provide accurate repositioning.

[0083] Examples of optical inspections that can be implemented by embodiments of the present invention are shown in Figure 12 and 13 As shown in the image. For example, Figure 13 This demonstrates the difference between images captured without a masking system and those captured using a circular dark-field-style stop.

[0084] like Figures 7 to 11 As specifically shown, a preferred embodiment of the invention also provides a culture dish comprising a basic structure containing multiple micropores for culturing samples, such as conjugates, embryos, oocytes, and pluripotent cells. The culture dish also includes usability-enhancing components, as described above, for allowing precise positioning of the culture dish within a modular device and improving patient safety.

[0085] The culture dish is designed to work, for example, with the modular instrument described in co-pending Australian Provisional Patent Application No. 2013900039 for the maintenance and imaging of conjugates, embryos, oocytes, and pluripotent cells, enabling high-throughput culture of these cells in a highly controlled, optimal environment. It includes a built-in microscope system with image acquisition and remote processing capabilities. The microscope system includes a unique elliptical rotating objective lens capable of performing multi-well scanning without disturbing the developing embryo.

[0086] exist Figure 8 The image shows an embodiment of the simplest form of a culture dish. The simplest form of a culture dish comprises a basic structure with multiple microwells for culturing samples such as zygotes, embryos, oocytes, and pluripotent cells. (See reference...) Figure 8 , Figure 9 and Figure 9 In a preferred embodiment, the micropores of this basic structure are arranged in a ring pattern, each micropore positioned at the bottom of channel 93 to allow the introduction of culture medium. These structures are surrounded by fluid control walls 91, which provide fluid control to maintain the culture medium in a desired area of ​​the culture dish. The bottom of channel 93 can slope upwards from the micropores to the fluid control walls 91, allowing gravity to help embryos move toward the micropores (if placed on this surface). The micropores have sufficient depth and geometry to ensure that embryos do not migrate out of the pores during transport of the culture dish or during aspiration or dispensing of the culture medium, while other embryos are placed or moved. Figure 9 A shows these characteristics in more detail. The culture medium can then be covered with a suitable oil, which is retained by the walls of the petri dish to limit evaporation of the medium during incubation.

[0087] The simplest form of this invention features a feature that allows the culture dish to be easily filled with culture medium and retained in the desired area. During culture, the culture medium can be removed from below the oil layer and replaced as needed, avoiding the need to equilibrate fresh media and transfer embryos to new dishes. Micropores ensure that embryos remain in a position that allows for observation using modular instruments in the preferred form of this invention and individual identification of the embryos. The design of the culture dish ensures that embryos can be observed using a stereomicroscope or inverted microscope using the preferred embodiment of the modular instruments of this invention. Because the material of the culture dish is transparent, embryos can be monitored without removing the cap.

[0088] In a preferred embodiment, the simplest form of the petri dish is incorporated into an improved design, such as... Figure 10 and Figure 11As shown. This embodiment has numerous usability-enhancing components that allow for precise positioning of the culture dish within the modular device and improve patient safety. The culture dish is provided with several gripping areas 101, which allow for safe handling of the culture dish in many configurations. A large area 102 provides a label location to ensure clear patient identification and traceability. Preferably, the culture dish is designed in such a way that it can only be placed in the modular instrument in one orientation, ensuring proper identification and visualization of the cultured sample (e.g., embryo) using the modular instrument. This is achieved by using components 111 on the culture dish aligned with positioning pins and latches on the modular instrument. The system also ensures precise positioning of the culture dish within the instrument. As described above, these components... Figure 10 and Figure 11 The figures shown are not identical to those depicted, but it will be apparent to any person skilled in the art that they may differ from these depictions.

[0089] In a preferred embodiment, the culture dish is constructed of a single type of plastic, preferably polystyrene. In alternative embodiments, any plastic used can be constructed for culture dishes that any person skilled in the art would consider suitable for conjugates, embryos, oocytes, and pluripotent cells. In further embodiments, all or some surfaces of the plastic culture dish can be treated using a process suitable for cell culture vessels, such as plasma treatment. Furthermore, the purpose of this surface treatment can be to improve surface wettability, thereby enhancing the filling of the culture dish with the culture medium. In alternative embodiments, the above-described improved design of the culture dish can be constructed from a variety of different types of plastics, wherein... Figure 8 The part depicted is constructed from one type of plastic, while the rest is constructed from another type.

[0090] In a preferred embodiment, the micropores used in this invention should meet the following requirements, which have the following advantages:

[0091] • Independent identification and grouping of cultures should be permitted.

[0092] • The micropores should be arranged in a circle or grouped around a circle for observation with a rotating lens.

[0093] • Sufficient depth / geometry to keep the embryo in the hole during disturbance.

[0094] • Components used to position petri dishes within the instrument.

[0095] • Allows for unique orientations.

[0096] • Accurate and precise positioning.

[0097] • Components designed for easy and safe operation - reducing the chance of spills.

[0098] • Fluid control wall used to hold the medium.

[0099] • Oil control wall.

[0100] • Preferably, the inclined walls are provided to assist the cultured sample in falling into the well.

[0101] • Markings / steps on the walls of the aperture to assist autofocus.

[0102] • Media replacement.

[0103] • Minimize the number of remaining parts in the petri dish.

[0104] • Components that enhance the flow of the medium through the "channel".

[0105] In a particularly preferred embodiment, the invention is used as a modular system for maintaining and imaging conjugates, embryos, oocytes, and pluripotent cells. Therefore, the device is provided as comprising modules, each module including components for maintaining suitable gas and temperature conditions conducive to cell viability, components for balancing chamber humidity, a microscope unit intended for use in the culture space, an elliptical drive mechanism capable of imaging multiple fields of view, an image capture unit, and components for transmitting images for further processing.

[0106] The system includes an image processing component integrated into the device.

[0107] Furthermore, a preferred embodiment provides a method for transmitting images of cells or tissues located in a culture space to a data processing component, comprising the following steps:

[0108] Cells or tissues are placed in culture containers on the shelf of the microscope / incubator module.

[0109] The microscope / incubator module is arranged inside the module shell.

[0110] Keeping cells or tissues essentially still during incubation

[0111] Imaging of individual cells or tissues within a culture vessel using an elliptical drive path lens system.

[0112] In other aspects, the preferred embodiment uses gamete fusion as a reference point for the timing assessment of subsequent embryonic developmental events. In this respect, using gamete fusion as a reference point for the timing assessment of subsequent embryonic developmental events provides an estimate of viability based on the assessment of embryos in culture, and it can be argued that this allows for more precise event timing compared to currently known IVF methods. Therefore, event timing derived from gamete fusion can improve the analysis of embryonic development.

[0113] In other aspects, prior to implantation back into the patient, the preferred implementation uses time-lapse as a measure to assess embryo expansion and viability during thawing. Time-lapse imaging is used to assess the viability of thawed embryos based on characteristics such as expansion. This novel approach to assessing thawed embryos has the potential to lead to improvements in the selection of optimal embryos.

[0114] This invention provides an embryo assessment capability that is easy to use and reduces the time spent by embryologists in evaluating embryos. In this regard, existing systems utilize complex assessment methods that require a significant time investment from embryologists. The preferred system according to the invention can utilize one or a combination of the following.

[0115] The creation or generation of a highlights package that displays images of embryonic development over time. This package can be user-defined, automatically generated based on identified events, or automatically generated based on measurements of embryonic structures / components. It can "bookend" a period using images from the beginning and end of a predefined time period or window to determine whether an event or combination of events occurred during that period.

[0116] Video clips can also be generated from key stages of embryonic development (such as gamete mating, cleavage, and blastocyst formation). Clips are edited based on default values ​​and / or user-defined "time windows." These clips are then evaluated individually or together according to the user's discretion.

[0117] Red, amber, and green can be used to determine fate and to record important events, such as choosing red to indicate that the embryo is not viable, choosing amber when adverse reactions are observed, and green to indicate good development.

[0118] Evaluations can be conducted during the training process (immediate evaluations), such as daily or when anticipated significant events have occurred. A summary of all events can then be provided at the end of the fate selection process.

[0119] The user interface is arranged to match the physical arrangement of the petri dishes to minimize errors and reduce the chance of selecting the wrong item. The physical layout of the chambers is reflected on the large display screen. The circular embryo layout of the petri dishes is represented by a circular arrangement on the large display screen.

[0120] RFID, barcodes, OCR, or other identifiers that can be read electronically (or optically and convertibly) on or in the culture dish can be used. This system is able to acquire all data associated with the culture dish (and therefore the embryo), thereby minimizing labeling errors and potential confusion that could lead to the transfer of the wrong embryo to the patient. The system can be used to correlate all time-lapse images with the correct patient ID.

[0121] Each room has an independent display screen that can present information such as, but not limited to, patient ID, environmental conditions, alarm status, warnings, or combinations thereof. The presentation of the patient ID on the screen minimizes the risk of potential confusion, in which case the wrong embryo might be transferred to the patient. The display screen can be an LCD screen, electronic paper, or other electronic display device. Preferably, it is an LCD screen.

[0122] During the culture process, the environment in each chamber can be automatically controlled and altered according to the overall situation, including temperature, gases, humidity, movement, sound, or combinations thereof. This automated process provides the opportunity to optimize embryo conditions throughout the culture period. The system can be implemented in the following ways.

[0123] Users predefine a general environmental profile for a given set of instruments and then apply it to all embryos cultured in that set of instruments. This profile can be based on physiological rhythm cycles.

[0124] The environmental profile is customized for each individual patient by the user. This customization can be based on patient measurements and / or observations, such as body temperature.

[0125] In addition, this customized profile can be automatically generated by the system using data provided by the patient, which may have been collected using some type of application or recorder.

[0126] Automated analysis of time-lapse images of patient embryos generates and / or modifies environmental profiles “in real time” during culture.

[0127] Images can be time-shifted for parallel capture across all chambers, thus reducing the time lag between images within the z-stack. This is important when traversing the z-stack to search for, for example, gamete ligations. This is also achieved by using a USB hub within the instrument, allowing multiple cameras to be connected to a PC via a single connection. The preferred number of cameras and chambers for parallel capture is six. In a preferred embodiment, the PC is included within the system.

[0128] It can provide many other functions, including:

[0129] Humidity is controlled independently in each room.

[0130] Each room has its own independent gas supply.

[0131] • Bubble detection is used to determine gas flow, thus preventing blockages.

[0132] • Controlling the positioning of the culture dish

[0133] Door latch lock

[0134] • CO2 sensing. Preferably, each chamber has at least one dedicated CO2 sensor.

[0135] • Condensing lens system for enhancing contrast and uniform illumination across all micropores

[0136] • Cover positioning pins are used to ensure the correct position of the lighting source, wherein the cover positioning pins in the platform of the room are used to ensure the correct position of the cover and the lighting source.

[0137] • Using PCB heating in embryo incubators

[0138] • As an institution integrating up to six cameras into a single system, it is conceivable that a USB hub could be provided within the instrument to allow all six cameras to be connected to a PC via a single connection. This could potentially enable the simultaneous management of cameras as a single system.

[0139] To minimize the time between z-stack and image capture, parallel image capture across six modules is provided. This reduces the time difference between images within the z-stack. This is important when traversing the z-stack to find, for example, gamete coordination.

[0140] In existing technologies, the time required to illuminate the embryo is much longer than the time needed for image capture. To address this issue, scheduling software can minimize LED on-time and bandwidth usage. One way to achieve this is by simultaneously adjusting the view while the illumination is turned off.

[0141] • Generate highlight packages based on user-defined events and / or automatically identified events.

[0142] • User-defined focal planes during playback, i.e., in this case, the focal plane may be located at a single position throughout the playback. In current systems, it is possible that the Z-stack can only be acquired intermittently, for example, the Z-stack is only acquired once every fourth image. As a solution, it is preferable to use images from previously captured Z-stacks during time-lapse playback to achieve viewing multiple focal planes. Advantageously, this ensures that the embryo is always in focus during playback, even if the embryo moves or grows. This also allows the user to analyze different parts of the embryo throughout its development. As a means of 'instantaneous' focusing, the user can manually adjust the focus during playback.

[0143] • Automatic focal plane selection during playback – Automatic focal plane selection during playback can be achieved based on time-lapse analysis. In this sense, the system automatically selects the focal plane to ensure the embryo remains in focus.

[0144] • Methods for determining cleavage can help identify events or absolute detection.

[0145] • 3D imaging is provided by merging z-stack images. Therefore, z-stack images are processed and transformed to provide 3D images of the embryo.

[0146] Environmental conditions are controlled independently in each chamber, such as independently controlling one or a combination of temperature, humidity, and / or gas supply in each chamber, thus enabling customized conditions for each patient. Furthermore, if one chamber malfunctions for any reason, the embryos of all other patients will not be affected.

[0147] A mechanism is provided by which embryos can be gently moved, such as, but not limited to, a platform or portion thereof in a moving / tilting incubation chamber, to achieve micro-movement or tilting of the platform / medium to mimic the in vivo microenvironment of oocytes / embryos. Therefore, it is possible to improve embryo culture performance by mimicking the in vivo microenvironment.

[0148] A mechanism can also be provided for rolling the embryo through the aperture for better evaluation. This allows the user to interact with the embryo to observe features that were not visible in the images prior to this operation.

[0149] Similarly, a mechanism could be provided through which embryos are exposed to sound and / or music within an incubation chamber. Therefore, exposing embryos to sound / music could potentially improve embryo culture performance.

[0150] To avoid errors or mistakes that may be introduced by human intervention, it is possible to embed electronically (or optically and convertibly) readable RFID, barcodes, or other identifiers in the petri dishes. Using such identifiers, all data associated with the petri dish can be accessed from a database when the dish is placed in the instrument.

[0151] Similarly, the instrument can associate all images (and other recorded data) with the corresponding patient ID, thereby avoiding human error in the entries of patient details associated with the images.

[0152] The instrument can also be equipped with a small display screen that connects to the display information of the culture dish being evaluated, such as the patient's name, individual environmental conditions, and / or other parameters related to the monitoring of room or alarm conditions. Providing this component means that the user does not need to interact with the instrument to understand its current status and does not need to externally record the information on a whiteboard or similar surface.

[0153] The inventors have noted that data on the passage of time and other recorded information is typically stored outside of laboratory databases. Preferably, directly outputting the data to a laboratory database will enable more uses for the data, simplify data access, and ensure a consistent approach to data access.

[0154] Additionally, environmental conditions can be automatically controlled during the incubation period, where the environment of each chamber can be altered throughout the incubation period, including temperature, gases, humidity, movement, sound, or combinations thereof. For example, the following conditions can be utilized:

[0155] Users can predefine profiles for all patients at a given location / clinic, whereby users set a common environmental profile for a given set of instruments and then apply it to all embryos cultured in that set of instruments.

[0156] Overviews can be based on physiological rhythm temperature cycles, using precise temperature control to simulate the in vivo microenvironment of oocytes and embryos.

[0157] A profile can be customized for an individual patient, where the environmental profile is set by the user and customized for the individual patient.

[0158] A summary can be based on the patient’s measurements and / or observations, such as body temperature.

[0159] The profile can be donor-based, aided by data obtained from the donor (application, recorder, etc.), wherein the customized profile is based on patient measurements and / or observations and is automatically generated by the system.

[0160] The profile can be based on the automated analysis, measurement, and / or observation of time-lapse images of the embryos recorded during culture, wherein the environmental profile is generated and / or modified “instantaneously” based on the automated analysis of time-lapse images of the patient’s embryos during culture.

[0161] The inventors recognized that the auditing and tracking of embryo interactions is sparse and paper-based. Sample security is also a significant concern. In this regard, for purposes such as QC, electronic signatures, and witnessing, the embryologist's identification can be recorded each time they interact with the system. Preferably, each embryologist is identified using one or a combination of RFID, barcodes (or other optically identifiable IDs), fingerprints on a scanner, retinal scans, or entered PINs, and all interactions with the instrument they interact with are recorded. Advantageously, auditing is provided for who, when, and what kind of interaction was performed on each culture dish. Advantageously, only appropriate interactions are allowed for each user of the instrument.

[0162] To expand on the above concept, in a preferred embodiment, embryologists can have their fingerprints scanned when they visit the incubator. Again, each embryologist uses one or a combination of RFID, barcodes (or other optically identifiable IDs), fingerprints on a scanner, retinal scans, or entered PINs to interact with the instruments approved for their use.

[0163] In the prior art, complex optics may be required to obtain good quality images. Therefore, in a preferred embodiment, the inventors have provided a condenser lens system for providing enhanced contrast and uniform illumination across all microapertures, which can be provided by a simple condenser lens system for providing enhanced contrast and uniform illumination across all microapertures.

[0164] Currently, training materials are assembled manually. However, by automatically generating QC training packages using recorded assessment / grading results, training can be conducted effortlessly. Furthermore, there is often insufficient QC training. The automatic generation of a QC image library defined by users regarding embryo events / types will automatically generate emails / internal websites, reminding all embryologists in the clinic to complete routine QC and training objectives.

[0165] In this system, patients cannot see the development of their embryos. However, in a preferred embodiment, remote monitoring is provided to patients using secure network interaction, allowing them to remotely observe approved embryos and thus view their development. In one preferred embodiment, a backup heater component is provided for each PCB, utilizing two heater circuits so that if one fails, the other can take over. The PCB circuitry has internal redundancy to ensure continued control of the ambient temperature even in the event of a heater failure. This can be automatically controlled via software to ensure that the embryonic environment is not compromised.

[0166] The petri dishes are designed to facilitate media preparation and replacement. Figure 8 In this design, the petri dish is equipped with a spare processing well for media preparation. Figure 9 In A, the culture dish is designed with a fluid control barrier 91 and channels 93 adjacent to the void 9 to facilitate the removal and replacement of the culture medium, while the embryos remain in the dish. Therefore, during the culture cycle, there is no need to move the embryos to a new culture dish, thus reducing disruption to their development. The design minimizes media residue while ensuring the embryos do not "dry out."

[0167] Modular software can be structured to allow firmware (F / W) upgrades for one module at a time and to be scheduled whenever an opportunity arises (i.e., during module downtime).

[0168] While the Z-stack is only applicable to image capture optimized for certain time-lapses, it allows all time-lapses to include the Z-stack. Therefore, the Z-stack can be observed for any frame of the time-lapse, and video playback can be performed at any point on the Z-stack. Furthermore, video playback can be performed using a Z-stack overview.

[0169] Furthermore, in the current system, events can only be found by viewing long videos. A semaphore of difference (difference between consecutive images) is established by plotting the graphical differences between time-lapse frames over time. This reduces the evaluation time.

[0170] Because recorded images consume a large amount of storage space, time-lapse images use one or a combination of temporal and spatial (intra-image and across the z-stack) compression in order to reduce the storage space required for time-lapse images.

[0171] The water level in the humidifier bottle can be measured using a liquid level sensor. This ensures that the water does not run out, resulting in a low humidity environment.

[0172] Precise positioning of the petri dish is achieved using alignment components or abutments 111, for example, in the form of three locating pins and a movable latch.

[0173] Furthermore, as a latch / locking mechanism, each module has a cover operated by a latch, which seals the incubation chamber relative to the external environment and allows independent access to the chamber. In this way, each chamber is completely sealed relative to the external environment, thereby ensuring that external influences are minimized and that gas concentrations are maintained at stable levels.

[0174] In existing systems, it may be unclear which physical item(s) is represented by which item(s) on the display screen. A preferred solution is to match the physical layout of the GUI to minimize errors. This is achieved by reflecting the chamber layout sequence onto a large display. The circular embryo layout of the culture dish is represented by a circular arrangement on the large display screen, which reduces the chance of selecting the incorrect item.

[0175] Alternative systems for culturing biological samples, including alternative embodiments of the present invention, are shown in [the figure]. Figures 14 to 18 In the middle, the same label is used to refer to Figures 1 to 11 The same components as the implementation scheme.

[0176] Reference Figure 14 Alternative embodiments of the present invention include a device, a biosampling device 10, which is similar to... Figure 1 The implementation scheme is a modular system for culturing or continuously monitoring biological samples or cultured samples and is particularly suitable for the culture and imaging of zygotes, embryos, oocytes and pluripotent cells.

[0177] Preferred devices include those having, for example Figure 14 The multiple incubator modules 20 shown, including the lid 13 and the open latch 12, can be operated and controlled independently. Each module is capable of monitoring and controlling temperature, monitoring and controlling gas, microscopic observation and image capture, time-lapse image processing, and connection to external data analysis equipment.

[0178] In such Figure 16 In further detail of a preferred form shown, each module 20 has a cover 33 operated by a cover latch 32, which seals the incubation chamber 36 relative to the external environment and allows independent access to the chamber 36. Figure 3 The moving mechanism 42 (preferably Z-stack and focused Y-axis movement control) is not shown.

[0179] Figure 17 It shows Figure 4 An alternative implementation of the camera assembly requires a spacer tube to ensure the CCD camera is positioned at the correct distance for optimal focusing.

[0180] exist Figure 18 It shows Figure 4a The alternative embodiment shown illustrates humidification of the gas by supplying the gas directly through a pipe into a vial containing an aqueous solution. Similar to... Figure 4a The diagram, Figure 18 A portion of module 20 is shown, comprising a vial 53 containing an aqueous solution. Moist gas rises through the aqueous solution in vial 53 into an incubation chamber or individual culture chamber 52. In one embodiment, the aqueous solution consists only of water. In an alternative embodiment, the aqueous solution may contain water and an additive such as glycerol. An optical sensor 54 is attached to the end of the tube or to the vial 53 to detect the presence of air bubbles, thereby ensuring no gas blockage.

[0181] Regarding minimizing patient confusion, the embodiments of the present invention can be used for:

[0182] Using culture dishes incorporating RFID, barcodes, or OCR enhances embryo and patient safety, allowing devices to automatically and programmatically read patient details, ensuring no patient confusion and no image confusion. This is achieved through a reading method that does not require user input of patient details.

[0183] The instrument reads and displays patient information on a separate LCD screen.

[0184] Regarding the improvement of embryo viability, the embodiments of the present invention can be used for:

[0185] This allows for individual control of each patient room (temperature, humidity, and gas).

[0186] Feedback from time-lapse images is fed directly into the incubator to customize the optimal development environment by adjusting temperature, humidity, and gas concentration levels.

[0187] o Utilizing sound and vibration to further enhance embryonic development, that is, utilizing / stimulating physiological rhythms.

[0188] o Customize optimal conditions for embryos based on feedback over time (sound, vibration, temperature, humidity, and gas).

[0189] o Combine bright field and dark field to improve the ability of embryo assessment.

[0190] Regarding minimizing environmental interference, the embodiments of the present invention can be used for:

[0191] Each patient has an independent environment and camera.

[0192] The patient samples were stationary during incubation.

[0193] The improved media replacement technology facilitates the removal and replacement of the culture medium while the embryo remains in the culture dish. This, in turn, allows for the automatic replacement of the culture medium into the instrument.

[0194] With automated media changing, the instrument has the potential to use feedback from over time to tailor optimal media conditions for embryos and further reduce patient sample interference.

[0195] While the invention has been described in conjunction with specific embodiments thereof, it should be understood that further modifications (in many places) are possible. This application is intended to cover any variations or modifications of the invention that generally follow the principles of the invention and include those falling within the field of the invention and known and customary practice that may deviate from the basic features set forth above.

[0196] Since the present invention can be implemented in several forms without departing from the essential spirit of the invention, it should be understood that the above embodiments do not limit the invention, unless otherwise stated, but should be interpreted broadly within the spirit and scope of the invention as defined by the appended claims. The described embodiments are to be considered in all respects only illustratively and not restrictively.

[0197] Various modifications and equivalent arrangements are intended to be included within the spirit and scope of this invention and the appended claims. Therefore, the specific embodiments should be understood as illustrations of many aspects in which the principles of the invention can be implemented. In the following claims, the means plus function clauses are intended to cover structures that perform the defined function, not only structural equivalents but also equivalent structures. For example, although nails and screws may not be structural equivalents because nails use a cylindrical surface to hold wooden parts together, while screws use a helical surface to hold wooden parts together, in the context of fastening wooden parts, nails and screws are equivalent structures.

[0198] It should be noted that when the terms "server," "secure server," or similar terms are used herein, communication devices that can be used in a communication system are described, unless the context requires otherwise, and should not be construed as limiting the invention to any particular type of communication device. Therefore, communication devices may include, but are not limited to, bridges, routers, bridging routers (routers), switches, nodes, or other communication devices, which may or may not be secure. Furthermore, those skilled in the art will understand that other software packages or applications may be utilized for possible implementations that will include cloud-based systems.

[0199] It should also be noted that while flowcharts are used herein to illustrate various aspects of the invention, this should not be construed as limiting the invention to any particular logical flow or implementation. The described logic may be divided into different logical blocks (e.g., programs, modules, functions, or subroutines) without altering the overall result or otherwise deviating from the true scope of the invention. Typically, logical elements may be added, modified, omitted, executed in a different order, or implemented using different logical structures (e.g., logic gates, loop primitives, conditional logic, and other logical structures) without altering the overall result or otherwise deviating from the true scope of the invention.

[0200] Various embodiments of the present invention can be embodied in many different forms, including for processors (e.g., microprocessors, microcontrollers, digital signal processors, or general-purpose computers), and in this regard, any commercial processor can be used to implement embodiments of the present invention, as a single processor in a system, or a group of processors in serial or parallel fashion. Therefore, examples of commercial processors include, but are not limited to, Merced. TM , galloping TM Pentium II TM Supreme TM Cygnus TM Pentium Pro TM Efficeon TM Velociraptor TM AMD TM Computer program logic for programmable logic devices (e.g., field-programmable gate arrays (FPGAs) or other PLDs), discrete components, integrated circuits (e.g., application-specific integrated circuits (ASICs)), or any other means, including any combination thereof. In an exemplary embodiment of the invention, the dominant approach is that all communication between the user and the server is implemented as a set of computer program instructions, which are translated into a computer-executable form, stored in a computer-readable medium, and executed by a microprocessor under the control of an operating system.

[0201] The computer program logic that implements all or part of the functions described herein can be manifested in various forms, including source code, computer executable, and various intermediate forms (e.g., forms produced by an assembler, compiler, linker, or locator). The source code may include a set of computer program instructions that can be executed in any of the following programming languages ​​(e.g., object code, assembly language, or high-level languages ​​such as Fortran, C, C++, JAVA, or HTML. Furthermore, there are hundreds of available computer languages ​​that can be used to implement embodiments of the invention, more commonly Ada; Algol; APL; awk; Basic; C; C++; Conol; Delphi; Eiffel; Euphoria; Forth; Fortran; HTML; Icon; Java; Javascript; Lisp; Logo; Mathematica; MatLab; Miranda; Modula-2; Oberon; Pascal; Perl; PL / I; Prolog; Python; Rexx; SAS; Scheme; sed; Simula; Smalltalk; Snobol; SQL; Visual Basic; Visual C++; Linux and XML, QT, Python.) for use with various operating systems or operating environments. The source code can define and use various data structures and communication messages. Source code can be in a computer-executable form (e.g., via an interpreter), or source code can be transformed (e.g., via a translator, assembler, or compiler) into a computer-executable form.

[0202] Computer programs can be permanently or temporarily embedded in tangible storage media in any form (e.g., source code, computer-executable, or intermediate form), such as semiconductor storage devices (e.g., RAM, ROM, PROM, EEPROM, or flash programmable RAM), magnetic storage devices (e.g., magnetic disks or fixed disks), optical storage devices (e.g., CD-ROMs or DVD-ROMs), PC cards (e.g., PCMCIA cards), or other storage devices. Computer programs can be embedded in signals that can be transmitted to a computer using any of the various communication technologies available, including but not limited to analog, digital, optical, wireless (e.g., Bluetooth), networking, and internetworking technologies. The computer program can be distributed as a removable storage medium in any form, accompanied by printed or electronic documentation (e.g., shrink-wrapped software), pre-loaded with a computer system (e.g., on a system ROM or fixed disk), or distributed by a server or electronic bulletin board on a communication system (e.g., the Internet or the World Wide Web).

[0203] The hardware logic (including programmable logic for programmable logic devices) that performs all or part of the functionality described herein can be designed using conventional manual methods, or it can be designed, captured, simulated, or electronically archived using various tools such as computer-aided design (CAD), hardware description languages ​​(e.g., VHDL or AHDL), or PLD programming languages ​​(e.g., PALASM, ABEL, or CUPL). The hardware logic can also be incorporated into a display screen for implementing embodiments of the invention, and this can be a segmented display screen, analog display screen, digital display screen, CRT, LED screen, plasma screen, liquid crystal diode screen, etc.

[0204] Programmable logic can be permanently or temporarily embedded in tangible storage media, such as semiconductor storage devices (e.g., RAM, ROM, PROM, EEPROM, or flash programmable RAM), magnetic storage devices (e.g., floppy disks or fixed disks), optical storage devices (e.g., CD-ROMs or DVD-ROMs), or other storage devices. Programmable logic can be embedded in any form in signals that can be transmitted to a computer using any of the various communication technologies available, including but not limited to analog, digital, optical, wireless (e.g., Bluetooth), networking, and internet technologies. Programmable logic can be distributed as a removable storage medium accompanied by printed or electronic documentation (e.g., shrink-wrapped software), pre-loaded with a computer system (e.g., on a system ROM or fixed disk), or distributed by a server or electronic bulletin board on a communication system (e.g., the internet or the world wide web).

[0205] The terms “comprising” and “includes” are used in this specification to indicate the presence of the stated feature, integer, step, or component, but do not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. Therefore, unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “including,” “including,” “comprising,” etc., are interpreted as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.”

[0206] References

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Claims

1. A time-lapse measurement device for evaluating the viability of cultured samples, the device comprising a plurality of independently accessible modules, each of the independently accessible modules being removable from the device independently of the other independently accessible modules without affecting the functionality of the other independently accessible modules, each of the independently accessible modules forming a culture chamber with a controlled environment, the culture chamber being adapted to incubate a plurality of individually identifiable samples placed in a substantially elliptical arrangement via at least one culture dish, the culture dish comprising a plurality of spaced microwells for accommodating the individually cultured samples, wherein, Each of a plurality of independently accessible modules is operatively associated with a light source and an optical detection component located outside the controlled environment and along the observation axis of the module. The optical detection component is adapted for movement limited to one or a combination of the following: an XY plane perpendicular to the observation axis and a Z direction including the observation axis. The optical detection component includes an objective lens system adapted for elliptical rotational movement of the objective lens independently of the optical detection component, thereby enabling scanning of an observation area around the observation axis encompassing each of the plurality of individually identifiable samples. The movement of the optical detection component, which is combined with the elliptical rotating objective lens, is essentially eccentric or orbital. The optical detection component includes a camera supported and spaced apart by a motor belt assembly, the motor belt assembly providing elliptical rotational motion of the objective lens.

2. The apparatus according to claim 1, wherein, The optical detection component includes a condenser lens system for enhancing the contrast and uniform illumination of the cultured sample.

3. The apparatus according to claim 1 or 2, wherein, At least one independently accessible module includes a lid and latching mechanism for sealing the culture chamber.

4. The apparatus according to claim 1, wherein, The module includes components for controlling one or more of the gas composition, humidity, and temperature within the culture chamber, which is used to hold the cultured sample.

5. The apparatus according to claim 1, wherein, At least one independently accessible module also includes a balancing component.

6. The apparatus according to claim 5 further includes an alignment member for precisely positioning the culture dish relative to the optical detection member.

7. The apparatus according to claim 6, wherein, The culture dish also includes a surface treatment to improve wettability for accommodating individual culture samples and / or processing fluids.

8. The apparatus according to claim 6, wherein, The culture dish is operatively associated with one or a combination of RFID, barcode, or OCR systems to capture details unique to each individual culture sample.

9. The apparatus of claim 8, further comprising a display for displaying the captured details.

10. The apparatus according to claim 4, wherein, A component for controlling one or more of the gas composition, humidity and temperature is suitable for controlling one or more of the gas composition, humidity and temperature of an individual cultured sample.

11. A method for assessing the viability of cultured samples, comprising the following steps: Individually identifiable biological samples are placed in a substantially elliptical arrangement in a culture chamber of an independently accessible module. The culture dish includes a plurality of spaced microwells for accommodating individual culture samples. Operablely associate an independently accessible module with a light source and an optical detection component, the optical detection component having an objective lens system and located outside the controlled environment and through the observation axis of the module, wherein the optical detection component is adapted to be limited to one or a combination of the following movements: in the XY plane perpendicular to the observation axis and in the Z direction including the observation axis; The objective system provides elliptical rotational movement of the objective lens, wherein the elliptical rotational movement of the objective lens is independent of the optical detection member, so as to enable scanning of an observation area around the observation axis, the observation area including each of the plurality of individually identifiable samples, wherein the movement of the optical detection member in conjunction with the elliptical rotation of the objective lens is substantially eccentric or orbital. Using an elliptical rotational movement of the objective lens independent of the optical detection element, the optical detection element images a substantially elliptical, individually identifiable sample by means of time-lapse measurements; The optical detection component includes a camera, which is supported by and spaced from a motor belt assembly, the motor belt assembly providing elliptical rotational motion of the objective lens; The independently accessible modules are multiple, and each independently accessible module can be removed from the device independently of the other independently accessible modules without affecting the functionality of the other independently accessible modules.

12. The method of claim 11, further comprising the step of: The culture chamber is precisely positioned relative to the optical detection component.

13. The method according to claim 11 or 12, further comprising the step of: Images of individually identifiable samples are sent to a data processing unit to obtain time-lapse measurements of the development of individual samples.

14. The method according to claim 11, wherein, The optical detection component includes a condenser lens system for enhancing the contrast and uniform illumination of the cultured sample.

15. The method of claim 11, further comprising the step of: Independently control one or a combination of temperature, gas supply, CO2 level and humidity in an independent culture chamber.

16. The method according to claim 11, wherein, The steps for imaging individually identifiable samples include using gamete coordination as a reference point for assessing subsequent sample developmental events in time-lapse measurements.

17. The method according to claim 11, wherein, The cultured samples include thawed embryos, and the imaging steps include measurements to assess embryo expansion and viability.

18. An apparatus suitable for providing automated evaluation of culture samples, the apparatus comprising: A processor component suitable for operating according to a predetermined instruction set, the means, in combination with the instruction set, being suitable for implementing the method of any one of claims 11 to 17.

19. A computer-readable storage medium having stored thereon computer program instructions that, when executed, perform the method according to any one of claims 11 to 17.

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