阅读说明：本技术 用于显微技术的方法与装置 (Method and apparatus for microscopy ) 是由 爱德华多·沃马 西蒙·乔纳森·思朋斯 塞缪尔·罗丝·加兰·蓝阳 本尼迪克特·约翰·斯图尔特-斯 于 2016-03-15 设计创作，主要内容包括：本发明提供利用通过在延时测量间隔内同时地捕获生物样本的亮视野图像与暗视野图像的显微技术来评估生物样本的发育活力。(The present invention provides for assessing the developmental viability of a biological sample using microscopy techniques by simultaneously capturing a bright field image and a dark field image of the biological sample within a time-delayed measurement interval.)

1. A method for assessing developmental viability of an in vitro fertilisation sample of an organism using microscopy techniques, the method comprising the steps of:

selectively activating a bright-field light source and a dark-field light source from a combined dark-field and bright-field illuminator, the dark-field light source being concentrically disposed about the bright-field light source;

selectively illuminating a dark-field light path and a bright-field light path, respectively, with a composite dark-field and bright-field lens system of the combined dark-field and bright-field illuminator, wherein the composite lens system includes a focal point common to the dark-field light path and the bright-field light path for positioning a biological sample thereat, and wherein the composite lens system includes: a first lens to focus bright field illumination from the bright field light source to form a bright field light path; and a second aspheric lens arrangement arranged concentrically with the bright field light path so as to focus dark field illumination from the dark field light source to form a dark field light path;

bright-field and dark-field time-lapse images of the biological sample are selectively captured within the time-lapse measurement interval.

2. A method as claimed in claim 1, wherein the dark field light source and the bright field light source are isolated from each other.

3. A method as claimed in claim 1 or 2, wherein the dark field light path and the bright field light path are isolated from each other.

4. A method as claimed in claim 2 or 3, wherein the isolation is one or a combination of:

performing optical isolation;

electrically isolating;

and (4) thermal isolation.

5. A method as claimed in any one of claims 1 to 4, wherein the dark field light source and the bright field light source are located on the same printed circuit assembly and are separated from each other by one or a set of gaskets and adhesive.

6. A method as claimed in any one of claims 1 to 5 wherein the step of selectively activating either the dark field light source or the bright field light source comprises independently controlling the light sources by one or a combination of a software controller, an electronic switch controller and a mechanical switch controller.

7. A method as claimed in any one of claims 1 to 6 further comprising the step of:

generating a data set comprising a combination of captured bright-field and dark-field images from a plurality of time delay measurements; and

one or a combination of the captured images from the data set is selectively displayed for analysis.

8. A method as claimed in any one of claims 1 to 7, wherein the delay measurement interval is approximately 5 minutes.

9. A microscope arrangement for assessing developmental viability of an in vitro fertilization sample of an organism, the microscope arrangement comprising:

a combined dark-field and bright-field illuminator having a dark-field light source concentrically disposed about a bright-field light source, wherein the dark-field light source and the bright-field light source are each adapted for selective activation;

a composite dark-field and bright-field lens system adapted to selectively illuminate a dark-field light path and a bright-field light path, respectively, by a combined illuminator, wherein the composite lens system comprises a focal point common to the dark-field light path and the bright-field light path, and is further adapted to position an in vitro fertilization sample at the common focal point, and wherein the composite dark-field and bright-field lens system further comprises: a first lens to focus bright field illumination from the bright field light source to form a bright field light path; and a second aspheric lens arrangement disposed concentrically with the bright field light path to focus the dark field illumination from the dark field light source to form a dark field light path and to allow selective capture of a bright field image and a dark field image of the biological sample.

10. An apparatus as claimed in claim 9, wherein the aspheric lens arrangement comprises a toroidal aspheric lens.

11. An apparatus as claimed in claim 9 or 10, wherein the dark field light path and the bright field light path are isolated from each other.

12. An apparatus as claimed in claim 9, 10 or 11, further comprising a conical reflector which, in combination with the aspheric lens arrangement, isolates the dark field illumination path from the bright field illumination path.

13. An apparatus as claimed in claim 9, wherein the bright field light source and the dark field light source of the combined dark field and bright field luminaire form a composite light source comprising at least one LED as a bright field light source and a series of LEDs as dark field light sources, the dark field light sources being arranged concentrically with respect to the bright field light sources.

14. The apparatus of any of claims 9-13, further comprising a specimen platform positioned at a focal point of the compound lens system.

15. The apparatus of any of claims 9-14, further comprising:

a switching device for selectively activating either the dark-field light source or the bright-field light source;

a delay measurement apparatus for capturing a delay image within a delay measurement interval of dark field illumination and/or bright field illumination of an in vitro fertilization sample, respectively, positioned at a focal point of a compound lens system, wherein the focal point is common to the dark field light path and the bright field light path.

16. An apparatus adapted to culture an in vitro fertilization sample of an organism, the apparatus comprising:

a processing device adapted to operate according to a predetermined set of instructions,

the apparatus, in combination with the set of instructions, is adapted to perform the method steps as claimed in any one of claims 1 to 8.

Technical Field

The present invention relates to the field of clinical testing and evaluation of biological samples using microscopy. In particular, the invention relates to the use of dark and bright field microscopy techniques in the evaluation of biological samples. It will be convenient to hereinafter describe the invention in relation to methods and apparatus which may be used to record or observe embryos during embryo development, but it will be understood that the invention is not limited to this use.

Background

It should be appreciated that any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the invention. Further, the discussion throughout this specification comes about as a result of the inventors' implementation and/or determination of certain related technical problems by the inventors. Further, any discussion of material such as documents, devices, acts or knowledge in this specification is included to explain the context of the invention in terms of the inventors' knowledge and experience, and accordingly, no admission is made that any such discussion forms part of the prior art base or the common general knowledge in the relevant art in australia or elsewhere on or before the priority date of the disclosure and claims herein.

In short, as seen in the common designation of wikipedia, bright field microscopy is the simplest of all optical microscopy illumination techniques. Transmissive sample illumination, e.g., white light, is illuminated from below and viewed from above, and contrast in the sample is caused by absorption of a portion of the transmitted light in a dense region of the sample. Bright field microscopy can be considered the simplest of the range of techniques for illuminating a specimen in an optical microscope, and its simplicity makes it a popular technique. A typical appearance of bright field microscope images is a dark sample on a bright background, hence the term "bright field".

Dark field microscopy, or "dark background microscopy", also known under this name under wikipedia, describes a method of microscopy in light and electron microscopy that excludes non-scattering light beams from the image. Thus, the field of view around the specimen, or in other words where there is no specimen to spread the beam, is typically dark.

Fig. 1 is a graph providing a schematic comparison between portions of bright field and dark field microscopy techniques as commonly understood in the prior art. The differences in sample illumination (shown by stippling) between the bright-field optical arrangement and the dark-field optical arrangement are emphasized in fig. 1. The dark field in the left-hand exploded view utilizes a dark field stop 001 shown by a "spider stop" arranged below the condenser 002. The stop 001 blocks the center of the beam to form a hollow cone of light 003. This light does not enter the objective lens 004 directly. Only light scattered by the sample and entering the objective is considered as an image in the dark field. In contrast, the right-hand exploded view of fig. 1 shows a solid cone of light 005, which illuminates and enters objective lens 004 in bright field.

In general, bright field microscopy can be applied to view live or stained cells, which requires a simple setup requiring little preparation. However, biological samples typically have low contrast with little natural staining, so the sample typically needs to be stained and staining may destroy or introduce artifacts. Furthermore, the resolution may be limited to about 0.2 μm. In another aspect, dark field microscopy techniques are generally applicable to view live, unstained specimens. This can also require only a simple setup for a suitable dark field optical arrangement and advantageously provides contrast with unstained tissue, so that live cells can be observed. However, the tissue may need to be strongly illuminated, which may damage delicate samples.

Another known technique, phase contrast microscopy, is most useful for viewing clear, unstained living cells. Phase contrast imaging provides excellent images for bright field optics, and fine details that are not visible under bright field optics are displayed at high contrast. However, phase contrast imaging is not ideal for thick samples, with which thick samples may exhibit distortion-producing "halo effects" or "phase artifacts," which may present distorted details around the periphery of the sample.

Dark field microscopy has advantages. In this regard, dark field microscopy is ideal for viewing objects that are undyed, transparent, and absorb little or no light. Therefore, these specimens typically have a similar refractive index to their surroundings, making it difficult to distinguish them with other illumination techniques. Dark field imaging can be used to study marine organisms such as algae and plankton, diatoms, insects, fibers, hair, yeast and protozoa, as well as some minerals and lenses, thin polymers and some ceramics. Dark field imaging can also be used in live and smear cell and tissue experiments. It is a more useful technique in examining external details such as contours, edges, grain boundaries and surface defects rather than internal structures. Dark field microscopy is generally not considered because of more modern viewing techniques such as phase contrast and DIC (differential interference contrast), which provide more accurate, higher contrast images and can be used to view a greater number of specimens. However, as noted above, these techniques have their own drawbacks, such as, for example, warping as described above. Currently, dark field microscopes recapture their popularity to some extent and, when combined with other illumination techniques such as fluorescence, expand their possible use in certain areas.

Although dark field microscopy can result in beautiful and brilliant images, this technique is associated with a number of drawbacks. First, dark field images tend to be degraded, distorted, and inaccurate. Thus, specimens that are not thin enough or whose density varies along the slide may appear to have artifacts through the image. The preparation and quality of the slide can greatly affect the contrast and accuracy of the dark field image, so it is important to be particularly careful that the slide, stage, nose and light source are protected from small particles, such as dust, that will be present as part of the image. Similarly, if oil or water is needed on the condenser and/or the slide, almost no complete bubbles can be avoided. These vacuoles will cause image degradation, luminescence and distortion and even reduce the contrast and detail of the specimen. Dark fields also require an intense amount of light to work and this, in conjunction with the fact that it relies only on scattered light, can cause luminescence and distortion. The dark field may thus not be a reliable tool for obtaining accurate measurements of the specimen. Finally, when using and using dark field microscopes, several problems can arise. For example, the light quantity and the position, size and arrangement of the intensity condenser and diaphragm of the light need to be corrected to avoid any aberrations. However, dark fields have a variety of applications and are very good viewing tools, particularly when used in conjunction with other technologies. However, when this technique is used as part of experimental studies, it is necessary to take into account the limitations and knowledge of artifacts that may not be desirable.

With respect to recording or observing biological specimens, more particularly with respect to embryos during morphological/developmental processes, optical wavelength microscopy is suitable for this purpose, but bright field imaging or dark field imaging each typically requires a separate system setup comprising an optical arrangement. The lighting methods generally require to be independent and isolated from each other. Dark field microscopy requires that the area behind the specimen is not illuminated, but that the observation requires light to be transmitted through the specimen/object. For bright field viewing, light is displayed directly behind the specimen and a direct beam is focused on the specimen/object. Dark field illumination may be created by a cone/ring beam that intersects at a specimen/object viewed through a microscope positioned on the other side of the specimen bed relative to the light source.

One known microtechnical system for biological samples is provided by the company auxygyn. The company auxygyn successfully provides clear imaging without obstacles in the vicinity of the specimen being observed/recorded. However, the drawback of the auxygyn company system is that it is a large unit that is not customizable for designs where other interchangeable standards are available on the market, and the cost of the auxygyn company system is very high compared to other customized designs. Furthermore, while the auxygyn company system, which provides clean and clear imaging of embryos, can obtain good clear images, in configurations using custom petri dish variants, there is insufficient illumination of all wells for a given petri dish design. Fig. 9 is an illustration of the Auxogyn system superimposed on a commercially available sample module as an independent access sample incubation module of the present application. Fig. 12 is a similar illustration of the auxynx corporation system superimposed on applicants' module with an envelope that allows for optics covered in this illustration to contrast with the form factor that is significantly oversized for the auxynx corporation system. The image in fig. 12 shows the module of Auxogyn in the required location on the applicant's known device. As can be seen, it is very large and may require significant modifications, including modifications to the appearance of the overall module product to fit the auxygyn company design. As is apparent from fig. 9 and 12, the auxygyn company system is not suitable for the current instrumentation.

The sample well imaged in fig. 10 is not an illumination well, as can be seen by the background, as the shadows appear on both sides of the edge. And thus not even across all wells. It is contemplated that this is formed by the height of the plastic ring around the culture well in the culture dish. This is an exemplary currently used culture dish. This image is a microwell within a well in a petri dish.

The image in fig. 11 shows a well without shading.

Generally, the non-invasive early embryo viability assessment test (Eeva) by Auxogyn is based on embryo assessment^TM) In Vitro Fertilization (IVF) results can be improved by providing objective information about embryo viability for IVF clinics and patients. Eeva, when used in traditional embryo evaluation techniques^TMThe system may provide IVF clinics and their patients with the possibility of improving clinical success. With greater chances of success, it is possible to reduce multiple births by allowing a single embryo transfer to be used for a large number of patients. Eeva^TMThe transfer software of the system automatically analyzes the embryo development for the cell division time parameters which are scientifically and clinically verified. By Eeva against the developmental prospects of each embryo^TMThe quantitative data of the system, the IVF clinic, can optimize the treatment path for their patients undergoing IVF surgery.

Eeva from Auxogyn Corp^TMThe system is designed to fit into a daily IVF laboratory workflow system. Eeva^TMThe culture dish comprises a microwell which enables Eeva^TMThe individual development of each embryo can be tracked and allowed to grow under tissue culture techniques. Dark field Eeva^TMMicroscope adapted for most standard IVFAn incubator, and provides automatic dark field image capture and cell division tracking without embryologist intervention or excessive light exposure to embryos. Eeva^TMThe microscope screen fits on the outside of the incubator and allows the embryologist to control each Eeva without opening the incubator or disturbing the embryos^TMThe patient converses and views the latest images. Eeva^TMEmbryo development was automatically analyzed for scientific, clinically validated cell division time parameters and the future viability of each embryo to day 2 was predicted. From Eeva^TMIn combination with standard morphological grading, enables the IVF clinic to make better informed decisions about embryo selection and optimal patient treatment paths. Using Eeva^TMThe station can easily check for each Eeva^TMImages and videos of a patient session. Furthermore, in Eeva^TMThe downloadable reports and videos provided in the system may assist and improve the overall patient experience when consulting with a patient.

For general reference, the role of delay monitoring in embryo selection is described in the RB & E article authored by PeterKovacs, which is incorporated herein by reference and a portion of the content of this article is reproduced herein below.

Various delay systems are currently used. Two of the most widely used techniques, PrimoVision (Vitroffe)^TM) And Embryoscope (Fertilitech)^TM) Systems, both using brightfield techniques, however the EEVA described above^TM(viability assessment of early embryos, Auxogyn) System, using dark field techniques. The overall system includes a digital inverted microscope that takes images of embryos at 5-20 minute time lapse intervals, where these time intervals are well understood by those skilled in the art as being time periods commonly used in the art of microscopic observation generally. This image is processed by a custom image acquisition technique and then displayed on a computer screen. Images acquired at preset or selected intervals are then connected to a short sheet, which can then be rewound and fast forwarded for detailed analysis.

EEVA^TMThe system utilizes dark field illumination, which allows more accurate observation of embryonic leavesA cell membrane; thus, differentiation can be accurately monitored, but this method provides little information about intracellular morphology and is limited in its ability to track embryos with increasing numbers of cells over 2 days. Automated systems may confuse large fragments with blastomere cells, which may thereby affect their selection accuracy. Table 1 below provides a comparison of these systems.

TABLE 1

Some exemplary known uses for bright-field and dark-field viewing are as follows.

Dark field illuminators are commonly used on reflective surfaces to create a sharp contrast between the background and specific features (for OCR detection) or to defects such as scratches and package tears. Forming dark areas with light may sound like a strange idea, but projecting light at an angle to the object surface will cause it to deviate from the camera unless surface variations cause the light to deviate into the lens. Accordingly, if there is no surface aberration, nothing is seen by the vision system.

The company Adcancedillummation provides their RL5064 model with a "combined" bright-field/dark-field illuminator. This device has the dual functionality of bright and dark field illumination in a compact housing where bright and dark fields can be used independently. However, the problems in imaging living cells are apparent. In this aspect, stained (i.e., dead) cells can "absorb" light, with the primers being largely transparent except for the bulk of the live cells, substantially non-absorbing and relatively little scattering. Accordingly, as noted above, the dark field image may appear to be self-luminous similar to the fluorescent image. This design does not use a lens to control or focus the light from the LED onto the specimen. This requires the device to be in close proximity to the specimen, which is inconvenient in complex systems.

It should be noted that all systems developed for time-lapse imaging of embryos for the purpose of embryo evaluation as described above currently rely only on one or the other imaging system, either bright-field or dark-field. There are many advances in brightfield in embryo evaluation, and thus all known, current embryo morphology description and quality ranking systems rely on the use of brightfield images. It is and has been the most common method of embryo evaluation under an inverted or stereomicroscope. It also allows (to some extent) observation of intracellular structures as well as intracellular tissues (e.g., the appearance of the inner cell mass and trophectoderm, as well as the extent and type of cell disruption).

When referring to observing the morphology and developmental viability of embryos, there are a number of desirable attributes for the morphological system. These include the following:

1. bright field and dark field microscopy techniques are utilized to illuminate the method to take advantage of the combined information that can be obtained from the respective datasets of the two techniques. While the use of dark field illumination allows for greater contrast, bright field illumination is an industry standard for the examination of biological specimens, which provides more accurate information for algorithms that detect edges and highlight embryos.

2. Economic and clinical standards may require specifically tailored microscopy systems to focus illumination at specific controlled and repeatable locations.

3. From a commercial point of view, low cost lighting solutions are highly desirable.

4. Furthermore, it is highly desirable that the microscopy system is preferably adapted for dark-field and bright-field microscopy without complex components.

5. Although two specific light sources may be required for each of dark-field and bright-field illumination, it is highly desirable to combine two separate light sources in a single component while remaining isolated from each other.

6. Space limitations are unavoidable and therefore solutions to address space limitations are also highly desirable.

7. Illumination of custom dish or biological sample capsule geometry is provided.

8. The wavelength of the light is safe for the embryo.

9. Given the nature of the microtechnical systems for biological samples, it may be necessary to bring the thermal management of the microscope lens structure into contact with the otherwise necessary humid environment.

Disclosure of Invention

It is an object of the embodiments described herein to overcome or mitigate at least one of the above-identified disadvantages in the related art systems or at least to provide a useful alternative to the related art systems.

In one aspect, the present invention provides a method for assessing developmental viability of a biological sample using microscopy, the method comprising the steps of:

a bright-field image and a dark-field image of the biological sample are captured simultaneously within the delay measurement interval.

Preferably, said step of simultaneously capturing bright field and dark field images comprises the steps of:

selectively activating a dark field light source or a bright field light source;

illuminating a bright-dark field light path or a bright field light path by the composite bright field and dark field lens systems, respectively;

capturing a time-lapse image of dark-field illumination or bright-field illumination of a biological specimen positioned at a focal point of the compound lens system, respectively, wherein the focal point is common to the dark-field light path and the bright-field light path.

Preferably, the dark-field light source and the bright-field light source are isolated from each other. Preferably, the dark-field light path and the bright-field light path are isolated from each other.

Preferably, this isolation is one or a combination of optical isolation, electrical isolation, thermal isolation:

preferably, the step of selectively activating either the dark-field light source or the bright-field light source comprises independently controlling the light sources by one or a combination of a software controller, an electronic switch controller, and a mechanical switch controller.

Preferably, the method herein further comprises the steps of:

generating a data set comprising a combination of captured bright-field and dark-field images from a plurality of time delay measurements; and

one or a combination of the captured images from the data set is selectively displayed for analysis.

In a preferred embodiment, the delay measurement interval is about 5 minutes.

In another aspect, the present invention provides a light path guide for selective bright field or dark field illumination of a biological sample, comprising:

a composite lens system having a first lens for focusing the brightfield illumination from the brightfield light source to form a brightfield light path; and a second aspheric lens arrangement arranged concentric with the bright-field optical path for focusing the dark-field illumination from the dark-field light source and for enabling simultaneous capture of a bright-field image and a dark-field image of the biological sample.

Preferably, the aspheric lens arrangement comprises an annular aspheric lens.

Preferably, the dark-field light path and the bright-field light path are isolated from each other.

Preferably, the light path guide further comprises a conical reflector in combination with the aspheric lens arrangement to isolate the dark-field illumination path from the bright-field illumination path.

Preferably, the focus of the compound lens system is common to the focus of the dark-field light path and the bright-field light path.

In another aspect, the present invention provides an apparatus for assessing the developmental competence of a biological sample using microscopy, comprising:

a composite light source arrangement comprising a bright field light source and a dark field light source, wherein the bright field light source and the dark field light source are isolated from each other; and

such as the light path guide described herein.

Preferably, the apparatus further comprises a specimen platform positioned at a focal point of the compound lens system of the light path guide.

Preferably the apparatus further comprises:

a switching device for selectively activating either the dark-field light source or the bright-field light source;

a latency measurement device for capturing a latency image within a latency measurement interval of dark field illumination or bright field illumination of a biological specimen positioned at a focal point of the composite lens system, respectively, wherein the focal point is common to the dark field light path and the bright field light path.

Other aspects and preferred forms are disclosed in the specification and/or defined in the appended claims, which form a part of the description of the invention.

The present invention stems from the realization that, despite the deficiencies in the prior art of attempting to observe embryos or more generally biological sample development using bright field microscopes in combination with dark field microscopes, combining dark field observation with bright field observation allows more information to be obtained, provided that dark field can detect important and subtle differences in good morphology embryos to improve embryo selection, and also allows software algorithms, while bright field allows morphological analysis.

Further scope of applicability of the embodiments of the present invention will become apparent from the detailed description set forth hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure herein will become apparent to those skilled in the art from this detailed description.

Drawings

Other disclosures, objects, advantages and aspects of the preferred embodiments of the present invention, as well as other embodiments, are better understood by those skilled in the relevant art by reference to the following description of the embodiments taken in conjunction with the accompanying drawings, which are provided by way of illustration only and thus do not limit the disclosure herein, and in which:

FIG. 1 shows a comparison of bright field and dark field illumination for microscopy according to a prior art system;

FIG. 2 is a cross-sectional view of a microscopy system showing a dark field illumination configuration according to a preferred embodiment of the present invention;

FIG. 3 is a cross-sectional view of a microscopy system showing a bright-field illumination configuration according to a preferred embodiment of the present invention;

FIG. 4 is an exploded view of system components of the microscopy system of FIGS. 2 and 3;

fig. 5 is an assembly module including the components of the exploded view shown in fig. 4, and is an in-situ view of the optical element of the embodiment of fig. 2, 3 and 4, but from an angle substantially 180 ° from that of fig. 4.

Fig. 6 shows dark field test images of wells 5 and 9 of a culture dish of a biological sample, respectively, taken by the microscopy system of fig. 2 and 3, according to a preferred embodiment of the invention;

fig. 7 shows a brightfield test image of wells 5 and 9 of a petri dish of a biological sample, obtained by the microscopy system of fig. 2 and 3, respectively, according to a preferred embodiment of the invention.

Fig. 8 illustrates a Printed Circuit Assembly (PCA) for an illumination source including centrally disposed LEDs for selective dark field and bright field illumination, according to an embodiment of the invention.

FIG. 9 illustrates an example prior art dark field illumination module applied to a current incubator assembly, identifying that the illumination module is not adapted in known incubators and incubation instruments.

Fig. 10 shows a sample well illuminated with a prior art dark field system, which provides poor image quality highlighted by shading on both sides of the edge.

FIG. 11 is an image captured with darkfield illumination providing sufficient quality or automated algorithms for user review to assist in assessing embryo viability, in accordance with a preferred embodiment.

FIG. 12 shows a dark field prior art microscopy module in a desired position on a known incubator and culture instrument. Highlighted in the center of the dark-field illumination source is the bright-field illumination spatial envelope.

Fig. 13 shows a microscope optical arrangement according to another embodiment of the invention with an alternative sealing option and illustrates the assembly process including a similar lens and illumination component.

Detailed Description

The following description of the preferred embodiments of the present invention provides one or a combination of dark field and bright field illumination to image embryos/biological specimens in a culture dish having a sample well for holding a plurality of biological samples.

Referring to fig. 2 and 3, a combined dark field and bright field illuminator 200 for use in a time delay incubator for embryos is shown. Bright field illumination (best shown as BF in fig. 3) includes a light source 175, preferably an LED, mounted on a PCA177 (where 'PCA' is considered a printed circuit assembly or PCBA printed circuit board assembly) directly over the specimen positioned in the petri dish 166. This bright field beam is contained in the shape of a cylinder to direct a column of light through lens 115 to be focused on the specimen.

In addition to the improved benefits from bright field microscopy, dark field microscopy is advantageous in embryo evaluation applications in that it is capable of detecting external details such as embryo/cell contours and edges, making it well suited for detecting cell numbers and sizes and embryo sizes. Both the brightfield and darkfield observation types can and have been used individually by themselves to assess embryo development potential, but combining them and allowing the development and application of algorithms that exploit the full information gathered makes embodiments of the present invention an exceptionally powerful tool for IVF clinics. With specific reference to fig. 2, for dark field illumination, a light source 180 mounted on the PCA177 illuminates light parallel to the bright field light beam, but does not interfere or encroach on the bright field illumination light path.

The Auxogyn system module as described in the preamble can be used to specifically form bright field illumination within the preferred microscopy detection system for the present invention.

According to a preferred embodiment, the dark field illumination source 180 is preferably 42 LEDs in two concentric rings around the bright field illumination source LEDs 175. Three 2mm breaks per 120 degrees in this arrangement of LEDs allows for the inclusion of structural ribs for support. This beam, best indicated as DF in fig. 2, is included on the outside of collar 181 for bright field illumination. This light is also contained by a slightly tapered mirror 170 to direct the light through an annular aspheric lens component 169. This forms an annular light path for dark field illumination. The lens 169 forms a conical and annular beam that is focused on the specimen/object. Allowing a dark field to be formed in the absence of the bright field illumination LED175, driven to thereby form a dark field behind the specimen without bright field illumination.

Lenses 115 and 169 are specifically selected to provide a uniform focal plane to provide a focal point for each illumination path on the biological sample. Accordingly, a lens is positioned in the assembly to direct the light path to a specific location consistent with the bright field situation and the dark field situation where the specimen/object is to be positioned.

Lenses 115 and 169 are low cost. In this regard, the center of the dark field aspheric lens assembly 169 needs to be changed to have a hole of about 14mm diameter in the center for installation of bright field illumination. The low cost lens configuration and number of lenses rather than multiple lenses allows for smaller and cheaper components to create the same illumination.

The dark field and bright field illumination are formed concentrically about the same axis, with the two separate beam paths isolated from each other. Optionally, the light source travels along two different paths as shown in fig. 2 and 3. By physical isolation, light from the dark field region cannot reach the bright field region. The LED light sources are independent and can be controlled individually. The configuration of the light paths is concentric with each other. The software is able to switch from dark to bright without changing focus through the microscope assembly.

The two light sources are positioned on the same PCA and are preferably isolated from each other by the use of a gasket. Typically, the light sources are isolated by using a gasket, since the machined surfaces of the two components (PCB and machined collar) are not completely flat, and it is not cost effective to make them completely flat. So that a gasket as a soft foam material can be arranged between them to fill the gap between the two parts.

Another way in which this isolation in both light paths can be achieved is by potting or filling the electronic components with adhesive, which is commonly used for corrosion protection and shock protection. The adhesive/filler will form a light and air seal, although it may not necessarily be easily reversible and may be a permanent change, further not allowing the two parts to be removed from each other.

Alternatively, different materials may be used for the gasket between the two components as long as the materials do not allow light to transmit between the individual focusing assemblies.

In these preferred embodiments, the insulation should also not allow air to transfer between the components.

Separation of the two light sources can also be achieved by arranging the LEDs on two separate PCBs or PCAs, which would allow the dark field PCA to be positioned so that the light is contained within a collar, and this collar has a cover on it and only a small space for some of the wires to pass through to make the LEDs electrically accessible.

An O-ring may also be used in place of the gasket that is clamped down against the PCB (since O-rings may be too stiff and are not typically used). The O-ring is pressed against the PCB and blocks the source bleed light between the dark field light path to the bright field light path, which needs to be kept dark for dark field operation. A single PCA allows for component cost reduction by having both light sources mounted to the same PCB (printed circuit board). As best shown in fig. 4, isolation of both the bright and dark field light sources may be achieved on one component by using a washer arranged and pressed by the intermediate support 3 to isolate the bright field light source from the dark field light source. Advantageously, this provides a simple means of allowing a single PCA to be used with multiple light sources.

The spatial constraints of the lens configuration require a tight arrangement of the lens to the specimen bed/dish. The use of an aspheric lens assembly removes the need for multiple lenses to focus the light to an appropriate focal point. The use of LEDs that are concentric with each other on the PCB, and thus on the same axis, makes the designed package significantly smaller than the reflective surfaces for dark field illumination with light paths following additional axes.

The working diameter of the microscope light path is about 14mm and the illumination is deteriorated within 1mm on either side thereof. This is used to define the aspherical lens as being critical to the clarity of the image due to the geometry of the specimen dish. The conical light source can be narrow or wide enough to illuminate all wells in the dish. This may be varied or selectable by the size and shape of the cone beam, the shape of the mirror, which is a machined component and is capable of directing light into the aspheric lens assembly. During the test several shapes of conical mirrors were tested to form the basic geometry now in use. The most effective inner dimension was found to be about 14mm in diameter and about 24mm in outer diameter. This diameter is interrupted only by structural ribs, which allows the brightfield lens to remain in place. The three ribs are located 120 apart and are approximately 2mm thick. This allows for a more uniform cone of dark field illumination with less thickness and reduced structural ribs.

It has been found that certain wavelengths are less harmful to the specimen than other wavelengths. In this regard, the closer the LED wavelength is to 625nm, the less damage the illumination has to the specimen. The LED thus selected is as close to 625nm as possible.

Thermal management of the lens is required. Due to the connection to the chamber, which is the same chamber as described in the GERI patent, this lighting module is screwed into the wall of its chamber and then sealed with O-rings. This O-ring seal serves to seal the chamber from moisture leakage to maintain the proper humidity range for the embryo, and also has a temperature locally, the temperature and humidity difference across the lens can cause condensation on the lens, which will diffract as light passes through, thereby distinguishing the light path. A portion of the chamber can be seen in the cross-sections of fig. 2 and 3. The white space is just above the culture dish 166. There is a change in humidity, and thus the PCA requires sealing from the humidity of the chamber. This seal is formed by using several O-rings 179. To maintain control of the temperature of the lens without forming condensation of the lens, we do not actively control the temperature of the lens by using heating elements, but we specifically select materials that will allow the temperature to pass through them to provide a gradual change in temperature rather than a sharp difference. The temperature difference is across the lens and the surrounding mounting member material is selected to provide good heat transfer around the lens to maintain a locally defined temperature change.

The test was performed to simulate illumination of the specimen/embryo in the dish by bright field illumination and dark field illumination. Several concepts of reflection patterns on dishes were tested by using a prototype reflection guide directed towards an aspheric lens assembly. The "steering" of the light source allows for variations in the geometry of the cone of light focused on the culture dish. The iteration of several prototypes allows the illumination to be uniform across all wells in the culture dish. In the development of the preferred embodiment, the pursuit of clarity or shading and the utilization of earlier adopted components, such as by iteration of the mirror 171 in tests to demonstrate the concept, allows the selection of the appropriate diameter of the aspheric lens assembly 169 and the central aperture. Within the aspheric lens assembly geometry, the best-fit aspheric lens is chosen as an off-the-shelf component, and then a hole is machined in its middle to form a bright-field light path. This is currently labeled BF, and serves as a path for blocking light to allow dark field measurements. Some test images are shown in the drawings, with figure 6 showing dark field images of wells 5 and 9 of a sample petri dish respectively, and figure 7 showing bright field images of wells 5 and 9 of the same petri dish respectively under test.

Illumination of the custom dish geometry is provided by using a specific light direction and focusing to the specimen around existing obstacles on the custom dish. Special lenses are used to create the beam geometry to illuminate all the specimens on the petri dish. Advantageously, this has been specifically tailored to the dish geometry to achieve the best lighting results. The custom culture dish geometry illustratively used in the preferred embodiment is disclosed in applicant's published international (PCT) patent specification No. wo 2014/131091. Another dish useful in a preferred embodiment of the invention is disclosed in applicant's published international (PCT) patent specification No. wo 2014/106286. By using a culture dish geometry as disclosed in WO2014/131091, it is necessary to use an illumination device to illuminate all microwells within one well on the culture dish. As described previously, the Auxogyn lighting module does not clearly illuminate all of the microwells in a well on a petri dish. The particular geometry of dark field illumination is used for all microwells on the boat.

Returning to fig. 2, a cross-sectional view of an embodiment of the present invention is shown and indicates a configuration for dark field illumination. The arrows shown in fig. 2 represent the path of the light beam in a rough estimation. The PCA177 has LEDs attached to the board. Light generated by the LEDs on the board of the PCA177 is directed through a dark field diffuser 172, which diffuses the light more uniformly than individual spots originating from multiple dark field source LEDs. The light beam is then directed through a conical outer portion 170, which directs the light into the surface of an aspheric annular lens assembly 169. This then focuses the light onto the culture dish 166.

Fig. 3 shows a cross-sectional view of the same embodiment of the invention as fig. 2, such that the arrows of fig. 3 depict in this case the light paths of the bright field illumination. In the bright field case, the PCA177 has a centrally mounted single LED that directs light for bright field illumination directly over the petri dish and well containing the specimen. Light emerges and is contained within the intermediate cylinder by an optical insulator 178 clamped against the PCA. The light is directed through a bright field diffuser 173, which diffuses to form a single propagation of light. The light beam is now contained within the collar and directed through the biconvex lens 115 which focuses the light onto the culture dish 166.

Fig. 4 shows an exploded view of the components of the preferred embodiment optical system as shown in fig. 2 and 3. In the exploded view of fig. 4, PCA177 shows 42 dark field source LEDs 180 in a circular configuration with three gaps of about 2mm each 120 degrees aligned with support ribs on the intermediate support 3. It will be appreciated by those skilled in the art that the arrangement of dark field source LEDs 180 or indeed bright field source LED/s 175 may be configured in other forms, but still provide illumination suitable for dark field illumination in accordance with embodiments of the present invention.

The cylindrical concentric design of the preferred embodiment with bright field and dark field illumination sources (175 and 180, respectively) on the same PCA plate 177 can be used in conjunction with an aspheric lens assembly 169 to provide an annular beam that is appropriately narrowed and focused on the specimen bed for both types of illumination. This allows a compact design that fits within a very small confined space while providing clear illumination of the specimen on the culture dish. The customized distance to the dish is adjusted by selecting two lens arrangements to provide focusing of light to the specimen bed. The location of the lens is important to the illumination target. The positioning position is required to be the same as the distance between the specimen and the predetermined focal length of the lens, so that the specimen is irradiated with light. The focal length is the distance from the lens where the light is focused. Locating the position is another way of specifying the exact position, or the position of the ledge on which the lens is located. This advantage is that low cost lenses are available and adapted to allow illumination of the specimen. In this respect, in a preferred embodiment that accommodates two sources of light (bright field and dark field), a low cost illumination solution is provided by the PCA 177. Further, the lens is preferably made of acrylic to reduce costs. The compact assembly reduces the required size of the lens and furthermore the number of lenses used for focusing the light. Thus advantageously, both the bright-field and dark-field light sources are on the same PCA to reduce parts count and cost.

The independent light sources may be controlled by software to allow switching between bright field illumination and dark field illumination methods. Advantageously, no moving mechanical parts are required. Furthermore, no filters or blocking members are required to move into position.

The concentric lens and illumination source arrangement of the preferred embodiment allows the system to be housed in a space of a defined area. Concentric illumination sources require that the bright-field and dark-field illumination be controlled independently to allow independent capture, viewing, or inspection of dark-field or bright-field images. In a preferred embodiment, the use of separate electronic and programmable software controls can be used to independently select each light source. By incorporating the illumination source in the same footprint as the lens, no mechanical movement is required to select and view either the bright-field or dark-field images.

In a preferred embodiment, the lighting assembly in fig. 3 is packaged in a vertical height of about 26 mm. It will be appreciated by those skilled in the art that the overall design factors, including focal length, target specimen location, and area to be illuminated, lens selection and design, may be varied to reduce or increase the size of the assembly. In a preferred embodiment, the components may be specifically designed and selected to illuminate and match the specific details of the overall instrument assembly, microscope, camera and embryo dish. The primary combination of geometrically concentric illumination sources, lens components, control PCA and software can be varied to suit a wide range of applications outside of this preferred embodiment.

In a preferred embodiment and referring to fig. 8, a single central Light Emitting Diode (LED)175 is used as the illumination source for bright field image capture, viewing or inspection. The bright field LEDs 175 are directed into the bright field diffuser 173 and through the lenticular lens 115. An array of LEDs 180 is centrally located around the center LED for producing illumination for dark field image capture, viewing or inspection. The outer ring of LEDs 180 is used to form a uniform light ring, which in the preferred embodiment passes through the diffuser 172 and into the annular non-spherical lens assembly 169.

Time lapse image capture can be performed in the same manner as conventional time lapse measurements by the composite dark field and bright field microscope lens assembly of the preferred embodiment, but the added benefit of selective switching is the ability to switch between dark field illumination of the sample on the one hand and dark field illumination on the other hand without moving the sample to an alternative optical arrangement, or having a time lapse to re-arrange the optical assembly for an alternative illumination area.

Furthermore, the fact that both bright field and dark field observations are actually achieved precisely at the same time (within seconds) and are continuously recorded, allows the development of advanced algorithms that take into account the rapidly developing nature of human embryos, which can undergo significant changes (e.g. prokaryotic membrane rupture) within minutes when at a moment of importance.

In a preferred form, the delay is such that the embryo incubator includes the following advantageous features:

combined dark-field/bright-field illumination source

Specific configurations of the combined lens assembly and the aspherical lens as described above

Dark field and bright field sources have independent controls that can be one of software, electronic, or mechanical

Ability to separate light sources so as not to allow cross-contamination of light

Ability to isolate electronics/optics from room environmental conditions

Describing a specific cone design for focusing dark field illumination

Selection of illumination wavelength specifically to reduce damage to embryos

Ring of LEDs for dark field illumination

Automatic capture of both bright-field and dark-field images

Ability to check bright and dark images on the incubator software platform

Position of the illuminator relative to the embryo

Selectively displaying the dark-field and bright-field images, which allows all images to be examined at substantially the same time point without requiring manual intervention or removal of the embryo from the environmentally controlled room. The capture and display of both DF/BF allows the user to manually examine both images for better results for viability of the embryo to be used.

Automated analysis of both bright and dark fields is used to assist in selecting the best embryo for implantation.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations uses or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

As the present invention may be embodied in several forms without departing from the spirit of the essential characteristics of the invention, it should also be understood that the above-described embodiments are not limiting of the present invention unless otherwise specifically noted, but rather should be construed broadly within its spirit and scope as defined in the appended claims. The described embodiments are to be considered in all respects only as illustrative and not restrictive.

Various modifications and equivalent arrangements are intended to be included within the spirit and scope of the invention and appended claims. Thus, the specific embodiments are to be understood as being illustrative of the various ways in which the principles of the invention may be practiced. In the following claims, means-plus-function limitations are intended to cover structures that perform the defined function and not structural equivalents, but equivalent structures. For example, although a nail and a screw may not be structural equivalents in that a nail employs a cylindrical surface to secure wooden parts together, whereas a screw employs a helical surface to secure wooden parts together, in the environment of fastening wooden parts, a nail and a screw are equivalent structures.

It should be noted that where the terms "server," "secure server," or similar terms are used herein, communication devices that may 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 communication device type. Thus, a communication device may include, without limitation, a bridge, router, bridge router (router), switch, node, or other communication device that may or may not be secure.

It should also be noted that flow charts are used herein to illustrate various aspects of the invention and should not be construed to limit the invention to any particular logic flow or logic implementation. The described logic may be partitioned into different logic modules (e.g., procedures, modules, functions, or subroutines) without changing the overall results or otherwise departing from the true scope of the invention. In general, logic elements (e.g., logic gates, looping primitives, conditional logic, and other logic structures) may be added, modified, omitted, performed in a different order, or performed with different logic structures without changing the overall results or otherwise departing from the true scope of the invention.

Embodiments of the invention may be embodied in many different forms, including for a processor(e.g., a microprocessor, microcontroller, digital signal processor, or general purpose computer, and in this regard, any commercial processor may be used to implement embodiments of the invention as a single processor, as a series or parallel set of processors in a system, and as such, examples of commercial processors include, but are not limited to, merceded^TM,Pentium^TM,Pentium II^TM,Xeon^TM,Celeron^TM,Pentium Pro^TM,Efficeon^TM,Athlon^TM,AMD^TMEtc.), a programmable logic device (e.g., an area programmable gate array (FPGA) or other PLD), a discrete component, an integrated circuit (e.g., an Application Specific Integrated Circuit (ASIC)), or any other device including any combination thereof. In an exemplary embodiment of the invention, significantly all communications between a user and a server are performed as a set of computer program instructions that are converted into a computer-executable form, stored per se in a computer-readable medium, and executed by a microprocessor under the control of an operating system.

Computer program logic that performs all or part of the functionality described herein may be embodied in a variety of forms, including source code forms, computer executable forms, and various intermediate forms (e.g., forms generated by an assembler, compiler, linker, or locator). Furthermore, there are hundreds of available computer languages that may be used to implement embodiments of the present invention, with the more common being Ada, Algol, APL, awk, Basic, C + +, Conol, Delphi, Eiffel, Euphoria, Forth, Fortran, HTML, Icon, Java, Javascript, Lisp, Logo, Mathimatia, MatLab, Miranda, Modula-2, Oberon, Pascal, Perl, PL/I, Prolog, Python, Rexx, SAS, Scheme, sed, Silula, Smalltalk, Snaol, SQL, visual Basic, SualC +, Linux, and any of the series of programming instructions. The source code may define and use a variety of data structures and communication information. The source code may be in computer-executable form (e.g., via an annotator), or the source code may be converted (e.g., via a translator, assembler, or editor) into computer-executable form.

The computer program may be fixed in any form (e.g., source code form, computer executable form, or intermediate form) either permanently or temporarily in a tangible storage medium such as a semiconductor memory device (e.g., RAM, ROM, PROM, EEPROM or flash programmable RAM), a magnetic memory device (e.g., a diskette or hard disk), an optical memory device (e.g., a CD-ROM or DVD-ROM), a PC card (e.g., a PCMCIA card), or other memory device. The computer programming may be embodied in any form of signals transmittable to a computer using any of a variety of communication technologies, including, but in no way limited to, analog technologies, digital technologies, optical technologies, wireless technologies (e.g., bluetooth), networking technologies, and internetworking technologies. The computer programming may be distributed in any form as a removable storage medium with accompanying printed or electronic documentation (e.g., shrink wrapped software), pre-loaded with the computer system (e.g., on system ROM or fixed disk), or distributed from a server or electronic bulletin board over a communication system (e.g., the internet or world wide web).

The hardware logic (including programmable logic for a programmable logic device) that performs all or part of the functionality described herein may be designed, captured, simulated, or recorded electronically using conventional manual methods of design, or may be designed using a variety of tools, such as computer-aided design (CAD), a hardware description language (e.g., VHDL or AHDL), or a PLD programming language (e.g., PALASM, ABEL, or CUPL). Hardware logic may also be incorporated into a display screen to perform embodiments of the present invention, and may be a segmented display screen, an analog display screen, a digital display screen, a CRT, an LED display screen, a plasma screen, a liquid crystal diode screen, and the like.

Programmable logic may be fixed permanently or temporarily in a tangible storage medium such as a semiconductor memory device (e.g., RAM, ROM, PROM, EEPROM, or flash programmable RAM), a magnetic memory device (e.g., a floppy disk or a hard disk), an optical memory device (e.g., a CD-ROM or a DVD-ROM), or other memory device. Programmable logic may be fixed in a signal that is transmittable to a computer using any of a variety of communication technologies, including, but in no way limited to, analog technologies, digital technologies, optical technologies, wireless technologies (e.g., bluetooth), networking technologies, and internetworking technologies. The programmable logic circuits may be distributed as a removable storage medium with accompanying printed or electronic files (e.g., shrink-wrapped software), pre-loaded with a computer system (e.g., on system ROM or fixed disk), or distributed over a communication system (e.g., the internet or world wide web) from a server or electronic bulletin board.

The terms "comprises," "comprising," "including," "has," "having," "includes," and "including" when used in this specification are taken to specify the presence of stated features, integers, steps, or components, but do not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. Thus, throughout the specification and claims, the terms "comprising," "including," "having," "including," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is, in a sense of "including, but not limited to," unless the context clearly requires otherwise.